Journal: International Journal of Molecular Sciences

Article Title: Breast Cancer-Derived Extracellular Vesicle miR-425-5p (miR-425) Promotes Brain Metastasis via Activating Astrocytes Through the Novel miR-425-ZNF24-CCL8 Signaling Axis

doi: 10.3390/ijms27073197

Figure Lengend Snippet: miR-425 suppresses transcription factor ZNF24 in astrocytes. ( A ) Predicted miR-425 binding sites within the CREB1, BCOR, and ZNF24 3′-UTRs from TargetScan. ( B ) ZNF24 mRNA expression is significantly decreased in astrocytes overexpressing miR-425. CREB1 and BCOR mRNA expression is unchanged as measured by RT-qPCR. ( C ) ZNF24 protein expression is decreased in astrocytes transfected with miR-425 mimic as indicated by Western blot analysis. ( D ) miR-425 suppresses ZNF24 3′-UTR activity as measured by dual luciferase reporter assay. ( E ) Ectopic expression of ZNF24 significantly decreases CCL8 and VEGFA mRNA expression, while KITLG is unchanged. mRNA expression measured by RT-qPCR. ( F ) Ectopic expression of ZNF24 suppresses CCL8 protein expression, but not SCF as indicated by Western blot analysis. ( G ) ZNF24 knockdown significantly increases CCL8 and VEGFA mRNA expression, but not KITLG. mRNA expression as indicated by RT-qPCR. ( H ) ZNF24 knockdown increases CCL8 and SCF protein expression as measured by Western blot analysis. Fold change was calculated in panels ( B , D , E , G ). Student’s t -test was used in panels ( B , D , E , G ). N = 3 experimental replicates unless otherwise indicated.

Article Snippet: Astrocytes were transfected with control or miR-425 mimic, control vector or ZNF24 3′-UTR (GeneCopoeia; Cs-HmiT119980-MT05-01), control vector or ZNF24 overexpression (GeneCopoeia; cat. #B221017-1240ueu), and control promoter or CCL8 promoter (GeneCopoeia; cat. #HPRM46246-PG02) constructs using X-tremeGENE HP DNA Transfection Reagent (Millipore Sigma; cat. #6366244001).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Activity Assay, Luciferase, Reporter Assay, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Breast Cancer-Derived Extracellular Vesicle miR-425-5p (miR-425) Promotes Brain Metastasis via Activating Astrocytes Through the Novel miR-425-ZNF24-CCL8 Signaling Axis

doi: 10.3390/ijms27073197

Figure Lengend Snippet: ZNF24 suppresses astrocyte activation and decreases CCL8 expression. ( A ) Human astrocytes activated by a cocktail of IL-1α, TNFα, and C1q. Astrocytes were serum-starved overnight before stimulation. Nuclei were stained with DAPI (blue), and GFAP was visualized in red. Representative 20× images. Scale bar indicates 100 µm. ( B ) Validation of GFAP mRNA expression in activated astrocytes as indicated by RT-qPCR. ( C ) Activated astrocytes transfected with ZNF24 plasmid have significantly reduced GFAP expression as indicated by GFAP IF. All slides were imaged at 4× or 20× using the Keyence BZ-X810 microscopes (cat. # BZ-X810 Keyence). At least 300 cells per slide were evaluated to identify GFAP+ cells. Representative 20× images. Scale bar indicates 100 µm. ( D ) Validation of GFAP reduction and ZNF24 overexpression with ectopic expression of ZNF24. mRNA expression measured by RT-qPCR. ( E ) ZNF24 binding to the CCL8 promoter in two regions (-69 and -205 bases upstream of the TSS) measured by ChIP-qPCR. ( F ) Ectopic expression of ZNF24 suppresses CCL8 promoter activity as indicated by dual luciferase reporter assays. ( G , H ) ZNF24 and CCL8 mRNA expression remains unchanged in breast cancer cells transfected with control or miR-425 mimic. mRNA expression measured by RT-qPCR. Fold change was calculated in panels ( B , D – H ). Student’s t -test was used in panels ( A – H ). N = 3 experimental replicates unless otherwise indicated.

Article Snippet: Astrocytes were transfected with control or miR-425 mimic, control vector or ZNF24 3′-UTR (GeneCopoeia; Cs-HmiT119980-MT05-01), control vector or ZNF24 overexpression (GeneCopoeia; cat. #B221017-1240ueu), and control promoter or CCL8 promoter (GeneCopoeia; cat. #HPRM46246-PG02) constructs using X-tremeGENE HP DNA Transfection Reagent (Millipore Sigma; cat. #6366244001).

Techniques: Activation Assay, Expressing, Staining, Biomarker Discovery, Quantitative RT-PCR, Transfection, Plasmid Preparation, Over Expression, Binding Assay, ChIP-qPCR, Activity Assay, Luciferase, Control

Journal: International Journal of Molecular Sciences

Article Title: Breast Cancer-Derived Extracellular Vesicle miR-425-5p (miR-425) Promotes Brain Metastasis via Activating Astrocytes Through the Novel miR-425-ZNF24-CCL8 Signaling Axis

doi: 10.3390/ijms27073197

Figure Lengend Snippet: The miR-425-ZNF24-CCL8 signaling pathway is upregulated in brain metastases and activated astrocytes of mice intracardially injected with breast cancer cells overexpressing miR-425. ( A ) Increased EV-miR-425 expression in mouse serum from the SKBR3-Luc-miR-425 group compared to the control group. miR-425 expression measured by RT-qPCR ( N = 5 per group). ( B ) SKBR3-Luc-miR-425 group mouse serum has significantly higher levels of mouse CCL8 as measured by ELISA ( N = 7 per group). ( C ) No significant difference in mouse SCF levels in SKBR3-Luc-miR-425 or control mice serum as measured by ELISA ( N = 5 per group). ( D , E ) GFAP H-score and intratumoral astrocytes are significantly higher in brain metastases from the SKBR3-Luc-miR-425 mouse group than the control in mouse brain sections as measured by IHC ( N = 5 per group). ( F ) Brain metastases from the SKBR3-Luc-miR-425 group have significantly increased Ki-67 positive cells as measured by IHC ( N = 5 per group). ( G ) Tumor-adjacent and infiltrative astrocytes in brain metastases from the SKBR3-Luc-miR-425 group have decreased ZNF24 staining measured by IHC ( N = 5 per group). ( H ) Representative IHC images at 20× magnification. Scale bar indicates 100 µm. ( I ) Co-staining IF of ZNF24 (green) and GFAP (red) in mouse brain sections containing brain metastases. Nuclei were stained with DAPI (blue). Representative images at 20× magnification. Scale bar indicates 100 µm. ( J ) Schematic of described mechanism by which breast cancer-derived EV-miR-425 activates astrocytes by suppressing ZNF24, increasing CCL8, and thereby promoting BCBM. Fold change was calculated in panel A. Student’s t -test used in panels ( A – G , I ).

Article Snippet: Astrocytes were transfected with control or miR-425 mimic, control vector or ZNF24 3′-UTR (GeneCopoeia; Cs-HmiT119980-MT05-01), control vector or ZNF24 overexpression (GeneCopoeia; cat. #B221017-1240ueu), and control promoter or CCL8 promoter (GeneCopoeia; cat. #HPRM46246-PG02) constructs using X-tremeGENE HP DNA Transfection Reagent (Millipore Sigma; cat. #6366244001).

Techniques: Injection, Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Derivative Assay

Journal: Diabetologia

Article Title: Human adipose microRNA-221 is upregulated in obesity and affects fat metabolism downstream of leptin and TNF-α

doi: 10.1007/s00125-013-2950-9

Figure Lengend Snippet: miR-221 targets the 3′ UTRs of ADIPOR1 and ETS1 and decreases their protein levels. ( a,b ) TargetScan conserved predicted miR-221 binding site on the 3′ UTRs of ADIPOR1 and ETS1 . ( c,d ) Quantification of dual-luciferase assay in HEK 293 cells co-transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars) and ADIPOR1/2 ( c ) or ETS1 ( d ) 3′ UTR reporter plasmids. ( d ) Two overlapping fragments of the ETS1 3′-UTR were subcloned into separate vectors, indicated (A) and (B). Firefly/renilla luciferase ratio (luc ratio) was normalised to no-UTR controls. Error bars indicate SD ( n = 5). * p < 0.05 (two-tailed Mann–Whitney U test). ( e ) Quantification of QRT-PCR for ETS1 mRNA in human pre-adipocytes transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars). Error bars indicate SD ( n = 3). ( f ) Immunoblots for ADIPOR1, ETS1 and β-actin in human pre-adipocytes transfected with miR-221 mimic (+) or control oligonucleotide. ( g ) Quantification of the immunoblots shown in ( f ). Background-subtracted mean signal ( n = 2) for ADIPOR1 and ETS1, normalised to the loading control (β-actin). Black bars, miR-221 mimic; white bars, control oligonucleotide

Article Snippet: For 3′ untranslated (UTR) reporter assays, JetPrime (Polyplus-transfection SA, Illkirch, France) was used to co-transfect HEK 293 cells (ATCC) with miRNA 3′ UTR target expression vectors for human ETS1 , ADIPOR1 , ADIPOR2 or control (Genecopoeia, Germantown, MD, USA), and human miR-221 mimic (Dharmacon, Lafayette, CO, USA) or scrambled control oligonucleotide, in 24-well plates.

Techniques: Binding Assay, Luciferase, Transfection, Control, Two Tailed Test, MANN-WHITNEY, Quantitative RT-PCR, Western Blot

Journal: Gut

Article Title: microRNA overexpression in slow transit constipation leads to reduced Na V 1.5 current and altered smooth muscle contractility

doi: 10.1136/gutjnl-2019-318747

Figure Lengend Snippet: Thirteen of 372 miRNA examined are differentially expressed in slow transit constipation. (A) Heatmap plotting negative Ct values from the miRNA qPCR array organised by increasing p values. The box indicates the ones with p<0.05. The expression level of miRNAs is colour-coded as indicated in the legend. (B) Blowup of highlighted group of miRNAs, with hierarchical clustering analysis on the left. (C) Map of SCN5A mRNA 3′-UTR with binding sites for miRNAs obtained from multiple prediction algorithms and literature. In the boxes are position and sequence of target region complementary to each miRNA seed. In red are miRNAs identified by qPCR array screening. (D) SCN5A mRNA expression is reduced in STC smooth muscle. Data are median±IQR. n=10–11. *p<0.05, Mann-Whitney test. CTRL, control; miRNA, microRNA; qPCR, quantitative PCR; STC, slow transit constipation.

Article Snippet: HEK293 cells were plated in six-well plates for 72 hours in minimum essential medium (MEM), supplemented with 10% fetal bovine serum and penicillin/streptomycin), then transfected by Lipofectamine 3000 (Invitrogen) with SCN5A full-length 3′ UTR (2259 bp) reporter vector (HmiT016601-MT05, Genecopoeia) and/or 50 nM of let-7f miRNA mimic (hsa-let7f-5p, Life Technologies) in a total volume of 2.4 mL per well.

Techniques: Expressing, Binding Assay, Sequencing, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Gut

Article Title: microRNA overexpression in slow transit constipation leads to reduced Na V 1.5 current and altered smooth muscle contractility

doi: 10.1136/gutjnl-2019-318747

Figure Lengend Snippet: HuSMCs have characteristics similar to GI smooth muscle cells. (A) Representative images of cell tractions’ colour maps generated by the same HuSMC in basal conditions (left) and response to carbachol (right). The colours correspond to magnitudes of traction forces generated as indicated in the colour bar. The RMS traction forces generated are quantified in the graph. Data are median±IQR, n=7, *p<0.05, Mann-Whitney test. (B) HuSMCs (passage 6, P6) are highly enriched for mRNA for smooth muscle cell marker genes, including SCN5A and CACNA1C, compared with later passages (>10, P10), which lose smooth muscle phenotype (n=4). (C) HuSMCs express Na V 1.5 protein by western blot. (D) Representative traces of Na + currents recorded from HuSMCs. (E) I/V plot with Na + currents recorded from HuSMCs. Time to peak (F) and time constants of inactivation (G) calculated from currents recorded at different voltages. ACTA2 , smooth muscle actin; CACNA1C, L-type calcium channel α1C; CARB, carbachol; CTRL, control; GAPDH , glyceraldehyde 3-phosphate dehydrogenase; HuSMC, human smooth muscle cells; MYH11 , myosin-11; pA, peak Na + currents; pF, whole cell capacitance; RMS, root mean square; SCN5A , sodium channel Na V 1.5; SMTN , smoothelin.

Article Snippet: HEK293 cells were plated in six-well plates for 72 hours in minimum essential medium (MEM), supplemented with 10% fetal bovine serum and penicillin/streptomycin), then transfected by Lipofectamine 3000 (Invitrogen) with SCN5A full-length 3′ UTR (2259 bp) reporter vector (HmiT016601-MT05, Genecopoeia) and/or 50 nM of let-7f miRNA mimic (hsa-let7f-5p, Life Technologies) in a total volume of 2.4 mL per well.

Techniques: Generated, MANN-WHITNEY, Marker, Western Blot

Journal: Gut

Article Title: microRNA overexpression in slow transit constipation leads to reduced Na V 1.5 current and altered smooth muscle contractility

doi: 10.1136/gutjnl-2019-318747

Figure Lengend Snippet: Overexpression of Let-7f significantly reduced Na + current density in HuSMCs and resulted in changes in the cells’ properties. (A) RT-qPCR show efficient delivery of miRNA after transfection of mimics in HuSMCs (means±SDs, n=6). (B) Representative patch-clamp traces for HuSMC±miRNA mimics. (C) Peak Na + current densities were reduced after transfection with let-7f (means±SEMs, n=5–17 cells, *p<0.05 by a one-way analysis of variance with Dunnett’s post-test). (D) NLUs of HEK-293 cells transfected with SCN5A 3′UTR vector alone ( control ) or cotransfected with the 3′UTR vector and miRNA mimic ( let-7f ) (means±SEMs, n=6 transfections, *p<0.05 to same-plate controls by a two-tailed paired t-test). GLuc, Gaussia luciferase; HuSMC, human smooth muscle cells; miRNA, microRNA; NLU, normalised luciferase unit; NT, non-targeted; pA, peak Na + currents; pF, whole cell capacitance; SEAP, secreted alkaline phosphatase.

Article Snippet: HEK293 cells were plated in six-well plates for 72 hours in minimum essential medium (MEM), supplemented with 10% fetal bovine serum and penicillin/streptomycin), then transfected by Lipofectamine 3000 (Invitrogen) with SCN5A full-length 3′ UTR (2259 bp) reporter vector (HmiT016601-MT05, Genecopoeia) and/or 50 nM of let-7f miRNA mimic (hsa-let7f-5p, Life Technologies) in a total volume of 2.4 mL per well.

Techniques: Over Expression, Quantitative RT-PCR, Transfection, Patch Clamp, Plasmid Preparation, Two Tailed Test, Luciferase

Journal: Cell Death & Disease

Article Title: Cell differentiation versus cell death: extracellular glucose is a key determinant of cell fate following oxidative stress exposure

doi: 10.1038/cddis.2014.52

Figure Lengend Snippet: Sirtuin 3 is a novel target of miR28-5p. RNA levels of ( a ) sirtuin 3 but not ( b ) sirtuin 1 were lower in peroxide-treated cells cultured in high glucose compared with untreated controls. Similarly, protein levels (as measured by relative quantification of the band density from western blots) of ( c ) sirtuin 3 were significantly lower in peroxide-treated cells cultured in high glucose compared with untreated controls or cells cultured in low glucose with or without peroxide treatment. ( d ) However, protein levels of sirtuin 1 were not significantly different between treatments. ( e ) No increase in the level of acetylated p53 was observed in peroxide-treated cells cultured in high glucose and infected with a sirtuin 3-bearing adenoviral vector (adSirt3). A GFP-bearing adenoviral vector (adGFP) was used as a control. ( f ) Levels of miR28-5p were significantly higher in peroxide-treated cells cultured in high but not low glucose compared with untreated controls. ( g ) Graph highlighting the difference in levels of miR28-5p in untreated cells in high and low glucose (note the y -axis scale differs from that in f ). Levels of miR28-5p were higher in untreated cells cultured in high glucose compared with in cells cultured in low glucose. ( h ) In cells cultured in high glucose, RNA levels of sirtuin 3 were significantly higher in peroxide-treated cells transfected with a miR28-5p inhibitor compared with peroxide-treated controls. ( i ) Conversely, in cells cultured in low glucose, RNA levels of sirtuin 3 were significantly lower in cells expressing a miR28-5p mimic compared with peroxide-treated controls. ( j ) Activity of a 3′-UTR sirtuin 3 reporter in which the 3′ UTR sequence of sirtuin 3 had been inserted downstream of the secreted GLuc reporter gene was significantly lower in cells transfected with a miR28-5p mimic compared with non-mimic expressing cells. A secreted alkaline phosphatase (SEAP) reporter driven by the CMV promoter cloned into the same vector served as an internal control for transfection efficiency. The miR28-5p mimic had no effect on the negative control 3′UTR construct, demonstrating its specificity for the sirtuin 3 3′-UTR. Statistically significant ( P ≤0.05) differences between treatments are indicated by *. Results are expressed as mean±S.D. Experiments were performed on cells isolated from a minimum of three different patients. Western blot images shown are representative of those obtained for all patients

Article Snippet: Cells were then transfected with 100 ng of either negative control 3′-UTR (cat. no. CmiT000001-MT05) or Sirt3 3′UTR (cat no. HmiT006079-MT05) GLuc-SEAP vector (GeneCopoeia, Rockville, MD, USA) with 30 nM of either miR28-5p mimic or mimic control using lipofectamine 2000 (all from Life Technologies).

Techniques: Cell Culture, Western Blot, Infection, Plasmid Preparation, Transfection, Expressing, Activity Assay, Sequencing, Clone Assay, Negative Control, Construct, Isolation

Journal: Cell Death & Disease

Article Title: Cell differentiation versus cell death: extracellular glucose is a key determinant of cell fate following oxidative stress exposure

doi: 10.1038/cddis.2014.52

Figure Lengend Snippet: Proposed mechanism for the differential response of tenocytes to oxidative stress under different extracellular glucose concentrations. Oxidative stress results in upregulation of both FOXO1 as well as HIF1 α . Under high-glucose conditions, miR28-5p levels are also upregulated, particularly in oxidative stress-exposed cells. miR28-5p directly inhibits expression of the p53 deacetylase sirtuin 3, allowing accumulation of acetylated p53. FOXO1 promotes transcription of bim , the gene product of which is a proapoptotic protein. p53 inhibits expression of miR17-92, a cluster of miRNAs including the bim repressor miR17-5p. Downregulation of miR17-92 by p53 coupled with increased bim transcription by FOXO1 allows accumulation of bim RNA levels and increased bim-mediated apoptosis. Under low-glucose conditions, however, the miR28-5p–sirt3-p53 pathway is not stimulated. Instead, p38 MAPK is activated and acts on both FOXO1 and HIF1 α , resulting in the inhibition of FOXO1 transcriptional activity and activation of HIF1 α . HIF1 α promotes the expression of sox9 and scleraxis , two genes whose products are essential for tenocyte differentiation

Article Snippet: Cells were then transfected with 100 ng of either negative control 3′-UTR (cat. no. CmiT000001-MT05) or Sirt3 3′UTR (cat no. HmiT006079-MT05) GLuc-SEAP vector (GeneCopoeia, Rockville, MD, USA) with 30 nM of either miR28-5p mimic or mimic control using lipofectamine 2000 (all from Life Technologies).

Techniques: Expressing, Histone Deacetylase Assay, Inhibition, Activity Assay, Activation Assay

Journal: Cell reports

Article Title: RNA-binding protein FXR1 drives cMYC translation by recruiting eIF4F complex to the translation start site

doi: 10.1016/j.celrep.2021.109934

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 3′UTR target expression clone for Human MYC (see ) , GeneCopoeia , Cat# HmiT102685-MT06.

Techniques: Control, Recombinant, Modification, Transfection, Protease Inhibitor, CCK-8 Assay, Bicinchoninic Acid Protein Assay, cDNA Synthesis, SYBR Green Assay, Caspase-Glo Assay, Immunoprecipitation, Plasmid Preparation, Staining, In Situ, Microarray, Western Blot, Expressing, Labeling, Negative Control, Mutagenesis, Software

Journal: Experimental and Therapeutic Medicine

Article Title: miR‑137 is a diagnostic tumor‑suppressive miRNA that targets SPHK2 to promote M1‑type tumor‑associated macrophage polarization

doi: 10.3892/etm.2023.12096

Figure Lengend Snippet: miR-137 inhibits the release of IL-13, and promotes the release of TNFα and IFNγ. HK was used as control group, and SH1, SH2, SH3 as knockdown groups. (A) miR-137 overexpression in glioma cells reduced IL-13 production. (B) miR-137 overexpression in glioma cells promoted TNFα production. (C) miR-137 overexpression in glioma cells promoted IFNγ production. * P<0.05, ** P<0.01 and *** P<0.001 vs. empty vector. (D and E) After transduction with lentiviral vectors, SPHK2 mRNA was detected using reverse transcription-quantitative PCR. ** P<0.01 vs. U373-HK. (F) SPHK2 protein level was assessed using western blot analysis. (G-I) The supernatant from NC and sh-SPHK2 was collected, and IL-13, TNF-α and IFN-γ were detected using ELISA. * P<0.05, ** P<0.01, and **** P<0.0001 vs. NC. miR, microRNA; SPHK2, sphingosine kinase 2; sh-, short hairpin; NC, negative control.

Article Snippet: The wild-type (p-WT) and mutant-type (p-MT) reporter vectors of the SPHK2 3'-UTR were constructed using the pEZX-MT01 Luciferase miRNA Expression Reporter Vector (GeneCopoeia, Inc.).

Techniques: Control, Knockdown, Over Expression, Plasmid Preparation, Transduction, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: miR‑137 is a diagnostic tumor‑suppressive miRNA that targets SPHK2 to promote M1‑type tumor‑associated macrophage polarization

doi: 10.3892/etm.2023.12096

Figure Lengend Snippet: SPHK2 is a direct target of miR-137. (A) miR-137 binding site in SPHK2 3'-UTR was predicted using TargetScan. SPHK2-3'-UTR-WT and SPHK2-3'-UTR-MT carried in recombinant luciferase mRNAs transcribed by p-WT and p-MT. (B) Reverse transcription-quantitative PCR analysis of SPHK2 mRNA expression in the cells as indicated. Their relative expression levels were normalized against GAPDH. The ratios of SPHK2/GAPDH in cells transfected with the scramble sequence were set to 1.0. (C) Western blot analysis of SPHK2 protein expression in the cells as indicated. (D and E) Dual-luciferase reporter assays were performed using (D) U373 and (E) U251 cells co-transfected with p-WT or p-MT and scramble sequence or miR-137 mimics. All experiments were performed at least in triplicate and the data in (B-E) are presented as the mean ± SD. * P<0.05 and ** P<0.01 vs. Scramble. SPHK2, sphingosine kinase 2; miR, microRNA; UTR, untranslated region; WT, wild-type; MUT, mutant.

Article Snippet: The wild-type (p-WT) and mutant-type (p-MT) reporter vectors of the SPHK2 3'-UTR were constructed using the pEZX-MT01 Luciferase miRNA Expression Reporter Vector (GeneCopoeia, Inc.).

Techniques: Binding Assay, Recombinant, Luciferase, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Transfection, Sequencing, Western Blot, Mutagenesis