pmir report vector  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pmir report vector
    Binding of miR-96 in the 3’ <t>UTR</t> of FOXO1. LNCaP cells were transfected with 10 nM pre-miR-96, anti-miR-96, pre-miR-NC #1 or combination of pre-miR-96 and anti-miR-96 as well as 500 ng <t>pMiR</t> report β-Galactosidase control plasmid and 500 ng pMiR report plasmid for FOXO1 3’ UTR A ) binding site 1 at position 264-270 and B) binding site 2 at position 2138-2145. * P
    Pmir Report Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmir report vector/product/Thermo Fisher
    Average 99 stars, based on 408 article reviews
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    pmir report vector - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "The Antiapoptotic Function of miR-96 in Prostate Cancer by Inhibition of FOXO1"

    Article Title: The Antiapoptotic Function of miR-96 in Prostate Cancer by Inhibition of FOXO1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080807

    Binding of miR-96 in the 3’ UTR of FOXO1. LNCaP cells were transfected with 10 nM pre-miR-96, anti-miR-96, pre-miR-NC #1 or combination of pre-miR-96 and anti-miR-96 as well as 500 ng pMiR report β-Galactosidase control plasmid and 500 ng pMiR report plasmid for FOXO1 3’ UTR A ) binding site 1 at position 264-270 and B) binding site 2 at position 2138-2145. * P
    Figure Legend Snippet: Binding of miR-96 in the 3’ UTR of FOXO1. LNCaP cells were transfected with 10 nM pre-miR-96, anti-miR-96, pre-miR-NC #1 or combination of pre-miR-96 and anti-miR-96 as well as 500 ng pMiR report β-Galactosidase control plasmid and 500 ng pMiR report plasmid for FOXO1 3’ UTR A ) binding site 1 at position 264-270 and B) binding site 2 at position 2138-2145. * P

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation

    2) Product Images from "miR-582-5p Is Upregulated in Patients with Active Tuberculosis and Inhibits Apoptosis of Monocytes by Targeting FOXO1"

    Article Title: miR-582-5p Is Upregulated in Patients with Active Tuberculosis and Inhibits Apoptosis of Monocytes by Targeting FOXO1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078381

    FOXO1 is a direct target of miR-582-5p. (A) Alignment between the predicted miR-582-5p target site of FOXO1 3′UTR region and miR-582-5p. (B) Real time RT-PCR and western blot analysis showing FOXO1 mRNA and protein expression levels in THP-1 cells transfected with miR-582-5p mimics or mimics NC, respectively. (C) Co-transfection of miR-582-5p mimics/mimics NC and FOXO1 3′UTR-luciferase reporter vector into HEK-293T cells demonstrated that significant decrease in luciferase activity was only found in reporter vector that contained a wild type sequence (FOXO1-3′UTR-wt), not in vector that contained a mutant sequence (FOXO1-3′UTR-mut) within the miR-582-5p binding site. Values were presented as relative luciferase activity after normalization to Renilla luciferase activity. FOXO1-3′UTR-wt: pMIR-FOXO1-3′UTR-wt vector; FOXO1-3′UTR-mut: pMIR-FOXO1-3′UTR-mut vector. (D) Representative flow cytometric plots showing apoptotic ratio of THP-1 transfected with FOXO1 siRNA or negative controls of siRNA (siRNA NC) (left panel). The apoptotic percentage of THP-1 cells transfected with FOXO1 siRNA were significantly lower than those transfected with negative control siRNA (siRNA NC) ( p
    Figure Legend Snippet: FOXO1 is a direct target of miR-582-5p. (A) Alignment between the predicted miR-582-5p target site of FOXO1 3′UTR region and miR-582-5p. (B) Real time RT-PCR and western blot analysis showing FOXO1 mRNA and protein expression levels in THP-1 cells transfected with miR-582-5p mimics or mimics NC, respectively. (C) Co-transfection of miR-582-5p mimics/mimics NC and FOXO1 3′UTR-luciferase reporter vector into HEK-293T cells demonstrated that significant decrease in luciferase activity was only found in reporter vector that contained a wild type sequence (FOXO1-3′UTR-wt), not in vector that contained a mutant sequence (FOXO1-3′UTR-mut) within the miR-582-5p binding site. Values were presented as relative luciferase activity after normalization to Renilla luciferase activity. FOXO1-3′UTR-wt: pMIR-FOXO1-3′UTR-wt vector; FOXO1-3′UTR-mut: pMIR-FOXO1-3′UTR-mut vector. (D) Representative flow cytometric plots showing apoptotic ratio of THP-1 transfected with FOXO1 siRNA or negative controls of siRNA (siRNA NC) (left panel). The apoptotic percentage of THP-1 cells transfected with FOXO1 siRNA were significantly lower than those transfected with negative control siRNA (siRNA NC) ( p

    Techniques Used: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Cotransfection, Luciferase, Plasmid Preparation, Activity Assay, Sequencing, Mutagenesis, Binding Assay, Negative Control

    3) Product Images from "miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1"

    Article Title: miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1

    Journal: Cancer Cell International

    doi: 10.1186/s12935-017-0398-6

    miR-335 negatively regulated POU5F1 gene expression. a Bioinformatics study indicated that miR-335 was partially complementary to the 3′-UTR of POU5F1 mRNA. b Different doses of pre-miR-335 were co-transfected with the specific pMIR-REPORT construct into HEK293 cells. Luciferase activities were measured and normalized to the phRL-TK activities. c The effect of pre-miR-335 gradually diminished in 2 weeks. d Osteosarcomas cells were transfected with pre-miR-335, anti-miR-335 or their control respectively. After induction for 7 and 14 days, the cells were harvested for measurement of POU5F1 mRNA using qRT-PCR and e the protein expression using Western blot. Untreated cells was set as control, and β-actin acts as an internal control. All experiments were carried out at least triplicates and the data were presented as the mean ± SD. Student t test was performed to evaluate the difference. *P
    Figure Legend Snippet: miR-335 negatively regulated POU5F1 gene expression. a Bioinformatics study indicated that miR-335 was partially complementary to the 3′-UTR of POU5F1 mRNA. b Different doses of pre-miR-335 were co-transfected with the specific pMIR-REPORT construct into HEK293 cells. Luciferase activities were measured and normalized to the phRL-TK activities. c The effect of pre-miR-335 gradually diminished in 2 weeks. d Osteosarcomas cells were transfected with pre-miR-335, anti-miR-335 or their control respectively. After induction for 7 and 14 days, the cells were harvested for measurement of POU5F1 mRNA using qRT-PCR and e the protein expression using Western blot. Untreated cells was set as control, and β-actin acts as an internal control. All experiments were carried out at least triplicates and the data were presented as the mean ± SD. Student t test was performed to evaluate the difference. *P

    Techniques Used: Expressing, Transfection, Construct, Luciferase, Quantitative RT-PCR, Western Blot

    4) Product Images from "MicroRNA-152 Regulates DNA Methyltransferase 1 and Is Involved in the Development and Lactation of Mammary Glands in Dairy Cows"

    Article Title: MicroRNA-152 Regulates DNA Methyltransferase 1 and Is Involved in the Development and Lactation of Mammary Glands in Dairy Cows

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0101358

    miR-152 regulates DNMT1 expression by binding its 3′ UTR. A: miR-152 binds to the 3′ UTR of DNMT1 in position 48–55. B, C: DNMT1 3′ UTR was inserted downstream of the luciferase pMIR-reporter vector. Luciferase activity was measured in DCMECs and HEK-293 cells. 1 phRL-TK and empty pMIR-REPORT vector cotransfection; 2 miR-152, phRL-TK, empty pMIR-REPORT vector cotransfection; 3 miR-NC, phRL-TK, DNMT1-pMIR-REPORT vector cotransfection and 4 miR-152, phRL-TK, DNMT1-pMIR-REPORT vector cotransfection. Luciferase activities of DNMT1-pMIR-REPORT are markedly decreased in cells transfected with miR-152 compared to those of reporter plasmids with miR-NC or empty vectors. D: Dnmt1 mRNA expression after treatment with miR-152, Anti-152, 5-Aza or their respective controls in DCMECs. E: DNMT1 protein expression of DCMECs treated with miR-152, Anti-152, 5-Aza or their respective controls. MiR-152 targets DNMT1 and represses the expression of DNMT1 mRNA and protein. The mRNA and protein expression level of DNMT1 are significantly decreased after 48 h treatment with 5-Aza in DCMECs. Values are means ± SD, * P
    Figure Legend Snippet: miR-152 regulates DNMT1 expression by binding its 3′ UTR. A: miR-152 binds to the 3′ UTR of DNMT1 in position 48–55. B, C: DNMT1 3′ UTR was inserted downstream of the luciferase pMIR-reporter vector. Luciferase activity was measured in DCMECs and HEK-293 cells. 1 phRL-TK and empty pMIR-REPORT vector cotransfection; 2 miR-152, phRL-TK, empty pMIR-REPORT vector cotransfection; 3 miR-NC, phRL-TK, DNMT1-pMIR-REPORT vector cotransfection and 4 miR-152, phRL-TK, DNMT1-pMIR-REPORT vector cotransfection. Luciferase activities of DNMT1-pMIR-REPORT are markedly decreased in cells transfected with miR-152 compared to those of reporter plasmids with miR-NC or empty vectors. D: Dnmt1 mRNA expression after treatment with miR-152, Anti-152, 5-Aza or their respective controls in DCMECs. E: DNMT1 protein expression of DCMECs treated with miR-152, Anti-152, 5-Aza or their respective controls. MiR-152 targets DNMT1 and represses the expression of DNMT1 mRNA and protein. The mRNA and protein expression level of DNMT1 are significantly decreased after 48 h treatment with 5-Aza in DCMECs. Values are means ± SD, * P

    Techniques Used: Expressing, Binding Assay, Luciferase, Plasmid Preparation, Activity Assay, Cotransfection, Transfection

    5) Product Images from "Decrease in Lymphoid Specific Helicase and 5-hydroxymethylcytosine Is Associated with Metastasis and Genome Instability"

    Article Title: Decrease in Lymphoid Specific Helicase and 5-hydroxymethylcytosine Is Associated with Metastasis and Genome Instability

    Journal: Theranostics

    doi: 10.7150/thno.21389

    Both miR-26b-5p and miR-29c-3p inhibited TET2 and TET3 expression and were silenced by LSH. (A) Ectopic expression of miR-26b-5p (Left) and miR-29C-3p (Right) in MCF-7 cells. Relative expression levels of TET2 (B) and TET3 (C) were detected by RT-PCR after the transfection of miRs as indicated. (D, E) Expression levels of TET2 and TET3 were analyzed after the transfection of miRs as indicated in HK1 (F) and HNE3 (G) cells, whereas LSH protein level unchanged. (F) Luciferase reporter assay in 293 cells together with miRs as indicated, and with transfection of pMiR-Report-TET2 3'-UTR sense or pMiR-Report-TET2 3'-UTR antisense as indicted. (G) ELISA of 5-hmC was used to assess 5-hmC levels from genomic DNA derived from HNE3 cells after introduction of miRs as indicated. (H, I) miR levels of miR-26b-5p and miR-29c-3p in HK1 (H) and HNE3 (I) cells after overexpression of LSH. (J-M) The recruitment of LSH the promoter regions of miR-26b-5p (J and L) and miR-29c-3p (K and M) was analyzed in HK1 and HNE3 cells after overexpression of LSH. * p
    Figure Legend Snippet: Both miR-26b-5p and miR-29c-3p inhibited TET2 and TET3 expression and were silenced by LSH. (A) Ectopic expression of miR-26b-5p (Left) and miR-29C-3p (Right) in MCF-7 cells. Relative expression levels of TET2 (B) and TET3 (C) were detected by RT-PCR after the transfection of miRs as indicated. (D, E) Expression levels of TET2 and TET3 were analyzed after the transfection of miRs as indicated in HK1 (F) and HNE3 (G) cells, whereas LSH protein level unchanged. (F) Luciferase reporter assay in 293 cells together with miRs as indicated, and with transfection of pMiR-Report-TET2 3'-UTR sense or pMiR-Report-TET2 3'-UTR antisense as indicted. (G) ELISA of 5-hmC was used to assess 5-hmC levels from genomic DNA derived from HNE3 cells after introduction of miRs as indicated. (H, I) miR levels of miR-26b-5p and miR-29c-3p in HK1 (H) and HNE3 (I) cells after overexpression of LSH. (J-M) The recruitment of LSH the promoter regions of miR-26b-5p (J and L) and miR-29c-3p (K and M) was analyzed in HK1 and HNE3 cells after overexpression of LSH. * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Reporter Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Over Expression

    6) Product Images from "Regulation of miR-146a by RelA/NFkB and p53 in STHdhQ111/HdhQ111 Cells, a Cell Model of Huntington's Disease"

    Article Title: Regulation of miR-146a by RelA/NFkB and p53 in STHdhQ111/HdhQ111 Cells, a Cell Model of Huntington's Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023837

    Endogenous expression of p53 in ST Hdh Q7 /Hdh Q7 and ST Hdh Q111 / Hdh Q111 cells: decreased miR-125b target p53. ( A ) Representative Western Blot showing increased p53 protein level in ST Hdh Q111 / Hdh Q111 cells compared to ST Hdh Q7 /Hdh Q7 cells; ( B ) Average integrated optical density (IOD) of p53 protein bands in A , normalized to β-actin level (n = 3, p = 0.024) in these cell lines. ( C ) Relative luciferase activity of cloned p53-3′UTR with miR-125b binding site (denoted by p53-UTR1) in ST Hdh Q111 / Hdh Q111 cells compared to ST Hdh Q7 /Hdh Q7 cells. Normalization of protein level between ST Hdh Q111 /Hdh Q111 cells and ST Hdh Q7 /Hdh Q7 cells was done by taking the ratio of RLU of cloned construct i.e. p53-UTR1 and empty vector pmiR. Relative luciferase activity of p53-UTR1 was found significantly higher (n = 3, p = 0.026) in ST Hdh Q111 /Hdh Q111 cells compared to ST Hdh Q7 /Hdh Q7 cells; ( D ) Reduced luciferase activity of p53-UTR1 co-transfected with pre-miR-125b in ST Hdh Q7 /Hdh Q7 cells (n = 3, p = 0.024) and ST Hdh Q111 /Hdh Q111 cells (n = 3, p = 0.0086) compared to those obtained in respective empty vector U61 tansfected cells; ( E ) Representative Western Blot showing reduction in p53 protein level in ST Hdh Q111 /Hdh Q111 cells 72 hours following transfection with pre-miR-125b compared to ST Hdh Q111 /Hdh Q111 cells transfected with empty vector U61, average IOD compared to β-actin (n = 3, p = 0.039) is shown in the adjacent bar diagram.
    Figure Legend Snippet: Endogenous expression of p53 in ST Hdh Q7 /Hdh Q7 and ST Hdh Q111 / Hdh Q111 cells: decreased miR-125b target p53. ( A ) Representative Western Blot showing increased p53 protein level in ST Hdh Q111 / Hdh Q111 cells compared to ST Hdh Q7 /Hdh Q7 cells; ( B ) Average integrated optical density (IOD) of p53 protein bands in A , normalized to β-actin level (n = 3, p = 0.024) in these cell lines. ( C ) Relative luciferase activity of cloned p53-3′UTR with miR-125b binding site (denoted by p53-UTR1) in ST Hdh Q111 / Hdh Q111 cells compared to ST Hdh Q7 /Hdh Q7 cells. Normalization of protein level between ST Hdh Q111 /Hdh Q111 cells and ST Hdh Q7 /Hdh Q7 cells was done by taking the ratio of RLU of cloned construct i.e. p53-UTR1 and empty vector pmiR. Relative luciferase activity of p53-UTR1 was found significantly higher (n = 3, p = 0.026) in ST Hdh Q111 /Hdh Q111 cells compared to ST Hdh Q7 /Hdh Q7 cells; ( D ) Reduced luciferase activity of p53-UTR1 co-transfected with pre-miR-125b in ST Hdh Q7 /Hdh Q7 cells (n = 3, p = 0.024) and ST Hdh Q111 /Hdh Q111 cells (n = 3, p = 0.0086) compared to those obtained in respective empty vector U61 tansfected cells; ( E ) Representative Western Blot showing reduction in p53 protein level in ST Hdh Q111 /Hdh Q111 cells 72 hours following transfection with pre-miR-125b compared to ST Hdh Q111 /Hdh Q111 cells transfected with empty vector U61, average IOD compared to β-actin (n = 3, p = 0.039) is shown in the adjacent bar diagram.

    Techniques Used: Expressing, Western Blot, Luciferase, Activity Assay, Clone Assay, Binding Assay, Construct, Plasmid Preparation, Transfection

    7) Product Images from "MiR-194 Regulates Chondrogenic Differentiation of Human Adipose-Derived Stem Cells by Targeting Sox5"

    Article Title: MiR-194 Regulates Chondrogenic Differentiation of Human Adipose-Derived Stem Cells by Targeting Sox5

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031861

    Sox5 is a target of miR-194. (A) Different doses of pre-miR-194 were co-transfected with the specific pMIR-REPORT construct into HEK293 cells. All cells were harvested at 48 h after transfection, and then luciferase activities were measured and normalized to the phRL-TK activities. The relative gene expression alteration was demonstrated by percentage decrease. Three independent transfection experiments were done. (B) The effect of pre-miR-194 gradually diminished in two weeks.
    Figure Legend Snippet: Sox5 is a target of miR-194. (A) Different doses of pre-miR-194 were co-transfected with the specific pMIR-REPORT construct into HEK293 cells. All cells were harvested at 48 h after transfection, and then luciferase activities were measured and normalized to the phRL-TK activities. The relative gene expression alteration was demonstrated by percentage decrease. Three independent transfection experiments were done. (B) The effect of pre-miR-194 gradually diminished in two weeks.

    Techniques Used: Transfection, Construct, Luciferase, Expressing

    8) Product Images from "MicroRNA-23b* targets proline oxidase, a mitochondrial tumor suppressor protein in renal cancer"

    Article Title: MicroRNA-23b* targets proline oxidase, a mitochondrial tumor suppressor protein in renal cancer

    Journal: Oncogene

    doi: 10.1038/onc.2010.237

    miR-23b* Directly Targets 3′ UTR of POX mRNA. (a) Schematic representation of miR-23b* target binding site in the POX mRNA 3′ UTR identified by the Microinspector prediction program. Mutations of 3′UTR and miR-23b* in the seed sequences were indicated. (b) The 3′UTR of POX mRNA was amplified by PCR and cloned downstream of a firefly luciferase gene of pMIR-REPORT to construct the pPOX 3′ UTR vector. TK10 renal cancer cells were transfected with pMIR-REPORT or pPOX 3′ UTR reporter with or without mimic miR-23b* or negative control siRNA. (c) The POX 3′UTR with mutations was constructed as the pPOX 3′UTR-MUT plasmid. TK10 renal cancer cells were transfected with pMIR-REPORT, pPOX 3′ UTR or pPOX 3′UTR-MUT reporter with or without 100nM mimic miR-23b* or negative control siRNA. (d) TK10 renal cancer cells were transfected with pPOX 3′ UTR reporter with 100nM mimic miR-23b*, mutant miR-23b* or negative control siRNA. All transfections used pRL-null renilla luciferase reporter as an internal control. Data are reported as relative luciferase activity normalized to that of pMIR-REPORT group (3b and 3c) and that of pPOX 3′ UTR (3d). All results were done in triplicates and repeated in two independent experiments. Values represent means ± SEM. p value was obtained in one-way ANOVA analysis.
    Figure Legend Snippet: miR-23b* Directly Targets 3′ UTR of POX mRNA. (a) Schematic representation of miR-23b* target binding site in the POX mRNA 3′ UTR identified by the Microinspector prediction program. Mutations of 3′UTR and miR-23b* in the seed sequences were indicated. (b) The 3′UTR of POX mRNA was amplified by PCR and cloned downstream of a firefly luciferase gene of pMIR-REPORT to construct the pPOX 3′ UTR vector. TK10 renal cancer cells were transfected with pMIR-REPORT or pPOX 3′ UTR reporter with or without mimic miR-23b* or negative control siRNA. (c) The POX 3′UTR with mutations was constructed as the pPOX 3′UTR-MUT plasmid. TK10 renal cancer cells were transfected with pMIR-REPORT, pPOX 3′ UTR or pPOX 3′UTR-MUT reporter with or without 100nM mimic miR-23b* or negative control siRNA. (d) TK10 renal cancer cells were transfected with pPOX 3′ UTR reporter with 100nM mimic miR-23b*, mutant miR-23b* or negative control siRNA. All transfections used pRL-null renilla luciferase reporter as an internal control. Data are reported as relative luciferase activity normalized to that of pMIR-REPORT group (3b and 3c) and that of pPOX 3′ UTR (3d). All results were done in triplicates and repeated in two independent experiments. Values represent means ± SEM. p value was obtained in one-way ANOVA analysis.

    Techniques Used: Binding Assay, Amplification, Polymerase Chain Reaction, Clone Assay, Luciferase, Construct, Plasmid Preparation, Transfection, Negative Control, Mutagenesis, Activity Assay

    9) Product Images from "RBM24 suppresses cancer progression by upregulating miR-25 to target MALAT1 in nasopharyngeal carcinoma"

    Article Title: RBM24 suppresses cancer progression by upregulating miR-25 to target MALAT1 in nasopharyngeal carcinoma

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.252

    miR-25 reduced the MALAT1 level. ( a ) Schematic constructions of the wild type and mutant pMIR-REPORT-MALAT1 miRNA expression vectors used in luciferase reporter assays. The five altered nucleotides in the mutant binding site are colored in red. ( b ) The relative luciferase activities in the 5-8 F and CNE-2 cells after transfection with the pMIR-REPORT-MALAT1 reporter and miR-25 mimic, NC mimic, miR-25 inhibitor or NC inhibitor. ( c ) QRT-PCR analysis of MALAT1 expression in 5-8 F and CNE-2 cells that were transfected with miR-25 mimic (50 nM) or NC mimic. ( d ) QRT-PCR analysis of MALAT1 expression in 5-8 F and CNE-2 Tet-Off-inducible RBM24-stable cells that were transfected with miR-25 inhibitor (100 nM) or NC inhibitor after the removal of doxycycline for 24 h. All data are shown as the mean±S.E.M. of three independent experiments (* P
    Figure Legend Snippet: miR-25 reduced the MALAT1 level. ( a ) Schematic constructions of the wild type and mutant pMIR-REPORT-MALAT1 miRNA expression vectors used in luciferase reporter assays. The five altered nucleotides in the mutant binding site are colored in red. ( b ) The relative luciferase activities in the 5-8 F and CNE-2 cells after transfection with the pMIR-REPORT-MALAT1 reporter and miR-25 mimic, NC mimic, miR-25 inhibitor or NC inhibitor. ( c ) QRT-PCR analysis of MALAT1 expression in 5-8 F and CNE-2 cells that were transfected with miR-25 mimic (50 nM) or NC mimic. ( d ) QRT-PCR analysis of MALAT1 expression in 5-8 F and CNE-2 Tet-Off-inducible RBM24-stable cells that were transfected with miR-25 inhibitor (100 nM) or NC inhibitor after the removal of doxycycline for 24 h. All data are shown as the mean±S.E.M. of three independent experiments (* P

    Techniques Used: Mutagenesis, Expressing, Luciferase, Binding Assay, Transfection, Quantitative RT-PCR

    10) Product Images from "MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells"

    Article Title: MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108997

    Luciferase assays for the identification of the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA. (A) Arrangement of rno-miR-376a and GRP78 mRNA and a schematic drawing of the predicted rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA. (B) Schematic drawings of the pMIR-REPORT luciferase vectors used in our experiment. To identify the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA, luciferase reporter vectors were generated as described in the Materials and Methods . (C) Luciferase activity was measured to identify the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA. HEK293 cells were prepared, and the cells were transfected with 200 ng of each reporter vector with 50 nM Pre-miR-376a (precursor) or Anti-miR-376a (inhibitor) as described in the Materials and Methods . For transfection normalization, the cells were also transfected with the pMIR-REPORT βgal vector. Luciferase activity was measured 24 h after transfection. The activity of the control (empty vector) was set at 1. Each value represents the mean ±SE of three independent experiments. *, significantly different from the control value, P
    Figure Legend Snippet: Luciferase assays for the identification of the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA. (A) Arrangement of rno-miR-376a and GRP78 mRNA and a schematic drawing of the predicted rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA. (B) Schematic drawings of the pMIR-REPORT luciferase vectors used in our experiment. To identify the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA, luciferase reporter vectors were generated as described in the Materials and Methods . (C) Luciferase activity was measured to identify the rno-miR-376a-binding site in the 3′-UTR of GRP78 mRNA. HEK293 cells were prepared, and the cells were transfected with 200 ng of each reporter vector with 50 nM Pre-miR-376a (precursor) or Anti-miR-376a (inhibitor) as described in the Materials and Methods . For transfection normalization, the cells were also transfected with the pMIR-REPORT βgal vector. Luciferase activity was measured 24 h after transfection. The activity of the control (empty vector) was set at 1. Each value represents the mean ±SE of three independent experiments. *, significantly different from the control value, P

    Techniques Used: Luciferase, Binding Assay, Generated, Activity Assay, Transfection, Plasmid Preparation

    11) Product Images from "MicroRNA-30a-5p suppresses proliferation, invasion and tumor growth of hepatocellular cancer cells via targeting FOXA1"

    Article Title: MicroRNA-30a-5p suppresses proliferation, invasion and tumor growth of hepatocellular cancer cells via targeting FOXA1

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.6745

    (A) FOXA1 was predicted to be a potential target of miR-30a-5p by Targetscan, and this is evolutionarily conserved. (B) Schematic diagram showing the generation of pMIR-Report vectors containing wild-type or mutant type of FOXA1 3′UTR. (C and D) In HepG2 and SMMC-7721 cells, luciferase reporter assay data indicated that the overexpression of miR-30a-5p decreased the luciferase activity significantly in the wild-type group, while no significant change in luciferase activity was observed in the mutant type group. (E and F) Quantitative reverse transcription polymerase reaction and (G and H) western blotting were used to examine the level of FOXA1 mRNA and protein in HepG2 and SMMC-7721 cells transfected with miR-30a-5p mimic or negative control miR, respectively. Non-transfected cells were used as the control group. *P
    Figure Legend Snippet: (A) FOXA1 was predicted to be a potential target of miR-30a-5p by Targetscan, and this is evolutionarily conserved. (B) Schematic diagram showing the generation of pMIR-Report vectors containing wild-type or mutant type of FOXA1 3′UTR. (C and D) In HepG2 and SMMC-7721 cells, luciferase reporter assay data indicated that the overexpression of miR-30a-5p decreased the luciferase activity significantly in the wild-type group, while no significant change in luciferase activity was observed in the mutant type group. (E and F) Quantitative reverse transcription polymerase reaction and (G and H) western blotting were used to examine the level of FOXA1 mRNA and protein in HepG2 and SMMC-7721 cells transfected with miR-30a-5p mimic or negative control miR, respectively. Non-transfected cells were used as the control group. *P

    Techniques Used: Mutagenesis, Luciferase, Reporter Assay, Over Expression, Activity Assay, Western Blot, Transfection, Negative Control

    12) Product Images from "MiR-451 is decreased in hypertrophic cardiomyopathy and regulates autophagy by targeting TSC1"

    Article Title: MiR-451 is decreased in hypertrophic cardiomyopathy and regulates autophagy by targeting TSC1

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12380

    MiR-451 directly targets tuberous sclerosis complex 1 in cardiomyocytes. ( A ) Tuberous sclerosis complex 1 (TSC1) is a potential target gene of miR-451 predicted by targetscan. The binding sites of miR-451 in wild-type (WT) and mutated (Mut) 3′UTR sequences of TSC1 are shown. ( B ) The WT and Mut 3′UTR of TSC1 were cloned into pMIR-REPORT vectors and cotransfected with pRL-TK, miR-451 mimics or scrambled control into H9c2 cells, respectively. The relative luciferase activity was measured and normalized to Renilla. ( C and D ) The expression of TSC1 in neonatal rat cardiomyocytes (NRCM) and HeLa cells transfected with miR-451 mimics or scrambled control ( C ). The relative expression of TSC1 was normalized with GAPDH ( D ). ( E and F ) The expression of TSC1 from 16 hypertrophic cardiomyopathy (HCM) patients and eight non-hypertrophic cardiomyopathy (NCM) donors, and representative graphs are shown ( E ). The expression of TSC1 was normalized with GAPDH. Values are expressed as means ± SD; * P
    Figure Legend Snippet: MiR-451 directly targets tuberous sclerosis complex 1 in cardiomyocytes. ( A ) Tuberous sclerosis complex 1 (TSC1) is a potential target gene of miR-451 predicted by targetscan. The binding sites of miR-451 in wild-type (WT) and mutated (Mut) 3′UTR sequences of TSC1 are shown. ( B ) The WT and Mut 3′UTR of TSC1 were cloned into pMIR-REPORT vectors and cotransfected with pRL-TK, miR-451 mimics or scrambled control into H9c2 cells, respectively. The relative luciferase activity was measured and normalized to Renilla. ( C and D ) The expression of TSC1 in neonatal rat cardiomyocytes (NRCM) and HeLa cells transfected with miR-451 mimics or scrambled control ( C ). The relative expression of TSC1 was normalized with GAPDH ( D ). ( E and F ) The expression of TSC1 from 16 hypertrophic cardiomyopathy (HCM) patients and eight non-hypertrophic cardiomyopathy (NCM) donors, and representative graphs are shown ( E ). The expression of TSC1 was normalized with GAPDH. Values are expressed as means ± SD; * P

    Techniques Used: Binding Assay, Clone Assay, Luciferase, Activity Assay, Expressing, Transfection

    13) Product Images from "MicroRNA-212 targets FOXA1 and suppresses the proliferation and invasion of intrahepatic cholangiocarcinoma cells"

    Article Title: MicroRNA-212 targets FOXA1 and suppresses the proliferation and invasion of intrahepatic cholangiocarcinoma cells

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2016.3824

    (A) Targetscan software predicted that FOXA1 was a putative target of miR-212. (B) The potential seed sequences of miR-212 within the wild-type and mutant 3′-UTR of FOXA1 mRNA. (C) A luciferase assay was performed using the pMIR-REPORT vector
    Figure Legend Snippet: (A) Targetscan software predicted that FOXA1 was a putative target of miR-212. (B) The potential seed sequences of miR-212 within the wild-type and mutant 3′-UTR of FOXA1 mRNA. (C) A luciferase assay was performed using the pMIR-REPORT vector

    Techniques Used: Software, Mutagenesis, Luciferase, Plasmid Preparation

    14) Product Images from "MiR-449a promotes breast cancer progression by targeting CRIP2"

    Article Title: MiR-449a promotes breast cancer progression by targeting CRIP2

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7753

    MiR-449a directly targeted the 3′-untranslated region (3′-UTR) of CRIP2 A. Schema depicting the luciferase reporter vectors carrying the predicted miR-449a binding sites downstream of the Firefly luciferase gene (pmiR-Report vector). B. Wild type or mutant reporter vector, with either scrambled control (SC; 40 nM) or anti-miR-449a (40 nM), were co-transfected into MDA-MB-231 cells, and luciferase activity was measured at 24 hours post-transfection. Vector and anti-miR-449a co-transfected luciferase activity was normalized to vector and SC co-transfected luciferase activity, with Renilla luciferase activity for transfection efficiency normalization. These data were then compared to the pmiR-Report control vector data. C. Western blot for CRIP2 in T47D and MDA-MB-231 cells, 48 hours after transfection. Data are presented as mean ± SEM; n=3; *p
    Figure Legend Snippet: MiR-449a directly targeted the 3′-untranslated region (3′-UTR) of CRIP2 A. Schema depicting the luciferase reporter vectors carrying the predicted miR-449a binding sites downstream of the Firefly luciferase gene (pmiR-Report vector). B. Wild type or mutant reporter vector, with either scrambled control (SC; 40 nM) or anti-miR-449a (40 nM), were co-transfected into MDA-MB-231 cells, and luciferase activity was measured at 24 hours post-transfection. Vector and anti-miR-449a co-transfected luciferase activity was normalized to vector and SC co-transfected luciferase activity, with Renilla luciferase activity for transfection efficiency normalization. These data were then compared to the pmiR-Report control vector data. C. Western blot for CRIP2 in T47D and MDA-MB-231 cells, 48 hours after transfection. Data are presented as mean ± SEM; n=3; *p

    Techniques Used: Luciferase, Binding Assay, Plasmid Preparation, Mutagenesis, Transfection, Multiple Displacement Amplification, Activity Assay, Western Blot

    15) Product Images from "MicroRNA-21 targets tumor suppressor genes ANP32A and SMARCA4"

    Article Title: MicroRNA-21 targets tumor suppressor genes ANP32A and SMARCA4

    Journal: Oncogene

    doi: 10.1038/onc.2011.15

    ANP32A and SMARCA4 are direct targets of miR-21. ( a ) Putative miR-21-binding sites within ANP32A (NM_006305) and SMARCA4 (NM_001128849) genes. Perfect matches are indicated by a line; G:U pairs by a colon. The frame marks nucleotides mutated for the reporter gene assays. ( b ) Comparison of nucleotides between the miR-21 sequence and its targets in various species. For the ANP32A site overlapping the open reading frame, amino acids are indicated. ( c ) Cloned ANP32A and SMARCA4sequences are targeted by miR-21 (left panel), and mutation of the putative binding sites abolishes this effect to wild type level (right panel). pMIR-REPORT vectors containing ANP32A-900-1112, SMARCA4-3′ untranslated region (3′UTR) and their mutated variants were co-transfected with miR-21 or control precursor into LNCaP cells. A vector containing three miR-21-binding sites was used as a positive control. Luciferase activity was assayed after 48 h. Data are normalized to the corresponding control sample. Values are the means±s.d. from six independent experiments. The P -values were determined using a two-tailed t -test.
    Figure Legend Snippet: ANP32A and SMARCA4 are direct targets of miR-21. ( a ) Putative miR-21-binding sites within ANP32A (NM_006305) and SMARCA4 (NM_001128849) genes. Perfect matches are indicated by a line; G:U pairs by a colon. The frame marks nucleotides mutated for the reporter gene assays. ( b ) Comparison of nucleotides between the miR-21 sequence and its targets in various species. For the ANP32A site overlapping the open reading frame, amino acids are indicated. ( c ) Cloned ANP32A and SMARCA4sequences are targeted by miR-21 (left panel), and mutation of the putative binding sites abolishes this effect to wild type level (right panel). pMIR-REPORT vectors containing ANP32A-900-1112, SMARCA4-3′ untranslated region (3′UTR) and their mutated variants were co-transfected with miR-21 or control precursor into LNCaP cells. A vector containing three miR-21-binding sites was used as a positive control. Luciferase activity was assayed after 48 h. Data are normalized to the corresponding control sample. Values are the means±s.d. from six independent experiments. The P -values were determined using a two-tailed t -test.

    Techniques Used: Binding Assay, Sequencing, Clone Assay, Mutagenesis, Transfection, Plasmid Preparation, Positive Control, Luciferase, Activity Assay, Two Tailed Test

    16) Product Images from "MiR-155 inhibits cell migration of human cardiomyocyte progenitor cells (hCMPCs) via targeting of MMP-16"

    Article Title: MiR-155 inhibits cell migration of human cardiomyocyte progenitor cells (hCMPCs) via targeting of MMP-16

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2012.01551.x

    MMP-16 expression in miRNAs transfected hCMPCs, on mRNA level (A) and on protein level (B), as demonstrated by western blotting, including densitometric quantification. Luciferase activity of either pMIR-REPORT vector, containing a part of the 3′-UTR of MMP-16 (pMIR MMP-16) or containing the MMP-16 3′-UTR with seed-mutated targets sites 1, 2 or both (C). Data are presented as mean ± S.E.M., N = 4 and * P
    Figure Legend Snippet: MMP-16 expression in miRNAs transfected hCMPCs, on mRNA level (A) and on protein level (B), as demonstrated by western blotting, including densitometric quantification. Luciferase activity of either pMIR-REPORT vector, containing a part of the 3′-UTR of MMP-16 (pMIR MMP-16) or containing the MMP-16 3′-UTR with seed-mutated targets sites 1, 2 or both (C). Data are presented as mean ± S.E.M., N = 4 and * P

    Techniques Used: Expressing, Transfection, Western Blot, Luciferase, Activity Assay, Plasmid Preparation

    17) Product Images from "Enhancer of Zeste homolog 2 (EZH2) is overexpressed in recurrent nasopharyngeal carcinoma and is regulated by miR-26a, miR-101, and miR-98"

    Article Title: Enhancer of Zeste homolog 2 (EZH2) is overexpressed in recurrent nasopharyngeal carcinoma and is regulated by miR-26a, miR-101, and miR-98

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2010.64

    EZH2 3′-UTR is a direct target of miR-26a, 98, and 101. ( a ) Schematic representation of the pMIR-REPORT-EZH2 UTR expression vector with the alignment of the indicated miRs with the EZH2 3′-UTR with the seed region highlighted in blue (miR), and red (UTR). ( b ) Percent luciferase activity at 48 h post-transfection with the indicated miR (100 nM), and reporter plasmid (100 ng). Data are presented as mean ± S.E. from two independent experiments, n =4; * P ≤0.05; ** P ≤0.005. ( c ) Inverse expression of EZH2 ( n =89) with miR-98 ( n =99) is observed in primary NPC specimens (cohort ‘C'), as measured by qRT-PCR and presented as fold-change expression in tumors compared with normal nasopharyngeal epithelial tissues ( n =6). *** P ≤0.0005. ( d ) A proposed model derived from these data, illustrating that underexpression of miR-26a, 98, and 101 lead to upregulation of EZH2, which in turn mediates several important cellular processes all driving the survival of NPC cells; (OS: oxidative stress)
    Figure Legend Snippet: EZH2 3′-UTR is a direct target of miR-26a, 98, and 101. ( a ) Schematic representation of the pMIR-REPORT-EZH2 UTR expression vector with the alignment of the indicated miRs with the EZH2 3′-UTR with the seed region highlighted in blue (miR), and red (UTR). ( b ) Percent luciferase activity at 48 h post-transfection with the indicated miR (100 nM), and reporter plasmid (100 ng). Data are presented as mean ± S.E. from two independent experiments, n =4; * P ≤0.05; ** P ≤0.005. ( c ) Inverse expression of EZH2 ( n =89) with miR-98 ( n =99) is observed in primary NPC specimens (cohort ‘C'), as measured by qRT-PCR and presented as fold-change expression in tumors compared with normal nasopharyngeal epithelial tissues ( n =6). *** P ≤0.0005. ( d ) A proposed model derived from these data, illustrating that underexpression of miR-26a, 98, and 101 lead to upregulation of EZH2, which in turn mediates several important cellular processes all driving the survival of NPC cells; (OS: oxidative stress)

    Techniques Used: Expressing, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Derivative Assay

    18) Product Images from "MicroRNA-30a-5p suppresses proliferation, invasion and tumor growth of hepatocellular cancer cells via targeting FOXA1"

    Article Title: MicroRNA-30a-5p suppresses proliferation, invasion and tumor growth of hepatocellular cancer cells via targeting FOXA1

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.6745

    (A) FOXA1 was predicted to be a potential target of miR-30a-5p by Targetscan, and this is evolutionarily conserved. (B) Schematic diagram showing the generation of pMIR-Report vectors containing wild-type or mutant type of FOXA1 3′UTR. (C and D) In HepG2 and SMMC-7721 cells, luciferase reporter assay data indicated that the overexpression of miR-30a-5p decreased the luciferase activity significantly in the wild-type group, while no significant change in luciferase activity was observed in the mutant type group. (E and F) Quantitative reverse transcription polymerase reaction and (G and H) western blotting were used to examine the level of FOXA1 mRNA and protein in HepG2 and SMMC-7721 cells transfected with miR-30a-5p mimic or negative control miR, respectively. Non-transfected cells were used as the control group. *P
    Figure Legend Snippet: (A) FOXA1 was predicted to be a potential target of miR-30a-5p by Targetscan, and this is evolutionarily conserved. (B) Schematic diagram showing the generation of pMIR-Report vectors containing wild-type or mutant type of FOXA1 3′UTR. (C and D) In HepG2 and SMMC-7721 cells, luciferase reporter assay data indicated that the overexpression of miR-30a-5p decreased the luciferase activity significantly in the wild-type group, while no significant change in luciferase activity was observed in the mutant type group. (E and F) Quantitative reverse transcription polymerase reaction and (G and H) western blotting were used to examine the level of FOXA1 mRNA and protein in HepG2 and SMMC-7721 cells transfected with miR-30a-5p mimic or negative control miR, respectively. Non-transfected cells were used as the control group. *P

    Techniques Used: Mutagenesis, Luciferase, Reporter Assay, Over Expression, Activity Assay, Western Blot, Transfection, Negative Control

    19) Product Images from "miR-186, a serum microRNA, induces endothelial cell apoptosis by targeting SMAD6 in Kawasaki disease"

    Article Title: miR-186, a serum microRNA, induces endothelial cell apoptosis by targeting SMAD6 in Kawasaki disease

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3397

    miR-186 directly targets SMAD6. (A) Venn diagram of the results from the gene target prediction algorithms. (B) Effect of miR-186 mimic overexpression on the endogenous SMAD6 mRNA levels (n=3). (C) Effect of miR-186 mimic overexpression on the endogenous SMAD6 protein levels (n=3). (D) Schematic of the predicted miR-186 binding sequence on the SMAD6 mRNA 3′-UTR. Luciferase reporter construct was made by cloning the human SMAD6 mRNA sequence into pMIR-Report construct. Wild-type or mutant SMAD6 mRNA fragments (from 2,769 to 2,792) were amplified and cloned into the luciferase reporter via Spe I and Hind III sites. (E) HUVECs were co-transfected with the reporter constructs bearing the wild-type and mutant SMAD6 sequences as indicated in Fig. 3D , and with miR-186 mimics or negative control oligonucleotides. After 36 h, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data are presented as the mean ± standard error of the mean. ** P
    Figure Legend Snippet: miR-186 directly targets SMAD6. (A) Venn diagram of the results from the gene target prediction algorithms. (B) Effect of miR-186 mimic overexpression on the endogenous SMAD6 mRNA levels (n=3). (C) Effect of miR-186 mimic overexpression on the endogenous SMAD6 protein levels (n=3). (D) Schematic of the predicted miR-186 binding sequence on the SMAD6 mRNA 3′-UTR. Luciferase reporter construct was made by cloning the human SMAD6 mRNA sequence into pMIR-Report construct. Wild-type or mutant SMAD6 mRNA fragments (from 2,769 to 2,792) were amplified and cloned into the luciferase reporter via Spe I and Hind III sites. (E) HUVECs were co-transfected with the reporter constructs bearing the wild-type and mutant SMAD6 sequences as indicated in Fig. 3D , and with miR-186 mimics or negative control oligonucleotides. After 36 h, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data are presented as the mean ± standard error of the mean. ** P

    Techniques Used: Over Expression, Binding Assay, Sequencing, Luciferase, Construct, Clone Assay, Mutagenesis, Amplification, Transfection, Negative Control, Activity Assay

    20) Product Images from "miR-198 Represses the Proliferation of HaCaT Cells by Targeting Cyclin D2"

    Article Title: miR-198 Represses the Proliferation of HaCaT Cells by Targeting Cyclin D2

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms160817018

    MiR-198 directly bound to the 3′-UTR of CCND2 mRNA. ( A ) Bioinformatics analyses showed that 10 potential target genes of miR-198 were predicted by three different databases; ( B ) The two distinct predicted binding sites of miR-198 in the 3′-UTR of CCND2 mRNA were allocated, and the fragments containing either mutated binding site were amplified according to the mature miR-198 sequence; ( C ) In pMIR-REPORT™ vector, CCND2 mRNA 3′-UTRfragment containing either the wild type or the mutated Site 1 was fused downstream the reporter gene. When the vectors were cotransfected with miR-198 mimic or mimic control, and the relative luciferase activity, normalised by β-gal, was significantly suppressed in vector with wild type Site 1 than that with mutated Site 1 (32.80% ± 6.89%); ( D ) In the presence of wild type Site 2, not the mutated one, miR-198 was able to significantly inhibit luciferase activity although to a less extent than wild type Site 1 (55.39% ± 8.48%). (NC, negative control; WT, wide-type; MT, mutated-type. * Compared with NC, p
    Figure Legend Snippet: MiR-198 directly bound to the 3′-UTR of CCND2 mRNA. ( A ) Bioinformatics analyses showed that 10 potential target genes of miR-198 were predicted by three different databases; ( B ) The two distinct predicted binding sites of miR-198 in the 3′-UTR of CCND2 mRNA were allocated, and the fragments containing either mutated binding site were amplified according to the mature miR-198 sequence; ( C ) In pMIR-REPORT™ vector, CCND2 mRNA 3′-UTRfragment containing either the wild type or the mutated Site 1 was fused downstream the reporter gene. When the vectors were cotransfected with miR-198 mimic or mimic control, and the relative luciferase activity, normalised by β-gal, was significantly suppressed in vector with wild type Site 1 than that with mutated Site 1 (32.80% ± 6.89%); ( D ) In the presence of wild type Site 2, not the mutated one, miR-198 was able to significantly inhibit luciferase activity although to a less extent than wild type Site 1 (55.39% ± 8.48%). (NC, negative control; WT, wide-type; MT, mutated-type. * Compared with NC, p

    Techniques Used: Binding Assay, Amplification, Sequencing, Plasmid Preparation, Luciferase, Activity Assay, Negative Control

    21) Product Images from "Micro-RNA-632 down regulates DNAJB6 in breast cancer"

    Article Title: Micro-RNA-632 down regulates DNAJB6 in breast cancer

    Journal: Laboratory investigation; a journal of technical methods and pathology

    doi: 10.1038/labinvest.2012.87

    miR-632 targets DNAJB6 for degradation (A) pMIR-REPORT-DNAJB6 (containing putative binding site of miR-632 from the ORF of DNAJB6) was co-transfected with miR-632 expression vector, miR-632-pIRES2EGFP or empty vector control. The assay was performed in triplicate and the experiment was performed twice. The luciferase activity readings were normalized with activity from a co-transfected β-gal expressing control. The error bars represent standard error of mean (SEM) (B) Levels of DNAJB6 and PTCH1 transcript were evaluated from MCF10A cells treated with miR-632-pIRES2EGFP or empty vector control. GAPDH was used as endorse control. The error bars represent standard error of mean (SEM). The reactions were performed in triplicate and the experiment was performed three times. (C) MDA-MB-231 cells were treated with X-miR-632 or control (50 and 100 nM). Total protein extract (30μg) was resolved using SDS-PAGE and subjected to western blot analysis for DNAJB6 levels. β-actin levels were determined as loading control.
    Figure Legend Snippet: miR-632 targets DNAJB6 for degradation (A) pMIR-REPORT-DNAJB6 (containing putative binding site of miR-632 from the ORF of DNAJB6) was co-transfected with miR-632 expression vector, miR-632-pIRES2EGFP or empty vector control. The assay was performed in triplicate and the experiment was performed twice. The luciferase activity readings were normalized with activity from a co-transfected β-gal expressing control. The error bars represent standard error of mean (SEM) (B) Levels of DNAJB6 and PTCH1 transcript were evaluated from MCF10A cells treated with miR-632-pIRES2EGFP or empty vector control. GAPDH was used as endorse control. The error bars represent standard error of mean (SEM). The reactions were performed in triplicate and the experiment was performed three times. (C) MDA-MB-231 cells were treated with X-miR-632 or control (50 and 100 nM). Total protein extract (30μg) was resolved using SDS-PAGE and subjected to western blot analysis for DNAJB6 levels. β-actin levels were determined as loading control.

    Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Multiple Displacement Amplification, SDS Page, Western Blot

    22) Product Images from "MicroRNA-212 targets FOXA1 and suppresses the proliferation and invasion of intrahepatic cholangiocarcinoma cells"

    Article Title: MicroRNA-212 targets FOXA1 and suppresses the proliferation and invasion of intrahepatic cholangiocarcinoma cells

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2016.3824

    (A) Targetscan software predicted that FOXA1 was a putative target of miR-212. (B) The potential seed sequences of miR-212 within the wild-type and mutant 3′-UTR of FOXA1 mRNA. (C) A luciferase assay was performed using the pMIR-REPORT vector
    Figure Legend Snippet: (A) Targetscan software predicted that FOXA1 was a putative target of miR-212. (B) The potential seed sequences of miR-212 within the wild-type and mutant 3′-UTR of FOXA1 mRNA. (C) A luciferase assay was performed using the pMIR-REPORT vector

    Techniques Used: Software, Mutagenesis, Luciferase, Plasmid Preparation

    23) Product Images from "Mycobacterium bovis BCG Triggered MyD88 Induces miR-124 Feedback Negatively Regulates Immune Response in Alveolar Epithelial Cells"

    Article Title: Mycobacterium bovis BCG Triggered MyD88 Induces miR-124 Feedback Negatively Regulates Immune Response in Alveolar Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092419

    Validation of potential targets of miR-124 by a dual-luciferase reporter assay. A: Sequences of potential binding sites of miR-124 in the 3′UTR of TLR6, MyD88, TRAF6 and TNF-α mRNA (top sequences). Mutations were introduced into the putative miR-124 binding sites to generate variant 3′UTRs (bottom sequence). B-E: Dual-luciferase reporter assays were designed to validate miR-124 specificity. Cells were transfected with miR-124 mimic, miR-124 inhibitor or miR-124 control and pMIR-Report/TLR6 containing wild type or mutant 3′UTR sequence (B), pMIR-Report/MyD88 containing wild type or mutant 3′UTR sequence (C), pMIR-Report/TRAF6 containing wild type or mutant 3′UTR sequence (D) or pMIR-Report/TNF-α containing wild type or mutant 3′UTR sequence (E). Compared with pSicoR/nc group, *: p
    Figure Legend Snippet: Validation of potential targets of miR-124 by a dual-luciferase reporter assay. A: Sequences of potential binding sites of miR-124 in the 3′UTR of TLR6, MyD88, TRAF6 and TNF-α mRNA (top sequences). Mutations were introduced into the putative miR-124 binding sites to generate variant 3′UTRs (bottom sequence). B-E: Dual-luciferase reporter assays were designed to validate miR-124 specificity. Cells were transfected with miR-124 mimic, miR-124 inhibitor or miR-124 control and pMIR-Report/TLR6 containing wild type or mutant 3′UTR sequence (B), pMIR-Report/MyD88 containing wild type or mutant 3′UTR sequence (C), pMIR-Report/TRAF6 containing wild type or mutant 3′UTR sequence (D) or pMIR-Report/TNF-α containing wild type or mutant 3′UTR sequence (E). Compared with pSicoR/nc group, *: p

    Techniques Used: Luciferase, Reporter Assay, Binding Assay, Variant Assay, Sequencing, Transfection, Mutagenesis

    24) Product Images from "Hepatitis B Virus X Protein Sensitizes TRAIL-Induced Hepatocyte Apoptosis by Inhibiting the E3 Ubiquitin Ligase A20"

    Article Title: Hepatitis B Virus X Protein Sensitizes TRAIL-Induced Hepatocyte Apoptosis by Inhibiting the E3 Ubiquitin Ligase A20

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127329

    HBx inhibits A20 expression by upregulating miR-125a. (A) The luciferase activity was measured in 293T cells transfected with miR-125a or the control plasmid (NC), along with reporter plasmids (pMIR-) containing the intact or mutant binding sites at the A20 ORF or UTR. (B) Luciferase activity was measured in 293T cells transfected with the miR-125a inhibitor or the control plasmid (NC), along with the pMIR- constructs as in A. (C) The expression of A20 was detected by western blot in L-O2 cells transfected with miR-125a, anti-miR-125a or their controls. Data are from three independent experiments and are presented as the mean ± SEM. *P
    Figure Legend Snippet: HBx inhibits A20 expression by upregulating miR-125a. (A) The luciferase activity was measured in 293T cells transfected with miR-125a or the control plasmid (NC), along with reporter plasmids (pMIR-) containing the intact or mutant binding sites at the A20 ORF or UTR. (B) Luciferase activity was measured in 293T cells transfected with the miR-125a inhibitor or the control plasmid (NC), along with the pMIR- constructs as in A. (C) The expression of A20 was detected by western blot in L-O2 cells transfected with miR-125a, anti-miR-125a or their controls. Data are from three independent experiments and are presented as the mean ± SEM. *P

    Techniques Used: Expressing, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Binding Assay, Construct, Western Blot

    25) Product Images from "Negative Correlation Between Hepatitis C Virus (HCV) and Let-7 MicroRNA Family in Transplanted Livers: The Role of rs868 Single-Nucleotide Polymorphism"

    Article Title: Negative Correlation Between Hepatitis C Virus (HCV) and Let-7 MicroRNA Family in Transplanted Livers: The Role of rs868 Single-Nucleotide Polymorphism

    Journal: Annals of Transplantation

    doi: 10.12659/AOT.905540

    Relative expression of luciferase reporter gene in HepG2 cells transfected with plasmids carrying empty pMIR-REPORT (poly) and A or G rs868 variant of the TGFBR1 gene. Data show medians and lower and upper quartiles from 9 independent experiments. P values were calculated by Kruskal-Wallis test and post hoc Mann-Whitney U test.
    Figure Legend Snippet: Relative expression of luciferase reporter gene in HepG2 cells transfected with plasmids carrying empty pMIR-REPORT (poly) and A or G rs868 variant of the TGFBR1 gene. Data show medians and lower and upper quartiles from 9 independent experiments. P values were calculated by Kruskal-Wallis test and post hoc Mann-Whitney U test.

    Techniques Used: Expressing, Luciferase, Transfection, Variant Assay, MANN-WHITNEY

    26) Product Images from "Simultaneous inhibition of multiple oncogenic miRNAs by a multi-potent microRNA sponge"

    Article Title: Simultaneous inhibition of multiple oncogenic miRNAs by a multi-potent microRNA sponge

    Journal: Oncotarget

    doi:

    The expression of multi-potent miRNA sponge exerts antitumor activity A, B. Effect of the miRNA sponge to cancer drug (doxorubicin) sensitivity. In A, Flp-In T-REx 293 cells were treated with doxycycline (sponge induction) and doxorubicin together. In B, sponge was induced in cells and after 24 hours, doxorubicin was treated for another 24 hrs. The cell viability was measured by AlamarBlue assay. C. Comparison of the multi-potent miRNA sponge with single miRNA sponge for the anti-proliferative function. MDA-MB-436 cells were transfected with each of the construct indicated and after 48 hrs the cell proliferation was measured by AlamarBlue assay. (pMIR :pmiR-Report vector only; 21, 155, 222 : miR-21, miR-155, miR-222 targeting sponge respectively. Combi : mixture of the 3 kinds of single sponge (miRNA 21, 155, 221/222)). D, E. Effect of the miRNA sponge to apoptosis analyzed by Annexin V-staining, after doxorubicin treatment (20 hrs). FACS analysis shows more Annexin V-positive cells after sponge expression (RFP: control without MBS, marked as black line; PF: perfect sponge marked as blue line in D; Bg: Bulged sponge marked as red line in E. F. Quantification of the results from D and E. * p
    Figure Legend Snippet: The expression of multi-potent miRNA sponge exerts antitumor activity A, B. Effect of the miRNA sponge to cancer drug (doxorubicin) sensitivity. In A, Flp-In T-REx 293 cells were treated with doxycycline (sponge induction) and doxorubicin together. In B, sponge was induced in cells and after 24 hours, doxorubicin was treated for another 24 hrs. The cell viability was measured by AlamarBlue assay. C. Comparison of the multi-potent miRNA sponge with single miRNA sponge for the anti-proliferative function. MDA-MB-436 cells were transfected with each of the construct indicated and after 48 hrs the cell proliferation was measured by AlamarBlue assay. (pMIR :pmiR-Report vector only; 21, 155, 222 : miR-21, miR-155, miR-222 targeting sponge respectively. Combi : mixture of the 3 kinds of single sponge (miRNA 21, 155, 221/222)). D, E. Effect of the miRNA sponge to apoptosis analyzed by Annexin V-staining, after doxorubicin treatment (20 hrs). FACS analysis shows more Annexin V-positive cells after sponge expression (RFP: control without MBS, marked as black line; PF: perfect sponge marked as blue line in D; Bg: Bulged sponge marked as red line in E. F. Quantification of the results from D and E. * p

    Techniques Used: Expressing, Activity Assay, Alamar Blue Assay, Multiple Displacement Amplification, Transfection, Construct, Plasmid Preparation, Staining, FACS

    27) Product Images from "Transcriptome association analysis identifies miR-375 as a major determinant of variable acetaminophen glucuronidation by human liver"

    Article Title: Transcriptome association analysis identifies miR-375 as a major determinant of variable acetaminophen glucuronidation by human liver

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2016.08.014

    Identification of a miR-375 binding site in the AhR 3'-UTR. Luciferase activities of cells transfected with the pMIR-REPORT vector carrying the triplicated putative miRNA response elements of HNF1A , HNF1B , Nrf2 , or AhR cloned in forward (A) or reverse
    Figure Legend Snippet: Identification of a miR-375 binding site in the AhR 3'-UTR. Luciferase activities of cells transfected with the pMIR-REPORT vector carrying the triplicated putative miRNA response elements of HNF1A , HNF1B , Nrf2 , or AhR cloned in forward (A) or reverse

    Techniques Used: Binding Assay, Luciferase, Transfection, Plasmid Preparation, Clone Assay

    miR-375 does not have a binding site in the UGT1A 3'-UTR. Luciferase activities of HEK293 cells transfected with the pMIR-REPORT vector carrying the full length UGT1A 3’-UTR (A) or the triplicated putative miRNA response elements of UGT1A (B).
    Figure Legend Snippet: miR-375 does not have a binding site in the UGT1A 3'-UTR. Luciferase activities of HEK293 cells transfected with the pMIR-REPORT vector carrying the full length UGT1A 3’-UTR (A) or the triplicated putative miRNA response elements of UGT1A (B).

    Techniques Used: Binding Assay, Luciferase, Transfection, Plasmid Preparation

    28) Product Images from "MicroRNA-125a Reduces Proliferation and Invasion of Oral Squamous Cell Carcinoma Cells by Targeting Estrogen-related Receptor α"

    Article Title: MicroRNA-125a Reduces Proliferation and Invasion of Oral Squamous Cell Carcinoma Cells by Targeting Estrogen-related Receptor α

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.584136

    miR-125a target sites in the 3′UTR of ESRRA and luciferase reporter assay. A, ClustalW alignment to show conservation of putative miR-125a TSs in the 3′UTR of ESRRA in different species. B, confirmation of miR-125a binding to the 3′UTR of ESRRA by the luciferase reporter assay. Note the significantly reduced luciferase activity in cells transfected with pmiR-125a and pmiR-REPORT-3′UTR-S in comparison with cells transfected with pmiR-REPORT-3′UTR-S and pcDNA3- EGFP , confirming the binding of miR-125a to the 3′UTR of ESRRA . *, p
    Figure Legend Snippet: miR-125a target sites in the 3′UTR of ESRRA and luciferase reporter assay. A, ClustalW alignment to show conservation of putative miR-125a TSs in the 3′UTR of ESRRA in different species. B, confirmation of miR-125a binding to the 3′UTR of ESRRA by the luciferase reporter assay. Note the significantly reduced luciferase activity in cells transfected with pmiR-125a and pmiR-REPORT-3′UTR-S in comparison with cells transfected with pmiR-REPORT-3′UTR-S and pcDNA3- EGFP , confirming the binding of miR-125a to the 3′UTR of ESRRA . *, p

    Techniques Used: Luciferase, Reporter Assay, Binding Assay, Activity Assay, Transfection

    29) Product Images from "MEF2A regulates the Gtl2-Dio3 microRNA mega-cluster to modulate WNT signaling in skeletal muscle regeneration"

    Article Title: MEF2A regulates the Gtl2-Dio3 microRNA mega-cluster to modulate WNT signaling in skeletal muscle regeneration

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.081851

    Gtl2-Dio3 miRNAs directly regulate the 3′UTR of Sfrp2 . ( A ) Schematic of miRs (colored diamonds) predicted to target the 3′UTRs of Sfrp1, Sfrp2 and Sfrp4 (MirANDA). Grouped miRNAs under each Sfrp transcript, listed from top to bottom, reflects their 5′ to 3′ order within the Gtl2-Dio3 locus. ( B ) Sfrp expression in WT and KO TA muscle, 7 days post-injury ( n =3). ( C ) sFRP2 protein expression in WT and KO TA muscle, 7 days post-injury ( n =2). Space indicates non-adjacent lanes from one blot. ( D ) miRNA and Sfrp2 changes in gene expression during regeneration. miR expression values represent the average change in gene expression of five miRs (miR-540-3p, miR-433, miR-495, miR-381, miR-410) predicted to target the 3′UTR of Sfrp2 ( n =3). At regeneration day 7, Sfrp2 levels in Mef2a KO mice are 4.5-fold higher than WT. At this timepoint, WT Sfrp2 levels are 27-fold higher than pre-injury levels. ( E ) Sequence alignments of miR-410 and miR-433 seed sequences and predicted 3′UTR sFRP2 target sites (MirANDA). ( F ) Luciferase analysis of pMIR-REPORT-3′UTR- sFRP2 (WT) and the miR-410 seed sequence mutant (MUT), co-transfected with miR-410 and miR-433 precursor miRNA mimics (150 nM, Ambion) in COS cells compared with a non-specific control (NSC) miR mimic ( n =6). Error bars represent s.e.m. * P
    Figure Legend Snippet: Gtl2-Dio3 miRNAs directly regulate the 3′UTR of Sfrp2 . ( A ) Schematic of miRs (colored diamonds) predicted to target the 3′UTRs of Sfrp1, Sfrp2 and Sfrp4 (MirANDA). Grouped miRNAs under each Sfrp transcript, listed from top to bottom, reflects their 5′ to 3′ order within the Gtl2-Dio3 locus. ( B ) Sfrp expression in WT and KO TA muscle, 7 days post-injury ( n =3). ( C ) sFRP2 protein expression in WT and KO TA muscle, 7 days post-injury ( n =2). Space indicates non-adjacent lanes from one blot. ( D ) miRNA and Sfrp2 changes in gene expression during regeneration. miR expression values represent the average change in gene expression of five miRs (miR-540-3p, miR-433, miR-495, miR-381, miR-410) predicted to target the 3′UTR of Sfrp2 ( n =3). At regeneration day 7, Sfrp2 levels in Mef2a KO mice are 4.5-fold higher than WT. At this timepoint, WT Sfrp2 levels are 27-fold higher than pre-injury levels. ( E ) Sequence alignments of miR-410 and miR-433 seed sequences and predicted 3′UTR sFRP2 target sites (MirANDA). ( F ) Luciferase analysis of pMIR-REPORT-3′UTR- sFRP2 (WT) and the miR-410 seed sequence mutant (MUT), co-transfected with miR-410 and miR-433 precursor miRNA mimics (150 nM, Ambion) in COS cells compared with a non-specific control (NSC) miR mimic ( n =6). Error bars represent s.e.m. * P

    Techniques Used: Expressing, Mouse Assay, Sequencing, Luciferase, Mutagenesis, Transfection

    30) Product Images from "MicroRNA-221 Induces Cell Survival and Cisplatin Resistance through PI3K/Akt Pathway in Human Osteosarcoma"

    Article Title: MicroRNA-221 Induces Cell Survival and Cisplatin Resistance through PI3K/Akt Pathway in Human Osteosarcoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053906

    miR-221 targets PTEN leading to activation of the Akt pathway. A , putative miR-221-binding sites (red) in the 3′-UTRs of PTEN ( top ). The middle panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The bottom shows a mutant of PTEN 3′-UTR for pMIR-REPORT. B , SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. C , SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using Lipofectamine 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). D , similar experiments were performed in MG63 cells as described in panel C except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p
    Figure Legend Snippet: miR-221 targets PTEN leading to activation of the Akt pathway. A , putative miR-221-binding sites (red) in the 3′-UTRs of PTEN ( top ). The middle panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The bottom shows a mutant of PTEN 3′-UTR for pMIR-REPORT. B , SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. C , SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using Lipofectamine 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). D , similar experiments were performed in MG63 cells as described in panel C except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p

    Techniques Used: Activation Assay, Binding Assay, Sequencing, Mutagenesis, Transfection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay

    31) Product Images from "MicroRNA-375 targets PAX6 and inhibits the viability, migration and invasion of human breast cancer MCF-7 cells"

    Article Title: MicroRNA-375 targets PAX6 and inhibits the viability, migration and invasion of human breast cancer MCF-7 cells

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2017.4593

    PAX6 is a direct target of miR-375. (A) Targetscan demonstrated that PAX6 was a putative target of miR-375 and (B) that this targeting association was evolutionally conserved. (C) The WT 3′-UTR or MUT 3′-UTR of PAX6 was inserted downstream of the luciferase reporter gene in a pMIR-REPORT vector. (D) MCF-7 cells were co-transfected with miR-375 mimics/miR-NC, WT-PAX6/MUT-PAX6 and pRL-SV40 expressing Renilla luciferase. In the control group, cells were co-transfected with WT-PAX6/MUT-PAX6 and pRL-SV40 expressing Renilla luciferase. Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System. **P
    Figure Legend Snippet: PAX6 is a direct target of miR-375. (A) Targetscan demonstrated that PAX6 was a putative target of miR-375 and (B) that this targeting association was evolutionally conserved. (C) The WT 3′-UTR or MUT 3′-UTR of PAX6 was inserted downstream of the luciferase reporter gene in a pMIR-REPORT vector. (D) MCF-7 cells were co-transfected with miR-375 mimics/miR-NC, WT-PAX6/MUT-PAX6 and pRL-SV40 expressing Renilla luciferase. In the control group, cells were co-transfected with WT-PAX6/MUT-PAX6 and pRL-SV40 expressing Renilla luciferase. Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System. **P

    Techniques Used: Luciferase, Plasmid Preparation, Transfection, Expressing, Activity Assay, Reporter Assay

    32) Product Images from "MicroRNA-196b Regulates the Homeobox B7-Vascular Endothelial Growth Factor Axis in Cervical Cancer"

    Article Title: MicroRNA-196b Regulates the Homeobox B7-Vascular Endothelial Growth Factor Axis in Cervical Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067846

    Identification of HOXB7 as an mRNA target of miR-196b. A) Relative luciferase activity of ME-180 or SiHa cells at 24 hours after co-transfection with pMIR-REPORT, pMIR-HOXB7 or pMIR-HOXB7-mut vectors and pre-miR-196b or NC (30 nmol/L). B) Relative HOXB7 mRNA expression levels in ME-180 or SiHa cells after transfection (48, and 72 hrs) with pre-miR-196b or NC (30 nmol/L), as measured by qRT-PCR. Expression levels were normalized to GAPDH expression. C) Representative Western blot image (top), and relative quantification of HOXB7 protein levels (bottom) after transfection (48, and 72 hrs) with pre-miR-196b or NC (30 nmol/L). All data represented the mean ± SEM from 3 independent experiments. OD, optical density; NC, pre-miR Negative Control; * P
    Figure Legend Snippet: Identification of HOXB7 as an mRNA target of miR-196b. A) Relative luciferase activity of ME-180 or SiHa cells at 24 hours after co-transfection with pMIR-REPORT, pMIR-HOXB7 or pMIR-HOXB7-mut vectors and pre-miR-196b or NC (30 nmol/L). B) Relative HOXB7 mRNA expression levels in ME-180 or SiHa cells after transfection (48, and 72 hrs) with pre-miR-196b or NC (30 nmol/L), as measured by qRT-PCR. Expression levels were normalized to GAPDH expression. C) Representative Western blot image (top), and relative quantification of HOXB7 protein levels (bottom) after transfection (48, and 72 hrs) with pre-miR-196b or NC (30 nmol/L). All data represented the mean ± SEM from 3 independent experiments. OD, optical density; NC, pre-miR Negative Control; * P

    Techniques Used: Luciferase, Activity Assay, Cotransfection, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Negative Control

    33) Product Images from "Cigarette Smoke Induces C/EBP-?-Mediated Activation of miR-31 in Normal Human Respiratory Epithelia and Lung Cancer Cells"

    Article Title: Cigarette Smoke Induces C/EBP-?-Mediated Activation of miR-31 in Normal Human Respiratory Epithelia and Lung Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013764

    WNT signaling pathway antagonists are direct target candidates of miR-31. A) Putative target sites of miR-31 within Dkk-1 3′ UTR (top) and DACT3 3′ UTR (bottom). B) miRNA-targeted transcripts in RISC were precipitated with AGO family antibodies after UV induced cross-linking of RNAs to their binding proteins, followed by RT-PCR amplification. Over-expression of miR-31 significantly increased precipitation of Dkk-1 and DACT3 target transcripts in SAEC cells. β-actin served as the negative control since no sequences in the 3′ UTR that can be targeted by miR-31. C) Luciferase assays demonstrating reduction in luciferase activity in SAEC transfected with pMiR-Report vectors with or without insertion 3′ UTR or mutated 3′ UTR of WNT signaling antagonists.
    Figure Legend Snippet: WNT signaling pathway antagonists are direct target candidates of miR-31. A) Putative target sites of miR-31 within Dkk-1 3′ UTR (top) and DACT3 3′ UTR (bottom). B) miRNA-targeted transcripts in RISC were precipitated with AGO family antibodies after UV induced cross-linking of RNAs to their binding proteins, followed by RT-PCR amplification. Over-expression of miR-31 significantly increased precipitation of Dkk-1 and DACT3 target transcripts in SAEC cells. β-actin served as the negative control since no sequences in the 3′ UTR that can be targeted by miR-31. C) Luciferase assays demonstrating reduction in luciferase activity in SAEC transfected with pMiR-Report vectors with or without insertion 3′ UTR or mutated 3′ UTR of WNT signaling antagonists.

    Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Over Expression, Negative Control, Luciferase, Activity Assay, Transfection

    34) Product Images from "MicroRNA-221 Induces Cell Survival and Cisplatin Resistance through PI3K/Akt Pathway in Human Osteosarcoma"

    Article Title: MicroRNA-221 Induces Cell Survival and Cisplatin Resistance through PI3K/Akt Pathway in Human Osteosarcoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053906

    miR-221 targets PTEN leading to activation of the Akt pathway. A , putative miR-221-binding sites (red) in the 3′-UTRs of PTEN ( top ). The middle panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The bottom shows a mutant of PTEN 3′-UTR for pMIR-REPORT. B , SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. C , SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using Lipofectamine 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). D , similar experiments were performed in MG63 cells as described in panel C except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p
    Figure Legend Snippet: miR-221 targets PTEN leading to activation of the Akt pathway. A , putative miR-221-binding sites (red) in the 3′-UTRs of PTEN ( top ). The middle panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The bottom shows a mutant of PTEN 3′-UTR for pMIR-REPORT. B , SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. C , SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using Lipofectamine 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). D , similar experiments were performed in MG63 cells as described in panel C except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p

    Techniques Used: Activation Assay, Binding Assay, Sequencing, Mutagenesis, Transfection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay

    35) Product Images from "A microenvironment-mediated c-Myc/miR-548m/HDAC6 amplification loop in non-Hodgkin B cell lymphomas"

    Article Title: A microenvironment-mediated c-Myc/miR-548m/HDAC6 amplification loop in non-Hodgkin B cell lymphomas

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI64210

    HDAC6 is a direct target of miR-548m, and cell adhesion–mediated HDAC6 induction is through miR-548m downregulation. ( A ) Overexpression of miR-548m decreased HDAC6 expression and inhibited cell adhesion–mediated HDAC6 expression. The lanes separated by white lines were run on the same gel but were noncontiguous. ( B ) Knockdown of miR-548m upregulated HDAC6 mRNA and protein expression. Jeko-1 cells were transfected with ( A ) pre–miR-548m or pre-miR control and ( B ) anti-miR control or anti–miR-548m, and miR-548m expression was analyzed by qRT-PCR. ( A and B ) The relative level of HDAC6 protein was measured by quantitative densitometry and is indicated below each lane. ( C ) Time course of miR-548m induction in HBL-2-i-miR-548m cells after addition of cumate (30 μg/ml) to cell culture medium. qRT-PCR was used to measure miR-548m expression levels at the indicated times. ( D ) The effect of miR-548m induction on HDAC6 protein expression in HBL-2-i-miR-548m cells after addition of cumate at 48 and 72 hours. ( E ) Schematic diagram showing putative miR-548m binding site on HDAC6 3′-UTR and the sequence of mutant-type reporter constructs. The white rectangle indicates putative miR-548m binding site in HDAC6 3′-UTR. The long black rectangle indicates the human HDAC6 3′-UTR. The red letters indicate the mutation sites of HDAC6 3′-UTR (in HDAC6 3′-UTRm reporter). ( F ) Jeko-1 or SUDHL-4 cells were transfected with pmiR-Report control vector (Ctrl) or pmiR-Report.HDAC6 3′-UTR wild-type plasmids or pmiR-Report.HDAC6 3′-UTR mutant plasmids (MUT) harboring point mutations in the target sites for miR-548m. These reporter plasmids were cotransfected with pre–miR-548m (50 nM) or control. The firefly luciferase activities of these transfected cells were measured and normalized to Renilla luciferase activities. Data are shown with the mean ± SD of at least 4 experiments. * P
    Figure Legend Snippet: HDAC6 is a direct target of miR-548m, and cell adhesion–mediated HDAC6 induction is through miR-548m downregulation. ( A ) Overexpression of miR-548m decreased HDAC6 expression and inhibited cell adhesion–mediated HDAC6 expression. The lanes separated by white lines were run on the same gel but were noncontiguous. ( B ) Knockdown of miR-548m upregulated HDAC6 mRNA and protein expression. Jeko-1 cells were transfected with ( A ) pre–miR-548m or pre-miR control and ( B ) anti-miR control or anti–miR-548m, and miR-548m expression was analyzed by qRT-PCR. ( A and B ) The relative level of HDAC6 protein was measured by quantitative densitometry and is indicated below each lane. ( C ) Time course of miR-548m induction in HBL-2-i-miR-548m cells after addition of cumate (30 μg/ml) to cell culture medium. qRT-PCR was used to measure miR-548m expression levels at the indicated times. ( D ) The effect of miR-548m induction on HDAC6 protein expression in HBL-2-i-miR-548m cells after addition of cumate at 48 and 72 hours. ( E ) Schematic diagram showing putative miR-548m binding site on HDAC6 3′-UTR and the sequence of mutant-type reporter constructs. The white rectangle indicates putative miR-548m binding site in HDAC6 3′-UTR. The long black rectangle indicates the human HDAC6 3′-UTR. The red letters indicate the mutation sites of HDAC6 3′-UTR (in HDAC6 3′-UTRm reporter). ( F ) Jeko-1 or SUDHL-4 cells were transfected with pmiR-Report control vector (Ctrl) or pmiR-Report.HDAC6 3′-UTR wild-type plasmids or pmiR-Report.HDAC6 3′-UTR mutant plasmids (MUT) harboring point mutations in the target sites for miR-548m. These reporter plasmids were cotransfected with pre–miR-548m (50 nM) or control. The firefly luciferase activities of these transfected cells were measured and normalized to Renilla luciferase activities. Data are shown with the mean ± SD of at least 4 experiments. * P

    Techniques Used: Over Expression, Expressing, Transfection, Quantitative RT-PCR, Cell Culture, Binding Assay, Sequencing, Mutagenesis, Construct, Plasmid Preparation, Luciferase

    c-Myc/miR-548m feed-forward circuit contributes to stroma-mediated c-Myc activation and miR-548m downregulation in lymphoma microenvironment. ( A ) miR-548m expression inversely correlated with c-Myc expression in tetracycline-treated (Myc off) and untreated (Myc on) P493-6 cells after addition and withdraw of tetracycline. ( B ) Inhibition of c-Myc and EZH2 induced miR-548m expression in HBL-2 and SUDHL-4 cells. ( C ) Schematic diagram of the location of Myc-binding sites of the miR-548m regulatory region. S1 and S2 represent Myc-binding sites with E-box sequence. The black bar represents the miR-548m locus. NC, negative control. Both S1 and S2 are highly conserved in their putative promoter region. ChIP assay shows c-Myc and EZH2 enrichment on miR-548m promoter in c-Myc on and c-Myc off P493-6 cells and Jeko-1 cells. ( D ) Ectopic expression of miR-548m downregulated c-Myc expression (left) and directly targeted 3′-UTR of c-Myc (right). Jeko-1 or SUDHL-4 cells were transfected with pmiR-Report control vector (Ctrl) or pmiR-Report.Myc 3′-UTR wild-type or pmiR-Report.Myc 3′-UTR mutant (Mut) plasmids harboring point mutations in the target sites for miR-548m. The luciferase activities were normalized against firefly luciferase activities. Data are representative of at least 3 independent experiments (mean ± SD). ( E ) Cell adhesion to HK cells induced upregulation of c-Myc and EZH2. ( F ) Silencing of c-Myc with JQ1 (1 μM, 48 hours) inhibited c-Myc and stroma-induced c-Myc upregulation and increased miR-548m expression. ( G ) Overexpression of miR-548m with pre–miR-548m decreased stroma-induced c-Myc expression, and knockdown of miR-548m by anti–miR-548m increased c-Myc expression. Results are mean ± SD or representative from at least 3 biological replicates.
    Figure Legend Snippet: c-Myc/miR-548m feed-forward circuit contributes to stroma-mediated c-Myc activation and miR-548m downregulation in lymphoma microenvironment. ( A ) miR-548m expression inversely correlated with c-Myc expression in tetracycline-treated (Myc off) and untreated (Myc on) P493-6 cells after addition and withdraw of tetracycline. ( B ) Inhibition of c-Myc and EZH2 induced miR-548m expression in HBL-2 and SUDHL-4 cells. ( C ) Schematic diagram of the location of Myc-binding sites of the miR-548m regulatory region. S1 and S2 represent Myc-binding sites with E-box sequence. The black bar represents the miR-548m locus. NC, negative control. Both S1 and S2 are highly conserved in their putative promoter region. ChIP assay shows c-Myc and EZH2 enrichment on miR-548m promoter in c-Myc on and c-Myc off P493-6 cells and Jeko-1 cells. ( D ) Ectopic expression of miR-548m downregulated c-Myc expression (left) and directly targeted 3′-UTR of c-Myc (right). Jeko-1 or SUDHL-4 cells were transfected with pmiR-Report control vector (Ctrl) or pmiR-Report.Myc 3′-UTR wild-type or pmiR-Report.Myc 3′-UTR mutant (Mut) plasmids harboring point mutations in the target sites for miR-548m. The luciferase activities were normalized against firefly luciferase activities. Data are representative of at least 3 independent experiments (mean ± SD). ( E ) Cell adhesion to HK cells induced upregulation of c-Myc and EZH2. ( F ) Silencing of c-Myc with JQ1 (1 μM, 48 hours) inhibited c-Myc and stroma-induced c-Myc upregulation and increased miR-548m expression. ( G ) Overexpression of miR-548m with pre–miR-548m decreased stroma-induced c-Myc expression, and knockdown of miR-548m by anti–miR-548m increased c-Myc expression. Results are mean ± SD or representative from at least 3 biological replicates.

    Techniques Used: Activation Assay, Expressing, Inhibition, Binding Assay, Sequencing, Negative Control, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Mutagenesis, Luciferase, Over Expression

    36) Product Images from "MicroRNA-221 Induces Cell Survival and Cisplatin Resistance through PI3K/Akt Pathway in Human Osteosarcoma"

    Article Title: MicroRNA-221 Induces Cell Survival and Cisplatin Resistance through PI3K/Akt Pathway in Human Osteosarcoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053906

    miR-221 targets PTEN leading to activation of the Akt pathway. A , putative miR-221-binding sites (red) in the 3′-UTRs of PTEN ( top ). The middle panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The bottom shows a mutant of PTEN 3′-UTR for pMIR-REPORT. B , SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. C , SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using Lipofectamine 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). D , similar experiments were performed in MG63 cells as described in panel C except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p
    Figure Legend Snippet: miR-221 targets PTEN leading to activation of the Akt pathway. A , putative miR-221-binding sites (red) in the 3′-UTRs of PTEN ( top ). The middle panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The bottom shows a mutant of PTEN 3′-UTR for pMIR-REPORT. B , SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. C , SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using Lipofectamine 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). D , similar experiments were performed in MG63 cells as described in panel C except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p

    Techniques Used: Activation Assay, Binding Assay, Sequencing, Mutagenesis, Transfection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay

    37) Product Images from "miR‐222 attenuates cisplatin‐induced cell death by targeting the PPP2R2A/Akt/ mTOR Axis in bladder cancer cells"

    Article Title: miR‐222 attenuates cisplatin‐induced cell death by targeting the PPP2R2A/Akt/ mTOR Axis in bladder cancer cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12760

    miR‐222 directly targets protein phosphatase 2A subunit B in bladder cancer cells. ( A – H ) The expression of protein phosphatase 2A subunit B ( PPP 2R2A) in T24 ( A – D ) and 5637 ( E – H ) cells transfected with the miR‐222 mimics ( A and E ) or antagomir ( B and F ). The relative expression of PPP 2R2A was normalized to GAPDH ( C , D , G and H ). ( I ) PPP 2R2A is predicted to be a potential target gene of miR‐222 by Targetscan. The miR‐222 binding sites in the wild‐type ( WT ) and mutated (mut) PPP 2R2A 3′ UTR sequences are shown. ( J and K ) The WT and Mut 3′ UTR of PPP 2R2A were cloned into pMIR ‐ REPORT vectors and cotransfected into T24 ( J ) and 5637 ( K ) cells with pRL ‐ TK and the miR‐222 mimics or scrambled RNA . The relative luciferase activity was measured and normalized to that of Renilla luciferase. ( L and M ) T24 cells were transfected as indicated and CCK ‐8 assays were performed ( M ). The expression of PPP 2R2A in each group is shown ( L ). Values are expressed as the means ± S.D.; ** P
    Figure Legend Snippet: miR‐222 directly targets protein phosphatase 2A subunit B in bladder cancer cells. ( A – H ) The expression of protein phosphatase 2A subunit B ( PPP 2R2A) in T24 ( A – D ) and 5637 ( E – H ) cells transfected with the miR‐222 mimics ( A and E ) or antagomir ( B and F ). The relative expression of PPP 2R2A was normalized to GAPDH ( C , D , G and H ). ( I ) PPP 2R2A is predicted to be a potential target gene of miR‐222 by Targetscan. The miR‐222 binding sites in the wild‐type ( WT ) and mutated (mut) PPP 2R2A 3′ UTR sequences are shown. ( J and K ) The WT and Mut 3′ UTR of PPP 2R2A were cloned into pMIR ‐ REPORT vectors and cotransfected into T24 ( J ) and 5637 ( K ) cells with pRL ‐ TK and the miR‐222 mimics or scrambled RNA . The relative luciferase activity was measured and normalized to that of Renilla luciferase. ( L and M ) T24 cells were transfected as indicated and CCK ‐8 assays were performed ( M ). The expression of PPP 2R2A in each group is shown ( L ). Values are expressed as the means ± S.D.; ** P

    Techniques Used: Expressing, Transfection, Binding Assay, Clone Assay, Luciferase, Activity Assay, CCK-8 Assay

    38) Product Images from "Modulation of NF-κB/miR-21/PTEN Pathway Sensitizes Non-Small Cell Lung Cancer to Cisplatin"

    Article Title: Modulation of NF-κB/miR-21/PTEN Pathway Sensitizes Non-Small Cell Lung Cancer to Cisplatin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121547

    miR-21 targeting 3’UTR of PTEN. (A) Construction of 3’UTR and mutated 3’UTR of PTEN into pMIR-REPORT vector. (B) In the cells transfected with pMIR-REPORT-PTEN-3’UTR, the luciferase activity was increased by miR-21 and decreased by miR-21 inhibitor. In the cells transfected with pMIR-REPORT-PTEN-mut-3’UTR, the luciferase activity remained no significant difference among the three groups. (C) The open reading frame of PTEN with or without 3’UTR was cloned into pCS2 vector. A Myc tag was attached to PTEN to distinguish the endogenous or exogenous PTEN. (D) Graphic representation of Myc showed that overexpression of miR-21 reduced Myc expression while miR-21 inhibitor increased its expression. While the PTEN 3’UTR was knocked out, the expression of PTEN was not influenced by miR-21. * compared with control group, # compared with miR-21 mimic group. P
    Figure Legend Snippet: miR-21 targeting 3’UTR of PTEN. (A) Construction of 3’UTR and mutated 3’UTR of PTEN into pMIR-REPORT vector. (B) In the cells transfected with pMIR-REPORT-PTEN-3’UTR, the luciferase activity was increased by miR-21 and decreased by miR-21 inhibitor. In the cells transfected with pMIR-REPORT-PTEN-mut-3’UTR, the luciferase activity remained no significant difference among the three groups. (C) The open reading frame of PTEN with or without 3’UTR was cloned into pCS2 vector. A Myc tag was attached to PTEN to distinguish the endogenous or exogenous PTEN. (D) Graphic representation of Myc showed that overexpression of miR-21 reduced Myc expression while miR-21 inhibitor increased its expression. While the PTEN 3’UTR was knocked out, the expression of PTEN was not influenced by miR-21. * compared with control group, # compared with miR-21 mimic group. P

    Techniques Used: Plasmid Preparation, Transfection, Luciferase, Activity Assay, Clone Assay, Over Expression, Expressing

    39) Product Images from "Posttranscriptional Regulation of Interleukin-33 Expression by MicroRNA-200 in Bronchial Asthma"

    Article Title: Posttranscriptional Regulation of Interleukin-33 Expression by MicroRNA-200 in Bronchial Asthma

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2018.04.016

    Confirmation of Target Site for miR-200b/c in IL-33 3′ UTR (A) Predicted binding site for miR-200b/c in 3′ UTR of IL-33. (B) Relative luciferase activity in Jurkat cells cotransfected with control firefly luciferase vector (pMIR-Report), or a firefly luciferase reporter vector containing the 3′ UTR of IL-33, or a firefly luciferase vector with perfect miR-200b/c binding site in the 3′ UTR (pMIR-200), and either the pre-miR-200 expression vector (pMIRNA1-Pre-miR-200) or control vector (pMIRNA1-Control). Firefly luciferase activity was normalized to the Renilla luciferase activity and then to the average of the control firefly luciferase reporter. n = 4 per group; data are representative of three experiments. An unpaired t test was used to compare reporter gene activity. (C) Jurkat, Raji, THP-1, and A549 cells were transfected with pMIR-REPORT-IL-33 3′ UTR to correlate the expression of miR-200b/c with IL-33 3′ UTR regulatory activity. (D) MRC5 cells were transfected with a miR-200b/c expression vector; 24 hr later, cells were stimulated with IL-13 to induce production of IL-33. Overexpression of miR-200b and miR-200c greatly reduced the induction of IL-33 production. (E) Silencing the endogenous miR-200b/c via transfection with inhibitory oligonucleotides increased IL-33 production. **p
    Figure Legend Snippet: Confirmation of Target Site for miR-200b/c in IL-33 3′ UTR (A) Predicted binding site for miR-200b/c in 3′ UTR of IL-33. (B) Relative luciferase activity in Jurkat cells cotransfected with control firefly luciferase vector (pMIR-Report), or a firefly luciferase reporter vector containing the 3′ UTR of IL-33, or a firefly luciferase vector with perfect miR-200b/c binding site in the 3′ UTR (pMIR-200), and either the pre-miR-200 expression vector (pMIRNA1-Pre-miR-200) or control vector (pMIRNA1-Control). Firefly luciferase activity was normalized to the Renilla luciferase activity and then to the average of the control firefly luciferase reporter. n = 4 per group; data are representative of three experiments. An unpaired t test was used to compare reporter gene activity. (C) Jurkat, Raji, THP-1, and A549 cells were transfected with pMIR-REPORT-IL-33 3′ UTR to correlate the expression of miR-200b/c with IL-33 3′ UTR regulatory activity. (D) MRC5 cells were transfected with a miR-200b/c expression vector; 24 hr later, cells were stimulated with IL-13 to induce production of IL-33. Overexpression of miR-200b and miR-200c greatly reduced the induction of IL-33 production. (E) Silencing the endogenous miR-200b/c via transfection with inhibitory oligonucleotides increased IL-33 production. **p

    Techniques Used: Binding Assay, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Transfection, Over Expression

    40) Product Images from "Haplotypes of the HLA-G 3’ Untranslated Region Respond to Endogenous Factors of HLA-G+ and HLA-G- Cell Lines Differentially"

    Article Title: Haplotypes of the HLA-G 3’ Untranslated Region Respond to Endogenous Factors of HLA-G+ and HLA-G- Cell Lines Differentially

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169032

    HLA-G 3’UTR polymorphisms. (A) Variations in HLA-G mRNA (red) along the 3’UTR. The less frequent variants are positioned below the most frequent one in the nucleotide sequence. Polymorphic sites are framed in bold when the frequency of the minor allele worldwide is higher than 1%. Purple single arrows point to the three predicted polyadenylation signals. Vertical lines with horizontal arrows indicate the 5’ end of the HLA-G nucleotide sequences for 1Fter, 2R and 4R primers used in pMIR-REPORT ™ constructions. (B) Sequence comparison of the most frequent 3’UTR haplotypes (21 worldwide populations; 2n = 3870) [ 25 ] analyzed in the present study. Frequencies are indicated on the right part. UTR-1 and UTR-2 differ at 5 positions (pink rectangles). UTR-6 and UTR-18 (analyzed in the present work) only differ at one position and depending on the UTR typing strategy that was used (i.e., targeting or not +3227 SNP), UTR-18 could be confused with UTR-6 in several studies.
    Figure Legend Snippet: HLA-G 3’UTR polymorphisms. (A) Variations in HLA-G mRNA (red) along the 3’UTR. The less frequent variants are positioned below the most frequent one in the nucleotide sequence. Polymorphic sites are framed in bold when the frequency of the minor allele worldwide is higher than 1%. Purple single arrows point to the three predicted polyadenylation signals. Vertical lines with horizontal arrows indicate the 5’ end of the HLA-G nucleotide sequences for 1Fter, 2R and 4R primers used in pMIR-REPORT ™ constructions. (B) Sequence comparison of the most frequent 3’UTR haplotypes (21 worldwide populations; 2n = 3870) [ 25 ] analyzed in the present study. Frequencies are indicated on the right part. UTR-1 and UTR-2 differ at 5 positions (pink rectangles). UTR-6 and UTR-18 (analyzed in the present work) only differ at one position and depending on the UTR typing strategy that was used (i.e., targeting or not +3227 SNP), UTR-18 could be confused with UTR-6 in several studies.

    Techniques Used: Sequencing

    Related Articles

    Clone Assay:

    Article Title: Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy
    Article Snippet: .. The 3′UTR luciferase reporter constructs of mTOR and TSC1 were generated by cloning the PCR-amplified human mTOR and TSC1 mRNA 3′UTRs (miR-99b and let-7g target sites, respectively) into the MluI/HindIII site of the pMIR-REPORT miRNA expression luciferase reporter plasmid (Ambion: AM5795). .. Mutant 3′UTRs of mTOR or TSC1 were used as a control in parallel.

    Luciferase:

    Article Title: Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy
    Article Snippet: .. The 3′UTR luciferase reporter constructs of mTOR and TSC1 were generated by cloning the PCR-amplified human mTOR and TSC1 mRNA 3′UTRs (miR-99b and let-7g target sites, respectively) into the MluI/HindIII site of the pMIR-REPORT miRNA expression luciferase reporter plasmid (Ambion: AM5795). .. Mutant 3′UTRs of mTOR or TSC1 were used as a control in parallel.

    Construct:

    Article Title: Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy
    Article Snippet: .. The 3′UTR luciferase reporter constructs of mTOR and TSC1 were generated by cloning the PCR-amplified human mTOR and TSC1 mRNA 3′UTRs (miR-99b and let-7g target sites, respectively) into the MluI/HindIII site of the pMIR-REPORT miRNA expression luciferase reporter plasmid (Ambion: AM5795). .. Mutant 3′UTRs of mTOR or TSC1 were used as a control in parallel.

    Real-time Polymerase Chain Reaction:

    Article Title: MicroRNA Profile of Circulating CD4-positive Regulatory T Cells in Human Adults and Impact of Differentially Expressed MicroRNAs on Expression of Two Genes Essential to Their Function
    Article Snippet: .. The real-time PCR results were analyzed and expressed as relative miRNA expression of Ct value, which was then converted to -fold changes. .. RT primer, PCR primers, and TaqMan probe for miR-24, miR-145, miR-210, and miR-95 were purchased from ABI.

    Article Title: MicroRNAs miR-30b, miR-30d, and miR-494 Regulate Human Endometrial Receptivity
    Article Snippet: .. The miRNA Array Validation by Real-Time PCRQuantitative real-time PCR was used as a validation tool for confirming the miRNA expression results obtained from microarray analysis. .. The microarrays were validated by predesigned and custom-made TaqMan MicroRNA assays (Applied Biosystems, Inc., Foster City, CA, USA).

    Microarray:

    Article Title: MicroRNAs miR-30b, miR-30d, and miR-494 Regulate Human Endometrial Receptivity
    Article Snippet: .. The miRNA Array Validation by Real-Time PCRQuantitative real-time PCR was used as a validation tool for confirming the miRNA expression results obtained from microarray analysis. .. The microarrays were validated by predesigned and custom-made TaqMan MicroRNA assays (Applied Biosystems, Inc., Foster City, CA, USA).

    Generated:

    Article Title: Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy
    Article Snippet: .. The 3′UTR luciferase reporter constructs of mTOR and TSC1 were generated by cloning the PCR-amplified human mTOR and TSC1 mRNA 3′UTRs (miR-99b and let-7g target sites, respectively) into the MluI/HindIII site of the pMIR-REPORT miRNA expression luciferase reporter plasmid (Ambion: AM5795). .. Mutant 3′UTRs of mTOR or TSC1 were used as a control in parallel.

    Quantitative RT-PCR:

    Article Title: Circulating miR-150 and miR-342 in plasma are novel potential biomarkers for acute myeloid leukemia
    Article Snippet: .. Due to a lack of generally accepted standards, all qRT-PCR data on single miRNA expression were analyzed as unadjusted Ct values and standardized to miR-16. ..

    Expressing:

    Article Title: TFPI Alpha and Beta Regulate mRNAs and microRNAs Involved in Cancer Biology and in the Immune System in Breast Cancer Cells
    Article Snippet: .. Results were normalized against the endogenous control U6 snRNA and changes in relative miRNA expression were calculated using the comparative Ct method and expressed as FC of empty vector pTOPO control. miRNAs with a FC of ≥|2| were considered differentially expressed. .. For the integrated miRNA and mRNA analysis, the lists of differentially expressed miRNAs were loaded into the Partek Genomics Suite software (Partek) already containing the analyzed microarray data.

    Article Title: MicroRNA Profile of Circulating CD4-positive Regulatory T Cells in Human Adults and Impact of Differentially Expressed MicroRNAs on Expression of Two Genes Essential to Their Function
    Article Snippet: .. The real-time PCR results were analyzed and expressed as relative miRNA expression of Ct value, which was then converted to -fold changes. .. RT primer, PCR primers, and TaqMan probe for miR-24, miR-145, miR-210, and miR-95 were purchased from ABI.

    Article Title: Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy
    Article Snippet: .. The 3′UTR luciferase reporter constructs of mTOR and TSC1 were generated by cloning the PCR-amplified human mTOR and TSC1 mRNA 3′UTRs (miR-99b and let-7g target sites, respectively) into the MluI/HindIII site of the pMIR-REPORT miRNA expression luciferase reporter plasmid (Ambion: AM5795). .. Mutant 3′UTRs of mTOR or TSC1 were used as a control in parallel.

    Article Title: MicroRNAs miR-30b, miR-30d, and miR-494 Regulate Human Endometrial Receptivity
    Article Snippet: .. The miRNA Array Validation by Real-Time PCRQuantitative real-time PCR was used as a validation tool for confirming the miRNA expression results obtained from microarray analysis. .. The microarrays were validated by predesigned and custom-made TaqMan MicroRNA assays (Applied Biosystems, Inc., Foster City, CA, USA).

    Article Title: Formaldehyde-Associated Changes in microRNAs: Tissue and Temporal Specificity in the Rat Nose, White Blood Cells, and Bone Marrow
    Article Snippet: .. Only the nose and WBC samples were assessed at the transcript level, as no changes in miRNA expression were detected in the BM. .. Transcript levels were measured using the Affymetrix GeneChip Rat Gene 1.0 ST Array, which assesses the expression levels of 27342 genes.

    Article Title: Circulating miR-150 and miR-342 in plasma are novel potential biomarkers for acute myeloid leukemia
    Article Snippet: .. Due to a lack of generally accepted standards, all qRT-PCR data on single miRNA expression were analyzed as unadjusted Ct values and standardized to miR-16. ..

    Polymerase Chain Reaction:

    Article Title: Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy
    Article Snippet: .. The 3′UTR luciferase reporter constructs of mTOR and TSC1 were generated by cloning the PCR-amplified human mTOR and TSC1 mRNA 3′UTRs (miR-99b and let-7g target sites, respectively) into the MluI/HindIII site of the pMIR-REPORT miRNA expression luciferase reporter plasmid (Ambion: AM5795). .. Mutant 3′UTRs of mTOR or TSC1 were used as a control in parallel.

    Plasmid Preparation:

    Article Title: TFPI Alpha and Beta Regulate mRNAs and microRNAs Involved in Cancer Biology and in the Immune System in Breast Cancer Cells
    Article Snippet: .. Results were normalized against the endogenous control U6 snRNA and changes in relative miRNA expression were calculated using the comparative Ct method and expressed as FC of empty vector pTOPO control. miRNAs with a FC of ≥|2| were considered differentially expressed. .. For the integrated miRNA and mRNA analysis, the lists of differentially expressed miRNAs were loaded into the Partek Genomics Suite software (Partek) already containing the analyzed microarray data.

    Article Title: MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells
    Article Snippet: .. The sense and antisense strands of the oligonucleotides were annealed by adding 2 µg of each oligonucleotide to 46 µL of annealing solution (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4 and 2 mM magnesium acetate) and incubating at 90°C for 5 min and then at 37°C for 1 h. The annealed oligonucleotides were digested with Hind III and Spe I and ligated into the Hind III and SpeI restriction sites of pMIR-REPORT vector. .. The sequences of the inserts were confirmed by sequence analysis using a PRISM 3100 genetic analyzer (Applied Biosystems).

    Article Title: Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy
    Article Snippet: .. The 3′UTR luciferase reporter constructs of mTOR and TSC1 were generated by cloning the PCR-amplified human mTOR and TSC1 mRNA 3′UTRs (miR-99b and let-7g target sites, respectively) into the MluI/HindIII site of the pMIR-REPORT miRNA expression luciferase reporter plasmid (Ambion: AM5795). .. Mutant 3′UTRs of mTOR or TSC1 were used as a control in parallel.

    Article Title: miR-582-5p Is Upregulated in Patients with Active Tuberculosis and Inhibits Apoptosis of Monocytes by Targeting FOXO1
    Article Snippet: .. The complementary oligos were annealed and inserted into pMIR-report vector, and the resulting vector was designated pMIR-FOXO1-3′UTR-mut. .. For luciferase assay, HEK-293T cells were seeded onto 96-well plates (1×104 cells per well) 24 h before transfection.

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  • 99
    Thermo Fisher pmir report mirna expression reporter vector system
    Identification of a miR-375 binding site in the AhR 3'-UTR. Luciferase activities of cells transfected with the <t>pMIR-REPORT</t> vector carrying the triplicated putative <t>miRNA</t> response elements of HNF1A , HNF1B , Nrf2 , or AhR cloned in forward (A) or reverse
    Pmir Report Mirna Expression Reporter Vector System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmir report mirna expression reporter vector system/product/Thermo Fisher
    Average 99 stars, based on 163 article reviews
    Price from $9.99 to $1999.99
    pmir report mirna expression reporter vector system - by Bioz Stars, 2020-09
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    84
    Thermo Fisher pmir luc fak plasmids
    HOTAIR acts as an endogenous competitive RNA sponge for miR-204. ( A ) <t>pmiR-LUC-HOTAIR</t> constructions showing the position of HOTAIR wild type and mutated sequences, and the containing seed region for miR-204 in grey box; ( B ) Firefly luciferase activity assays in MDA-MB-231 cells co-transfected with pmiR-LUC-HOTAIR wild type and mutated plasmids and miR-204 mimics; ( C ) qRT-PCR assays for miR-204 in MDA-MB-231 and Hs-578t cells transfected with DsiRNA HOTAIR inhibitor and untreated control. Experiments were performed three times by triplicate and data were expressed as mean ± S.D. ** p
    Pmir Luc Fak Plasmids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmir luc fak plasmids/product/Thermo Fisher
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    Image Search Results


    Identification of a miR-375 binding site in the AhR 3'-UTR. Luciferase activities of cells transfected with the pMIR-REPORT vector carrying the triplicated putative miRNA response elements of HNF1A , HNF1B , Nrf2 , or AhR cloned in forward (A) or reverse

    Journal: Biochemical pharmacology

    Article Title: Transcriptome association analysis identifies miR-375 as a major determinant of variable acetaminophen glucuronidation by human liver

    doi: 10.1016/j.bcp.2016.08.014

    Figure Lengend Snippet: Identification of a miR-375 binding site in the AhR 3'-UTR. Luciferase activities of cells transfected with the pMIR-REPORT vector carrying the triplicated putative miRNA response elements of HNF1A , HNF1B , Nrf2 , or AhR cloned in forward (A) or reverse

    Article Snippet: After overnight culture, cells were co-transfected in quadruplicate with a pMIR-REPORT vector carrying the triplicated miR-375 putative binding sequences of UGT1A (UGT1A 3x F/R), HNF1A (HNF1A 3x F/R), HNF1B (HNF1B 3x F/R), Nrf2 (Nrf2 3x F/R) or AhR (AhR 3x F/R) (10 ng/well), the pRL-CMV reporter vector (5 ng/well) and pre-miR for miR-375 or pre-miR negative controls 1 and 2 at 30 nM using Lipofectamine 2000 (0.25 μL/well).

    Techniques: Binding Assay, Luciferase, Transfection, Plasmid Preparation, Clone Assay

    miR-375 does not have a binding site in the UGT1A 3'-UTR. Luciferase activities of HEK293 cells transfected with the pMIR-REPORT vector carrying the full length UGT1A 3’-UTR (A) or the triplicated putative miRNA response elements of UGT1A (B).

    Journal: Biochemical pharmacology

    Article Title: Transcriptome association analysis identifies miR-375 as a major determinant of variable acetaminophen glucuronidation by human liver

    doi: 10.1016/j.bcp.2016.08.014

    Figure Lengend Snippet: miR-375 does not have a binding site in the UGT1A 3'-UTR. Luciferase activities of HEK293 cells transfected with the pMIR-REPORT vector carrying the full length UGT1A 3’-UTR (A) or the triplicated putative miRNA response elements of UGT1A (B).

    Article Snippet: After overnight culture, cells were co-transfected in quadruplicate with a pMIR-REPORT vector carrying the triplicated miR-375 putative binding sequences of UGT1A (UGT1A 3x F/R), HNF1A (HNF1A 3x F/R), HNF1B (HNF1B 3x F/R), Nrf2 (Nrf2 3x F/R) or AhR (AhR 3x F/R) (10 ng/well), the pRL-CMV reporter vector (5 ng/well) and pre-miR for miR-375 or pre-miR negative controls 1 and 2 at 30 nM using Lipofectamine 2000 (0.25 μL/well).

    Techniques: Binding Assay, Luciferase, Transfection, Plasmid Preparation

    Plasmids used for Transient assay systems to validate computationally predicted targets of miRNA. The plasmid pGrDL_SPb contain the renilla LUC expression cassette which acts as an internal control to standardize expression between replicates, while the firefly LUC expression cassette containing the predicted target sequence cloned between SalI and PstI is used to report miRNA interaction. Expression plasmid pKENminusbar with multiple cloning sites is used for miRNA precursor insertion.

    Journal: bioRxiv

    Article Title: Recent hybridization and allopolyploidy reprogrammed Spartina microRNA expression under xenobiotic induced stress

    doi: 10.1101/2019.12.13.875138

    Figure Lengend Snippet: Plasmids used for Transient assay systems to validate computationally predicted targets of miRNA. The plasmid pGrDL_SPb contain the renilla LUC expression cassette which acts as an internal control to standardize expression between replicates, while the firefly LUC expression cassette containing the predicted target sequence cloned between SalI and PstI is used to report miRNA interaction. Expression plasmid pKENminusbar with multiple cloning sites is used for miRNA precursor insertion.

    Article Snippet: Forward primers used for specific miRNA amplifications were designed manually, according to mature miRNA sequences.

    Techniques: Plasmid Preparation, Expressing, Sequencing, Clone Assay

    miR-150 and miR-342 expression levels do not vary between the first and second sampling. Plasma miR-150 and miR-342 expression levels in the first sampling and second sampling were assessed by qRT-PCR. The expression level of these two microRNAs was normalized to miR-16. Shown are the relative levels (mean ± S.D.) of five independent experiments performed on all participants, each done in triplicate.

    Journal: Journal of Translational Medicine

    Article Title: Circulating miR-150 and miR-342 in plasma are novel potential biomarkers for acute myeloid leukemia

    doi: 10.1186/1479-5876-11-31

    Figure Lengend Snippet: miR-150 and miR-342 expression levels do not vary between the first and second sampling. Plasma miR-150 and miR-342 expression levels in the first sampling and second sampling were assessed by qRT-PCR. The expression level of these two microRNAs was normalized to miR-16. Shown are the relative levels (mean ± S.D.) of five independent experiments performed on all participants, each done in triplicate.

    Article Snippet: Due to a lack of generally accepted standards, all qRT-PCR data on single miRNA expression were analyzed as unadjusted Ct values and standardized to miR-16.

    Techniques: Expressing, Sampling, Quantitative RT-PCR

    miR-150 and miR-342 expression levels in Remission AML patients resembles that of healthy controls. Plasma miR-150 and miR-342 expression levels in remission AML patients and healthy controls were assessed by qRT-PCR. The expression level of these two microRNAs was normalized to miR-16. Shown are the relative levels (mean ± S.D.) of five independent experiments performed on all controls, each done in triplicate. Statistical significance was determined by Student’s t test and is denoted as follows: ** p

    Journal: Journal of Translational Medicine

    Article Title: Circulating miR-150 and miR-342 in plasma are novel potential biomarkers for acute myeloid leukemia

    doi: 10.1186/1479-5876-11-31

    Figure Lengend Snippet: miR-150 and miR-342 expression levels in Remission AML patients resembles that of healthy controls. Plasma miR-150 and miR-342 expression levels in remission AML patients and healthy controls were assessed by qRT-PCR. The expression level of these two microRNAs was normalized to miR-16. Shown are the relative levels (mean ± S.D.) of five independent experiments performed on all controls, each done in triplicate. Statistical significance was determined by Student’s t test and is denoted as follows: ** p

    Article Snippet: Due to a lack of generally accepted standards, all qRT-PCR data on single miRNA expression were analyzed as unadjusted Ct values and standardized to miR-16.

    Techniques: Expressing, Quantitative RT-PCR

    Relative expression levels of the six differentially expressed miRs. Six miRNAs are significantly differentially expressed in the plasma of AML patients when compared to healthy controls (n=20). Data obtained by quantitative RT-PCR amplification of miRs are plotted. p-values for each miRNA are shown. Boxes represent SE. Error bars represent SD; pooled data from five independent experiments. *p

    Journal: Journal of Translational Medicine

    Article Title: Circulating miR-150 and miR-342 in plasma are novel potential biomarkers for acute myeloid leukemia

    doi: 10.1186/1479-5876-11-31

    Figure Lengend Snippet: Relative expression levels of the six differentially expressed miRs. Six miRNAs are significantly differentially expressed in the plasma of AML patients when compared to healthy controls (n=20). Data obtained by quantitative RT-PCR amplification of miRs are plotted. p-values for each miRNA are shown. Boxes represent SE. Error bars represent SD; pooled data from five independent experiments. *p

    Article Snippet: Due to a lack of generally accepted standards, all qRT-PCR data on single miRNA expression were analyzed as unadjusted Ct values and standardized to miR-16.

    Techniques: Expressing, Quantitative RT-PCR, Amplification

    HOTAIR acts as an endogenous competitive RNA sponge for miR-204. ( A ) pmiR-LUC-HOTAIR constructions showing the position of HOTAIR wild type and mutated sequences, and the containing seed region for miR-204 in grey box; ( B ) Firefly luciferase activity assays in MDA-MB-231 cells co-transfected with pmiR-LUC-HOTAIR wild type and mutated plasmids and miR-204 mimics; ( C ) qRT-PCR assays for miR-204 in MDA-MB-231 and Hs-578t cells transfected with DsiRNA HOTAIR inhibitor and untreated control. Experiments were performed three times by triplicate and data were expressed as mean ± S.D. ** p

    Journal: Non-Coding RNA

    Article Title: HOX Transcript Antisense RNA HOTAIR Abrogates Vasculogenic Mimicry by Targeting the AngiomiR-204/FAK Axis in Triple Negative Breast Cancer Cells

    doi: 10.3390/ncrna6020019

    Figure Lengend Snippet: HOTAIR acts as an endogenous competitive RNA sponge for miR-204. ( A ) pmiR-LUC-HOTAIR constructions showing the position of HOTAIR wild type and mutated sequences, and the containing seed region for miR-204 in grey box; ( B ) Firefly luciferase activity assays in MDA-MB-231 cells co-transfected with pmiR-LUC-HOTAIR wild type and mutated plasmids and miR-204 mimics; ( C ) qRT-PCR assays for miR-204 in MDA-MB-231 and Hs-578t cells transfected with DsiRNA HOTAIR inhibitor and untreated control. Experiments were performed three times by triplicate and data were expressed as mean ± S.D. ** p

    Article Snippet: Then, pmiR-LUC and pmiR-LUC-FAK plasmids were transfected into MDA-MB-231 cells using a lipofectamine 2000 (Thermo Fisher Scientific).

    Techniques: Luciferase, Activity Assay, Multiple Displacement Amplification, Transfection, Quantitative RT-PCR

    FAK is a novel target of miR-204. ( A ) pmiR-LUC-FAK plasmids showing the seed region for miR-204 in the 3′UTR of wild type and mutated FAK sequences; ( B ) Firefly luciferase activity assays in MDA-MB-231 cells co-transfected with wild type and mutated pmiR-LUC-FAK plasmids and miR-204 mimics; ( C ) Western blot assays FAK protein in MDA-MB-231 cells treated with miR-204 mimics (lane 3), scramble (lane 2), and non-transfected control (lane 1); ( D , E ) Immunofluorescence and confocal microscopy assays using anti-FAK antibodies (Alexa-488, green channel), rhodamine-phalloidin (TRITC, red channel) for cytoskeleton staining, and DAPI for nuclear DNA staining (blue channel) in MDA-MB-231 cells treated with ( E ) DsiRNA HOTAIR and ( D ) non-transfected control. Experiments were performed three times by triplicate and data were expressed as mean ± S.D. ** p

    Journal: Non-Coding RNA

    Article Title: HOX Transcript Antisense RNA HOTAIR Abrogates Vasculogenic Mimicry by Targeting the AngiomiR-204/FAK Axis in Triple Negative Breast Cancer Cells

    doi: 10.3390/ncrna6020019

    Figure Lengend Snippet: FAK is a novel target of miR-204. ( A ) pmiR-LUC-FAK plasmids showing the seed region for miR-204 in the 3′UTR of wild type and mutated FAK sequences; ( B ) Firefly luciferase activity assays in MDA-MB-231 cells co-transfected with wild type and mutated pmiR-LUC-FAK plasmids and miR-204 mimics; ( C ) Western blot assays FAK protein in MDA-MB-231 cells treated with miR-204 mimics (lane 3), scramble (lane 2), and non-transfected control (lane 1); ( D , E ) Immunofluorescence and confocal microscopy assays using anti-FAK antibodies (Alexa-488, green channel), rhodamine-phalloidin (TRITC, red channel) for cytoskeleton staining, and DAPI for nuclear DNA staining (blue channel) in MDA-MB-231 cells treated with ( E ) DsiRNA HOTAIR and ( D ) non-transfected control. Experiments were performed three times by triplicate and data were expressed as mean ± S.D. ** p

    Article Snippet: Then, pmiR-LUC and pmiR-LUC-FAK plasmids were transfected into MDA-MB-231 cells using a lipofectamine 2000 (Thermo Fisher Scientific).

    Techniques: Luciferase, Activity Assay, Multiple Displacement Amplification, Transfection, Western Blot, Immunofluorescence, Confocal Microscopy, Staining