Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: Volcano plot (A) and heat map of hierarchical gene clustering (B) demonstrating IGF-1-regulated microRNA profiles. After U87 MG cells were treated with 200 ng/ml IGF-1 for 48 h, total RNAs were extracted for a microarray analysis. For all array analyses, an adjusted (adj.) p value of <0.05 and a |log2 (ratio)| of ≥0.58 (±1.5 multiples of change) cutoff were applied. Volcano plots show the multiples of change (log 2 ratio) and probability (-log 10 adj. p values) of individual microRNAs from the microarray assay. The red dot shows the miR-513a-5p location. The heat map depicts the 241 microRNAs differentially expressed between IGF-1 subsets. A color was assigned to each microRNA based on its relative expression level across samples. (C) Detection of endogenous miR-513a-5p levels in human normal astrocytes and three different glioma cell lines. Endogenous miR-513a-5p levels were measured by a real-time PCR. The miR-191-5p level was used as an internal control. Data are the mean ± SD of three independent experiments. * p <0.05. Increased miR-513a levels in GBM patients and glioma stem cells (GSCs) compared to normal controls from TCGA miRNA microarray data (D), and GEO DataSets GSE61710 (E), GSE37366, and GSE41032 (F). (E and F) miR-513a-5p expression changes were analyzed by GEO2R software.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Microarray, Expressing, Real-time Polymerase Chain Reaction, Control, Software

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) Dose-dependent effects of IGF-1 on miR-513a-5p expression levels in U87-MG and M059K cells. (B) Effects of IGF-1 on primary (pri)-miR-513a-1 and pri-miR-513a-2 levels. The PI3K inhibitor, LY294002, but not the mitogen-activated protein kinase (MAPK) kinase inhibitor, U0126, attenuated IGF-1-stimulated miR-513a-5p (C) and pri-miR-513a-2 (D) levels. After cells were treated with the indicated doses of IGF-1 for 48 h or 200 ng/ml IGF-1 treatment for 48 h and pretreatment with 5 μM LY294002 and 10 μM U0126 for 1 h, endogenous miR-513a-5p, pri-miR-513a-1, and pri-miR-513a-2 levels were respectively measured by a real-time PCR. miR-191-5p and GAPDH levels were respectively used as internal controls for the mature and primary miR-513a-5p levels. Data are the mean ± SD of three independent experiments. * p <0.05.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) The inverse correlation between miR-513a and NEDD4L expression levels in TCGA microarray data. Using the median cutoff for miR-513a expression levels, patients were divided into two groups. A correlation was found in the group highly expressing miR-513a ( n = 259) by Pearson's correlation analysis. (B) Schematic diagram of potential miR-513a-5p-targeted sites in the NEDD4L 3’-untranslated region (UTR). (C and D) Effects of miR-513a-5p on NEDD4L 3’-UTR luciferase activity. To test for miR-513a-5p's effect, different doses of the miR-513a-5p plasmid were co-transfected with 500 ng of the pmiRGlo-NEDD4L 3’-UTR or mutant 3’-UTR. Luciferase activity was measured in these cells 24 h after transfection. Effects of miR-513a-5p overexpression on NEDD4L mRNA (E) and protein (F) expressions. After cells were transfected with the indicated dose of the miR-513a-5p plasmid for 24 h, relative mRNA and protein levels of NEDD4L were analyzed using a real-time PCR and an immunoblotting assay. (G) Dose-dependent effects of IGF-1 on reducing NEDD4L levels. (H) miR-513a-5p is involved in the IGF-1-reduced NEDD4L expression mechanism. After cells were treated with indicated doses of IGF-1 for 48 h or transfected with 1 μg of the miR-513a-5p plasmid combined with IGF-1 treatment for 48 h, the NEDD4L protein level was analyzed by an immunoblotting assay. Data are the mean ± SD of three experiments. * p <0.05. Inhibitory effects of miR-513a-5p on IGF-1-induced β-catenin nuclear accumulation (I) and -reduced NEDD4L levels (J). The protocol for nuclear protein extraction was showed in the method section. The β-catenin, lamni A/C, GAPDH, and NEDD4L protein levels were analyzed by immunoblotting assays.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Expressing, Microarray, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Mutagenesis, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Protein Extraction

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) Effects of the overexpression of miR-513a-5p on downstream protein expressions of NEDD4L-inhibited WNT/β-catenin signaling. (B) Overexpression of miR-513a-5p enhanced the IGF-1-activated WNT/β-catenin pathway. (C) Overexpression of NEDD4L repressed the IGF-1-activated WNT/β-catenin pathway. After U87-MG cells were transfected with the indicated doses of the miR-513a-5p plasmid for 24 h, 1 μg of the miR-513a-5p plasmid combined with 200 ng/ml IGF-1 treatment for 48 h, or 2 μg NEDD4L plasmids combined with 200 ng/ml IGF-1 treatment for 48 h, WNT/β-catenin pathway downstream protein expressions were measured by an immunoblotting assay. Effects of miR-513a-5p (D) and NEDD4L (E) on IGF-1-reduced glioma sensitivity to TMZ. After U87-MG cells were transfected with 1 μg of the miR-513a-5p plasmid or 2 μg of the NEDD4L plasmid combined with 200 ng/ml IGF-1 and 200 μM TMZ treatment for 48 h, cell viability was measured by an MTT assay. Data are the mean ± SD of three independent experiments. * p <0.05. (F) Knockdown effects of miR-513a-5p on IGF-1-insensitized TMZ cytotoxicity. Cell viability was measured by a cell counting assay. Data are the mean ± SD of three independent experiments. * p <0.05.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, MTT Assay, Knockdown, Cell Counting

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: Volcano plot (A) and heat map of hierarchical gene clustering (B) demonstrating IGF-1-regulated microRNA profiles. After U87 MG cells were treated with 200 ng/ml IGF-1 for 48 h, total RNAs were extracted for a microarray analysis. For all array analyses, an adjusted (adj.) p value of <0.05 and a |log2 (ratio)| of ≥0.58 (±1.5 multiples of change) cutoff were applied. Volcano plots show the multiples of change (log 2 ratio) and probability (-log 10 adj. p values) of individual microRNAs from the microarray assay. The red dot shows the miR-513a-5p location. The heat map depicts the 241 microRNAs differentially expressed between IGF-1 subsets. A color was assigned to each microRNA based on its relative expression level across samples. (C) Detection of endogenous miR-513a-5p levels in human normal astrocytes and three different glioma cell lines. Endogenous miR-513a-5p levels were measured by a real-time PCR. The miR-191-5p level was used as an internal control. Data are the mean ± SD of three independent experiments. * p <0.05. Increased miR-513a levels in GBM patients and glioma stem cells (GSCs) compared to normal controls from TCGA miRNA microarray data (D), and GEO DataSets GSE61710 (E), GSE37366, and GSE41032 (F). (E and F) miR-513a-5p expression changes were analyzed by GEO2R software.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Microarray, Expressing, Real-time Polymerase Chain Reaction, Control, Software

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) Dose-dependent effects of IGF-1 on miR-513a-5p expression levels in U87-MG and M059K cells. (B) Effects of IGF-1 on primary (pri)-miR-513a-1 and pri-miR-513a-2 levels. The PI3K inhibitor, LY294002, but not the mitogen-activated protein kinase (MAPK) kinase inhibitor, U0126, attenuated IGF-1-stimulated miR-513a-5p (C) and pri-miR-513a-2 (D) levels. After cells were treated with the indicated doses of IGF-1 for 48 h or 200 ng/ml IGF-1 treatment for 48 h and pretreatment with 5 μM LY294002 and 10 μM U0126 for 1 h, endogenous miR-513a-5p, pri-miR-513a-1, and pri-miR-513a-2 levels were respectively measured by a real-time PCR. miR-191-5p and GAPDH levels were respectively used as internal controls for the mature and primary miR-513a-5p levels. Data are the mean ± SD of three independent experiments. * p <0.05.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) The inverse correlation between miR-513a and NEDD4L expression levels in TCGA microarray data. Using the median cutoff for miR-513a expression levels, patients were divided into two groups. A correlation was found in the group highly expressing miR-513a ( n = 259) by Pearson's correlation analysis. (B) Schematic diagram of potential miR-513a-5p-targeted sites in the NEDD4L 3’-untranslated region (UTR). (C and D) Effects of miR-513a-5p on NEDD4L 3’-UTR luciferase activity. To test for miR-513a-5p's effect, different doses of the miR-513a-5p plasmid were co-transfected with 500 ng of the pmiRGlo-NEDD4L 3’-UTR or mutant 3’-UTR. Luciferase activity was measured in these cells 24 h after transfection. Effects of miR-513a-5p overexpression on NEDD4L mRNA (E) and protein (F) expressions. After cells were transfected with the indicated dose of the miR-513a-5p plasmid for 24 h, relative mRNA and protein levels of NEDD4L were analyzed using a real-time PCR and an immunoblotting assay. (G) Dose-dependent effects of IGF-1 on reducing NEDD4L levels. (H) miR-513a-5p is involved in the IGF-1-reduced NEDD4L expression mechanism. After cells were treated with indicated doses of IGF-1 for 48 h or transfected with 1 μg of the miR-513a-5p plasmid combined with IGF-1 treatment for 48 h, the NEDD4L protein level was analyzed by an immunoblotting assay. Data are the mean ± SD of three experiments. * p <0.05. Inhibitory effects of miR-513a-5p on IGF-1-induced β-catenin nuclear accumulation (I) and -reduced NEDD4L levels (J). The protocol for nuclear protein extraction was showed in the method section. The β-catenin, lamni A/C, GAPDH, and NEDD4L protein levels were analyzed by immunoblotting assays.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Expressing, Microarray, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Mutagenesis, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Protein Extraction

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) Effects of the overexpression of miR-513a-5p on downstream protein expressions of NEDD4L-inhibited WNT/β-catenin signaling. (B) Overexpression of miR-513a-5p enhanced the IGF-1-activated WNT/β-catenin pathway. (C) Overexpression of NEDD4L repressed the IGF-1-activated WNT/β-catenin pathway. After U87-MG cells were transfected with the indicated doses of the miR-513a-5p plasmid for 24 h, 1 μg of the miR-513a-5p plasmid combined with 200 ng/ml IGF-1 treatment for 48 h, or 2 μg NEDD4L plasmids combined with 200 ng/ml IGF-1 treatment for 48 h, WNT/β-catenin pathway downstream protein expressions were measured by an immunoblotting assay. Effects of miR-513a-5p (D) and NEDD4L (E) on IGF-1-reduced glioma sensitivity to TMZ. After U87-MG cells were transfected with 1 μg of the miR-513a-5p plasmid or 2 μg of the NEDD4L plasmid combined with 200 ng/ml IGF-1 and 200 μM TMZ treatment for 48 h, cell viability was measured by an MTT assay. Data are the mean ± SD of three independent experiments. * p <0.05. (F) Knockdown effects of miR-513a-5p on IGF-1-insensitized TMZ cytotoxicity. Cell viability was measured by a cell counting assay. Data are the mean ± SD of three independent experiments. * p <0.05.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, MTT Assay, Knockdown, Cell Counting

Journal: PLOS ONE

Article Title: A systematic review of non-coding RNA genes with differential expression profiles associated with autism spectrum disorders

doi: 10.1371/journal.pone.0287131

Figure Lengend Snippet: Studies examining ncRNA gene expression in ASD using tissue samples and laboratory methodologies that could be feasibly implemented into clinical practice (N = 33).

Article Snippet: 13:2 , 4.3 , Not stated: Sex and age matched , , 5 miRNA genes examined by qPCR on CFX96 BioRad , miR-16-5p.

Techniques: Gene Expression, Biomarker Discovery, Control, RNA Extraction, Gene Assay, Microarray, TLDA Assay, Sample Prep, Standard Deviation, SYBR Green Assay, Sequencing, Expressing, Isolation, Reverse Transcription, Clinical Proteomics, TaqMan microRNA Assay, Multiplex Assay, Construct, Fluorescence

Journal: Obesity Medicine

Article Title: Relationship between microRNA expression profiling and gestational diabetes: A systematic review

doi: 10.1016/j.obmed.2023.100487

Figure Lengend Snippet: Fig. 2. HeatMap and cluster patterns from gene expression pathways affected by miRNAs. A: Target of upregulated miRNAs in placental tissue, placenta-derived cells (Mononuclear cells), peripheral blood, plasma, plasma Evs, and serum B: Target of downregulated miRNAs in placental tissue and placenta-derived exosomes.

Article Snippet: RT-qPCR U6 n/a n/a HUANG et al., 2021* Trizol n/a RT-qPCR GAPDH n/a n/a Li 2015ɨ** RecoverAllTM Total Nucleic Acid Isolation kit (Ambion) y TRNzol reagent (TIANGEN Biotech, Beijing, China) miRNA first-strand cDNA kit (TIANGEN Biotech, Beijing, China) miRcute miRNA qPCR detection kit (TIANGEN Biotech, Beijing, China) U6 Agilent Human miRNA Microarray (Agilent) n/a Li et al., 2018** KingFisher Automated Purification Kits (Thermo Fisher) miRCURY LNATM microRNA Array Hi-Power Labeling kit (Qiagen) n/a Hairpin-itTMmiRNAs RealTime PCR Quantization Kit (GenePharma) n/a n/a n/a Li W 2022* TRIzol reagent (Magen Biotechnology Co.) TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa) miScript SYBR Green PCR Kit (TaKaRa) U6 n/a n/a Peng et al., 2019* Trizol (Ambion, USA) miRcute miRNA FirstStrand cDNA Synthesis Kit (TianGen, Beijing, China) miRcute miRNA qPCR Detection Kit (SYBR Green) (TianGen, Beijing China) U6 n/a n/a Sun et al., 2020* Trizol reagent (Invitrogen, Carlsbad, CA, USA) TaqMan MicroRNA Reverse Transcription Kit TaqMan Univel PCR Master Mix (Applied Biosystems, Foster City, CA, USA) U6 n/a n/a Tang 2020ᵻ* Trizol Reagent (Invitrogen, Carlsbad, CA, USA) M-MLV Reverse Transcriptase (Invitrogen) qRT–PCR (BIONEER, Daejeon, Korea) U6 n/a NEBNext Multiplex Small RNA Library Prep Set for Illumina_x0003_ (NEB, Ipswich, MA, USA) Wang S 2020* Trizol reagent (Invitrogen) Transcriptase Kit (Takara, Otsu, Japan) SYBR Green PCR Master Mix (Takara) U6 n/a n/a Zhang Lu 2021* Trizol n/a RT-qPCR U6 n/a n/a Zhao et al., 2014* Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) TaqMan MicroRNA Reverse Transcription kit TaqMan microRNA assay (Applied Biosystems, Branchburg, NJ, USA) U6 n/a n/a Serum Abdeltawab et al., 2020** miRVana PARIS kits (Life Technologies, Grand Island, NY, USA) TaqMan MicroRNA Reverse Transcription Kit TaqMan quantitative RTPCR.

Techniques: Gene Expression, Derivative Assay, Clinical Proteomics

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: Expression of miR‐107, miR‐146b‐5p, miR‐346 and miR‐1287‐5p in astrocytic gliomas of different WHO grades. Relative miRNA expression levels were determined by real‐time RT‐PCR using let‐7a as reference. Box plots are depicted indicating normalized expression values according to Tukey's method. Asterisks indicate significant expression differences compared with non‐neoplastic brain tissue samples (*P < 0.05; **P < 0.01, ***P < 0.001; Kruskal–Wallis test and Dunn's post‐hoc test). AII, diffuse astrocytoma, WHO grade II; AAIII, anaplastic astrocytoma, WHO grade III; sGBIV, secondary glioblastoma, WHO grade IV; pGBIV, primary glioblastoma, WHO grade IV; NB, non‐neoplastic brain tissue; n, number of investigated tissue samples.

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: Expressing, Quantitative RT-PCR

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: miR‐146b‐5p expression in 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) treated glioma cell lines (A) as well as methylation of CpG sites located in putative SP1 transcription factor‐binding sites associated with the miR‐146b‐5p locus (B). (A) Relative expression levels of miR‐146b‐5p were determined by real‐time RT‐PCR using let‐7a as reference gene. Expression of the miRNA in the 5‐AZA‐dCR/TSA samples was calculated relative to miRNA expression in the controls (Co). A + T1: cell lines were grown in medium supplemented with 500 nM 5‐AZA‐dCR for 48 h, washed and grown for another 24 h in medium with 500 nM 5‐AZA‐dcr and 1 µM TSA; A + T2: like A + T1, except for a higher 5‐AZA‐dCR concentration (1 µM). Asterisks indicate significant difference when compared with the respective non‐treated controls (*P < 0.05; **P < 0.01; paired t‐test). Bars represent the mean, and error bars represent the standard deviation of three independent experiments, except for U138MG A + T2 with two independent experiments. (B) Methylation analysis of the seven CpG sites located from nt 104,195,358 to nt 104,195,483 on chr10 (UCSC genome browser, GRCh37/hg19 assembly) was performed by pyrosequencing of two PCR products generated from sodium bisulfite‐modified DNA. The three CpG sites located within each of the three putative SP1‐binding sequences are labeled by hachures. The methylation status at each CpG site was gray scale‐coded as follows: white square, 0–25% methylated alleles; light gray square, 26–50% methylated alleles; gray square, 51–75% methylated alleles; black square, 76–100% methylated alleles. methCo, methylation positive control (CpGenome™ Universal Methylated DNA, Merck Millipore, Darmstadt, Germany); AII, diffuse astrocytoma WHO grade II; AAIII, anaplastic astrocytoma WHO grade III; sGBIV, secondary glioblastoma WHO grade IV; pGBIV, primary glioblastoma WHO grade IV; NB, non‐neoplastic brain tissue; Meth% (mean), mean of the percentages of methylated allele derived from pyrosequencing at the seven investigated CpG sites. IDH mut: gray square, IDH1 or IDH2 mutation detected; white square, no IDH1 or IDH2 mutation detected. TERT mut: gray square, TERT promoter mutation detected; white square, no TERT promoter mutation detected. Note a significant difference in the mean percentage of methylated alleles between primary glioblastoma (mean: 5% methylated alleles) and secondary glioblastoma (mean: 30% methylated alleles; P < 0.01; Student's t‐test, two‐sided).

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: Expressing, Methylation, Binding Assay, Quantitative RT-PCR, Gene Expression, Concentration Assay, Standard Deviation, Generated, Modification, Labeling, Positive Control, Derivative Assay, Mutagenesis

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: In vitro effects of miR‐107, miR‐146b‐5p, miR‐346 or miR‐1287‐5p overexpression on apoptotic activity (A) and proliferation (B) of glioma cells. A172 and T98G cells were transfected with either 10 nM, 25 nM or 50 nM pre‐miR™ precursor molecules (pre‐107, pre‐146‐5p, pre‐346, pre‐1287‐5p) or negative control pre‐miR™ miRNA precursors (pre‐NC1; Life Technologies). Apoptotic activity was determined by a luminescence‐based caspase‐3/7 assay. Proliferation was measured using a luminescence‐based BrdU incorporation assay. Dots represent the mean of five replicates. P‐values were calculated using paired t‐tests. Mean fold‐expression changes (FC) are also provided.

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: In Vitro, Over Expression, Activity Assay, Transfection, Negative Control, BrdU Incorporation Assay, Expressing

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: Validation of predicted miR‐107, miR‐146b‐5p, miR‐346 and miR‐1287‐5p target genes. (A) Results of mRNA analyses. Twenty‐four hours after seeding, cells were transfected with 25 nm pre‐miR™ precursor molecules (pre‐107, pre‐146b‐5p, pre‐346, pre‐1287‐5p; Life Technologies) or negative control pre‐miR™ miRNA precursors (pre‐NC1, Life Technologies). Total RNA was extracted 48 h after transfection and mRNA levels of the putative targets were determined by RT‐PCR using ARF1 (CDK6, EGFR), B2M (SEMA6A) or ACTG1 (TERT) as reference genes and universal human reference RNA (Stratagene) as calibrator. Data represent the mean of three independent transfection experiments. Error bars show standard deviations. Asterisks indicate significant difference as compared with the respective pre‐NC1 controls (paired t‐test, one‐sided). (B–C) Results of Western blot analyses. Cells were transfected with 50 nM pre‐miR™ precursor molecules (pre‐107, pre‐146b‐5p, pre‐1287‐5p) or negative control pre‐miR™ miRNA precursors (pre‐NC1). The expression levels of CDK6 (B), EGFR (C), α‐tubulin—as a loading control—were determined 72 h post‐transfection. Approximate sizes of the protein bands in kilo‐dalton (kDa) are indicated on the right. Below each exemplary Western blot, the results of protein quantification from three independent experiments are depicted. P‐values were calculated by a two‐sided paired t‐test. (D) Results of 3′‐UTR luciferase gene reporter assays. Relative luciferase activity in T98G cells were determined 48 h post‐transfection with 100 nm pre‐miR™ precursor molecules (pre‐107, pre‐146b‐5p, pre‐346, pre‐1287‐5p; Life Technologies) or negative control pre‐miR™ miRNA precursors (pre‐NC1, Life Technologies) in combination with either wild‐type (‐3′WT) or deleted/mutated (‐3'del, ‐3'mut) psiCHECK2 constructs. Renilla luciferase activity was normalized to F irefly luciferase activity. Vertical bars represent the mean of three independent experiments with each performed in quintuplicate. Asterisks indicate significant differences compared with pre‐NC1 transfected cells (paired t‐test, two‐sided).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: Biomarker Discovery, Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Luciferase, Activity Assay, Construct

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: mRNA expression levels of the 10q‐miRNA targets CDK6, EGFR, SEMA6A, and TERT in astrocytic glioma. (A) The data for CDK6, EGFR, and SEMA6A expression were obtained from a published microarray dataset 42. Box plots are depicted indicating normalized expression values according to Tukey's method; n, number of tissue samples investigated. The mRNA expression levels of TERT were determined by real‐time RT‐PCR using ARF1 as reference. Asterisks indicate significant expression differences compared with non‐neoplastic brain tissue samples (*P < 0.05; **P < 0.01; Kruskal–Wallis test and Dunn's post‐hoc test). AII, diffuse astrocytoma WHO grade II; AAIII, anaplastic astrocytoma WHO grade III; sGBIV, secondary glioblastoma WHO grade IV; pGBIV, primary glioblastoma WHO grade IV; NB, non‐neoplastic brain tissue. (B) MiR‐346 expression in TERT‐mutant and wild‐type gliomas. MiR‐346 expression is significantly different between TERT promoter‐mutant compared with TERT promoter wild‐type glioma samples (Mann–Whitney test, ****P < 0.0001). (C) The two cases (GB113 and A151) with highest TERT expression in the absence of a detectable TERT promoter mutation showed evidence for a low‐level copy number gain of TERT (A151) or TERT amplification (GB113). APRT was used as a reference gene in the duplex PCR analysis. NB, non‐neoplastic brain tissue; NTC, no template control; bp, base pairs.

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Mutagenesis, MANN-WHITNEY, Amplification, Control

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: Relationship between miRNA expression and IDH mutation status in our institutional glioma series (A) and the TCGA glioblastoma cohort (B). (A) MiR‐107 (P < 0.0001) and miR‐346 (P < 0.001) expression is increased, whereas the expression of miR‐146b‐5p (P < 0.01) and miR‐1287‐5p (P < 0.05) is decreased in astrocytic gliomas with IDH1/IDH2 mutation (IDH mut) compared with IDH1/IDH2 wild‐type (IDH wt) tumors (Mann–Whitney test). Box plots are depicted indicating normalized expression values according to Tukey's method; n, number of tissue samples investigated. (B) MiR‐146b‐5p expression is decreased in IDH1‐mutant (IDH1 mut) as compared with IDH1 wild‐type (IDH1 wt) glioblastomas of the TCGA cohort. MiRNA expression z‐scores and IDH1 mutation data were obtained from 158 TCGA glioblastoma cases via the cBio Cancer Genomics portal. P‐values were calculated using a two‐sided Student's t‐test and corrected for multiple testing (Bonferroni‐correction for 17 comparisons). IDH1 ‐mutant glioblastomas carry missense mutations affecting arginine 132; IDH1 wild‐type glioblastomas carry the regular arginine at codon 132; n, number of investigated cases.

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: Expressing, Mutagenesis, MANN-WHITNEY

Journal: Cell Reports

Article Title: Endogenous Control of Immunity against Infection: Tenascin-C Regulates TLR4-Mediated Inflammation via MicroRNA-155

doi: 10.1016/j.celrep.2012.09.005

Figure Lengend Snippet: Tenascin-C Is Required for Induction of miR-155 in Activated BMDMs and in Septic Mice (A) Microarray analysis of miRNA profile in tnc +/+ and tnc − / − BMDMs stimulated for 8 hr with 100 ng/ml LPS. The heatmap shows suppression (green) and induction (red) of expression as fold change on a log 2 scale, relative to nonstimulated BMDMs (n = 3 mice per genotype). (B and C) Expression of TLR-induced miRNAs in tnc +/+ and tnc − / − BMDMs (identified in A) classified as early (B) and late (C) response genes (n = 3 per genotype; mean ± SEM). ∗∗ p < 0.01; ns, not significant. (D) Time course analysis of miR-155, pri-miR-155, miR-146a, and miR-21 expression in tnc +/+ and tnc − / − BMDMs stimulated for 4–72 hr (miR-155), 0.5–48 hr (pri-miR-155), or 4–96 hr (miR-146a, miR-21) with 100 ng/ml LPS. RNA used in (A) (n = 3 mice per group) and RNA isolated from BMDMs derived from two (miR-146a, miR-21) and four (miR-155, pri-miR-155) additional mice per genotype was analyzed by quantitative RT-PCR (mean ± SEM). ∗∗ p < 0.01 compared to tnc − / − BMDMs at the same time point. (E) Correlation of miR-155 expression with TNF-α synthesis in tnc +/+ and tnc − / − BMDMs nonstimulated or stimulated with 100 ng/ml LPS for 4, 8, and 24 hr (n = 5 mice per genotype; R = 1.0; p < 0.05). (F) Quantitative RT-PCR analysis of miR-155 expression in spleen from tnc +/+ and tnc − / − mice 1.5 hr after LPS injection (n = 3 per genotype; mean ± SEM). ∗ p < 0.05. Data are from two independent experiments.

Article Snippet: For pri-miRNA detection, RNA was reverse transcribed with AffinityScript reverse transcriptase and random primers (Stratagene), followed by real-time PCR with TaqMan primer sets for mouse pri-miR155 (Mm03306395_pri) and HPRT1 (Applied Biosystems).

Techniques: Microarray, Expressing, Isolation, Derivative Assay, Quantitative RT-PCR, Injection

Journal: Cell Reports

Article Title: Endogenous Control of Immunity against Infection: Tenascin-C Regulates TLR4-Mediated Inflammation via MicroRNA-155

doi: 10.1016/j.celrep.2012.09.005

Figure Lengend Snippet: miR-155 Overexpression Rescues TNF-α Release in Tnc − / − BMDMs in Response to LPS (A) Quantitative RT-PCR analysis of miR-155 expression in tnc +/+ untransfected BMDMs and tnc − / − untransfected, mock transfected, and transfected with control precursor or miR-155 precursor BMDMs stimulated for 8 hr with 100 ng/ml LPS. Results are presented relative to those of nonstimulated BMDMs (n = 3; mean ± SEM). ∗ p < 0.05; ns, not significant. Data are representative of four independent experiments each with cells obtained from three mice per genotype. (B) ELISA of TNF-α secreted by tnc +/+ untransfected BMDMs and tnc − / − untransfected, mock transfected, and transfected with control precursor or miR-155 precursor BMDMs stimulated for 8 hr with 100 ng/ml LPS (n = 6; mean ± SEM). ∗∗∗ p < 0.001, ∗∗ p < 0.01; ns, not significant. Data are from two independent experiments each with cells obtained from three mice per genotype. See also Figure S4 .

Article Snippet: For pri-miRNA detection, RNA was reverse transcribed with AffinityScript reverse transcriptase and random primers (Stratagene), followed by real-time PCR with TaqMan primer sets for mouse pri-miR155 (Mm03306395_pri) and HPRT1 (Applied Biosystems).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports

Article Title: Endogenous Control of Immunity against Infection: Tenascin-C Regulates TLR4-Mediated Inflammation via MicroRNA-155

doi: 10.1016/j.celrep.2012.09.005

Figure Lengend Snippet: TNF-α Production in Tnc − / − BMDMs Is Rescued by miR-155 Overexpression but Not Soluble Tenascin-C, Related to Figure 7 (A) Quantitative RT-PCR analysis of miR-155 expression in tnc +/+ untransfected BMDMs and tnc −/− untransfected, mock transfected and transfected with control precursor or miR-155 precursor BMDMs, all in the absence of any stimulation with LPS (n = 3; mean ± SEM). Data are representative of four independent experiments each with cells obtained from three mice per genotype. (B) ELISA of TNF-α secreted by tnc +/+ untransfected BMDMs and tnc −/− untransfected, mock transfected and transfected with control precursor or miR-155 precursor BMDMs, all in the absence of any stimulation with LPS (n = 6; mean ± SEM). Data are from two independent experiments each with cells obtained from three mice per genotype. (C) ELISA of TNF-α and IL-6 secreted by tnc +/+ and tnc −/− BMDMs stimulated for 24 hr with 100ng/ml LPS without (-) or with recombinant purified tenascin-C (TN-C) (n = 3; mean ± SEM). ∗∗∗ = p < 0.001 compared to tnc +/+ BMDMs stimulated with LPS. Data are representative of three independent experiments each with cells obtained from three mice per genotype. The same results were obtained when we added equivalent amounts of commercially purified human tenascin-C.

Article Snippet: For pri-miRNA detection, RNA was reverse transcribed with AffinityScript reverse transcriptase and random primers (Stratagene), followed by real-time PCR with TaqMan primer sets for mouse pri-miR155 (Mm03306395_pri) and HPRT1 (Applied Biosystems).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay, Recombinant, Purification

Journal: PLoS ONE

Article Title: Altered Expression of Circulating MicroRNA in Plasma of Patients with Primary Osteoarthritis and In Silico Analysis of Their Pathways

doi: 10.1371/journal.pone.0097690

Figure Lengend Snippet: (A) Microarray analysis for miRNA levels was performed using RNA extracts from plasma of healthy subjects as control and patients with OA of different OA disease stages; (B) Unsupervised hierarchical clustering of the differentially expressed miRNA. Bright green: down-regulation; black, no change; bright red: up-regulation.

Article Snippet: Total RNA concentration was determined by spectrophotometry with the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) and the RNA samples were stored at –80°C until further processing. miRNA complementary DNA (cDNA) was synthesized with the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and Megaplex RT primers (Human Pool A, Applied Biosystems) following the manufacturer’s protocol.

Techniques: Microarray, Clinical Proteomics, Control

Journal: PLoS ONE

Article Title: Altered Expression of Circulating MicroRNA in Plasma of Patients with Primary Osteoarthritis and In Silico Analysis of Their Pathways

doi: 10.1371/journal.pone.0097690

Figure Lengend Snippet: (A) Tabulation of the number of transcripts targeted of 10 miRNA; (B) Tabulation of the number of predicted targets for 150 genes involved in OA; (C) Predicted targets that have been confirmed previously in other entities and OA; (D) Percentage of predicted target genes involved in OA.

Article Snippet: Total RNA concentration was determined by spectrophotometry with the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) and the RNA samples were stored at –80°C until further processing. miRNA complementary DNA (cDNA) was synthesized with the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and Megaplex RT primers (Human Pool A, Applied Biosystems) following the manufacturer’s protocol.

Techniques:

Journal: PLoS ONE

Article Title: Altered Expression of Circulating MicroRNA in Plasma of Patients with Primary Osteoarthritis and In Silico Analysis of Their Pathways

doi: 10.1371/journal.pone.0097690

Figure Lengend Snippet: Enriched biological processes among the 12 up-regulated miRNA. Red shows the e-value as more significant, while less significance is denoted in blue.

Article Snippet: Total RNA concentration was determined by spectrophotometry with the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) and the RNA samples were stored at –80°C until further processing. miRNA complementary DNA (cDNA) was synthesized with the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and Megaplex RT primers (Human Pool A, Applied Biosystems) following the manufacturer’s protocol.

Techniques:

Journal: Molecular Therapy

Article Title: Sustained miRNA-mediated Knockdown of Mutant AAT With Simultaneous Augmentation of Wild-type AAT Has Minimal Effect on Global Liver miRNA Profiles

doi: 10.1038/mt.2011.292

Figure Lengend Snippet: MicroRNA mediated knockdown of human AAT. HEK-293 cells were contrasfected with human Z-AAT plasmid and either a plasmid expressing three anti-AAT shRNAs from a U6 promoter or a plasmid expressing three anti-AAT miRNA from a hybrid chicken β-actin promoter. ( a ) Culture media was harvested at 24, 48, and 72 hours and was analyzed for the AAT concentration by ELISA. ( b ) At 72 hours cells were harvested and lysed for AAT concentration by ELISA. * ≤0.05 as determined by a two-way unpaired Student's t -test. ( c ) Transgenic mice expressing the human PiZ allele were injected with 5 × 10 11 virus particles of a rAAV9 control GFP vector or rAAV9 expressing miRNAs against AAT under the control of the hybrid chicken β-actin promoter via the tail vein. Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. Serum Z-AAT levels at each timepoint are expressed as a percent knockdown as compared to the rAAV9-GFP cohort. Data are expressed as group means ± SEM ( n = 6). Statistical significance was set at * ≤0.05 as determined by a two-way ANOVA comparing each treatment group to the control rAAV-GFP group. AAT, α-1 antitrypsin; ANOVA, analysis of variance; CMV, cytomegalovirus; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; miRNA, microRNA; rAAV, recombinant adeno-associated virus; shRNA, short hairpin RNA.

Article Snippet: Real-time RT-PCR RNA extraction : Flash frozen mouse liver tissue was ground in a pestle and mortar and used to extract either small or total RNA using the mirVana miRNA RNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions. microRNA qRT-PCR : mircoRNA was primed and reverse-transcribed with TagMan MicroRNA reverse transcription Kit (Applied Biosystems, Foster City, CA).

Techniques: Plasmid Preparation, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Injection, Recombinant, shRNA

Journal: Molecular Therapy

Article Title: Sustained miRNA-mediated Knockdown of Mutant AAT With Simultaneous Augmentation of Wild-type AAT Has Minimal Effect on Global Liver miRNA Profiles

doi: 10.1038/mt.2011.292

Figure Lengend Snippet: In vivo optimization of anti-AAT miRNA delivery within rAAV9 vectors . ( a ) Transgenic mice expressing the human PiZ allele were injected with 5 × 10 11 virus particles or rAAV9 expressing miRNAs against AAT under the control of the hybrid chicken β-actin promoter via the tail vein. Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. ( b ) Quantitative RT-PCR for artificial miRNA was quantified from total RNA obtained from mouse livers. RT-PCR was used to assay for the presence of the three artificial anti-AAT miRNAs from mice receiving rAAV9-miRNA vectors. * ≤0.05 as determined by a two-way unpaired Student's t -test. AAT, α-1 antitrypsin; CMV, cytomegalovirus; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; miRNA, microRNA; rAAV, recombinant adeno-associated virus; RT-PCR, reverse transcriptase-PCR.

Article Snippet: Real-time RT-PCR RNA extraction : Flash frozen mouse liver tissue was ground in a pestle and mortar and used to extract either small or total RNA using the mirVana miRNA RNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions. microRNA qRT-PCR : mircoRNA was primed and reverse-transcribed with TagMan MicroRNA reverse transcription Kit (Applied Biosystems, Foster City, CA).

Techniques: In Vivo, Transgenic Assay, Expressing, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Recombinant

Journal: Molecular Therapy

Article Title: Sustained miRNA-mediated Knockdown of Mutant AAT With Simultaneous Augmentation of Wild-type AAT Has Minimal Effect on Global Liver miRNA Profiles

doi: 10.1038/mt.2011.292

Figure Lengend Snippet: In vivo knockdown of Z-AAT with simultaneous augmentation of M-AAT after rAAV9 dual-function vector delivery . Transgenic mice expressing the human PiZ allele were injected with 1 × 10 12 virus particles or rAAV9 expressing miRNAs against AAT and a de-targeted cMyc tagged wild-type M-AAT cDNA under the control of the hybrid chicken β-actin promoter via the tail vein. ( a ) Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. Serum Z-AAT levels at each timepoint are expressed as a percent knockdown as compared to the rAAV9-GFP cohort by using a Z-specific AAT ELISA and M-AAT levels are calculated by using an ELISA to quantify the cMYC tag on the wild-type protein. Data are expressed as group means ±SEM ( n = 6). Statistical significance was set at * ≤0.05 as determined by a two-way ANOVA comparing each treatment group to the control rAAV-GFP group. Blue dashed line in the upper panel indicates therapeutic levels of wild-type PiM AAT as determined by the FDA and therapeutic knockdown of PiZ protein in the lower panel as determined by achieving levels expected in a PiZ heterozygous status. Total RNA from mouse livers was used to assay for the presence of the either ( b ) Z-AAT mRNA or ( c ) M-AAT mRNA by qRT-PCR. Data are expressed as group means ±SEM ( n = 6). * ≤0.05 as determined by a two-way unpaired Student's t -test. AAT, α-1 antitrypsin; ANOVA, analysis of variance; cDNA, complementary DNA; ELISA, enzyme-linked immunosorbent assay; CB-GFP, chicken β-actin–green fluorescent protein; FDA, Food and Drug Administration; mRNA, messenger RNA; miRNA, microRNA; ND, not detected; qRT-PCR, quantitative reverse transcriptase-PCR; rAAV, recombinant adeno-associated virus.

Article Snippet: Real-time RT-PCR RNA extraction : Flash frozen mouse liver tissue was ground in a pestle and mortar and used to extract either small or total RNA using the mirVana miRNA RNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions. microRNA qRT-PCR : mircoRNA was primed and reverse-transcribed with TagMan MicroRNA reverse transcription Kit (Applied Biosystems, Foster City, CA).

Techniques: In Vivo, Plasmid Preparation, Transgenic Assay, Expressing, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Recombinant

Journal: Molecular Therapy

Article Title: Sustained miRNA-mediated Knockdown of Mutant AAT With Simultaneous Augmentation of Wild-type AAT Has Minimal Effect on Global Liver miRNA Profiles

doi: 10.1038/mt.2011.292

Figure Lengend Snippet: Artificial miRNA have minimal impact on endogenous miRNA liver profiles . Liver RNA was harvested 3 months post delivery from animals injected with the following vectors: intron CB-3xmiR-GFP, PolyA -3XmiR-GFP, Double -6XmiR-GFP, CB-GFP along with RNA from untreated PiZ mice and wild-type C57Bl6 mice was used to run a miRNA microarray. Each group consisted of five mouse RNA samples and was run independently with a single color (Cy5) microarray. CB, chicken β-actin; GFP, green fluorescent protein; miRNA, microRNA.

Article Snippet: Real-time RT-PCR RNA extraction : Flash frozen mouse liver tissue was ground in a pestle and mortar and used to extract either small or total RNA using the mirVana miRNA RNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions. microRNA qRT-PCR : mircoRNA was primed and reverse-transcribed with TagMan MicroRNA reverse transcription Kit (Applied Biosystems, Foster City, CA).

Techniques: Injection, Microarray

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

doi: 10.1016/j.trsl.2018.06.005

Figure Lengend Snippet: Viability assays and reactive oxygen species detection after CDDP treatment in H23 cell lines. (A) Viability to CDDP in H23S (left) and H23R (right) at 24, 48, and 72 hours and 6 concentrations of CDDP measured by MTT assay; (B) Increment of ROS production in H23S (left) and H23R (right) after 24, 48, and 72 hours of CDDP treatment; For (A) and (B), bars represent the mean of at least two independent experiments measured by duplicate ± SD. $: P ≤ 0.05 versus basal; $$: P ≤ 0.01 versus basal; $$$: P ≤ 0.001 were considered as significant change in CDDP resistance and ROS production; (C) Semiquantitative mRNA expression analysis of downstream genes involved in ROS detoxification regulated by MAFG, which showed expression changes for H23 in the microarray data (GSE84201, ) (left panel). Representative images of MAFG and HMOX1 RT-PCR comparing sensitive and resistant subtypes. Each assay was performed at least three times to confirm the results. (Right panel) Relative intensity quantification of the amplified band for each gene measured by ImageJ Software. Bars represent the mean of the three independent experiments using the intensity of GAPDH as endogenous control and the sensitive subtype of each cell line as calibrator. (D) Immunoblots of HMOX1, NRF2 , and -actin. Images correspond to a representative experiment that was repeated four times with similar results. Scion Image program was used to quantify band intensities corresponding to immunoblot detection of protein samples. CDDP, cisplatin; MAFG, musculoaponeurotic fibrosarcoma oncogene family, protein G.

Article Snippet: Primers and probes for expression analysis were purchased from Applied Biosystems ( MAFG :Hs 01034678_g1; GAPDH : Hs03929097_g1; miRNA-7: 000268; and RNU48: 001006).

Techniques: MTT Assay, Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Amplification, Software, Control, Western Blot

Journal: Translational research : the journal of laboratory and clinical medicine

Article Title: MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

doi: 10.1016/j.trsl.2018.06.005

Figure Lengend Snippet: Effect of aptamer apMAFG6F on response to CDDP and ROS production. (A) Viability and ROS production assay in H23R cells after apMAFG6F transfection. Each experimental group was exposed for 72 h to 6 different CDDP concentrations, and data were normalized to each untreated control, set to 100%. The data represent the mean ± SD of at least four independent experiments performed in duplicate at each drug concentration for each cell line analyzed. * P < 0.05 was considered as significant change in CDDP resistance and ROS production; (B) Expression analysis of downstream genes regulated by MAFG involved in ROS detoxification in H23R and H23R transfected with apMAFG6F. Left, representative images of MAFG , HMOX1 , and GAPDH RT-PCR. Each assay was performed at least three times to confirm the results. Right, relative intensity quantification of the amplified band for each gene measured by ImageJ Software. Bars represent the mean of three independent experiments using the intensity of GAPDH as endogenous control and the resistant subtype of each cell line as calibrator ± SD. CDDP, cisplatin; ROS, reactive oxygen species; MAFG, musculoaponeurotic fibrosarcoma oncogene family, protein G.

Article Snippet: Primers and probes for expression analysis were purchased from Applied Biosystems ( MAFG :Hs 01034678_g1; GAPDH : Hs03929097_g1; miRNA-7: 000268; and RNU48: 001006).

Techniques: Transfection, Control, Concentration Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Software