MicroRNA‐101 Protects Against Cardiac Remodeling Following Myocardial Infarction via Downregulation of Runt‐Related Transcription Factor 1

Figures

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  Figure 1. MicroRNA‐101 (miR‐101) could target runt‐related transcription factor 1 (RUNX1), which was an upregulated gene in myocardial infarction (MI). A, The heatmap of the top 15 differentially expressed genes (DEGs) in the microarray data GSE46395. The abscissa refers to the sample number, the ordinate refers to the DEGs, and the upper right histogram represents color grade. Each rectangle in the image represents the expression of a sample. B, Gene interaction network of the DEGs and MI genes, in which the diamonds refer to MI genes and the circles refer to DEGs. C, Expression of 8 nonoverlapping miRs in myocardial tissues in the sham‐operated and MI rats determined by reverse transcription quantitative polymerase chain reaction. BCR indicates B cell receptor; DBN1, Drebrin; FLNC, filamin C; LTB, heat‐lable enterotoxin B unit; RTN4, reticulon 4; SH2D5, SH2 domain‐containing 5; SLFN2, Schlafen; TTC9, Tetratricopeptide repeat domain 9; Tubb6, beta 6 class V β‐tubulin.

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  Figure 2. MicroRNA‐101 (miR‐101) could directly bind to runt‐related transcription factor 1 (RUNX1) gene. A, The binding site of miR‐101 in the RUNX1‐3′untranslated region (3′UTR) predicted in the biological website. B, Luciferase activity of RUNX1‐3′UTR‐wild‐type (WT) and RUNX1‐3′UTR‐mutant (MUT) followed by the cotransfection with miR‐101 mimic detected by dual luciferase reporter assay. The data were expressed as mean±SD and tested using independent sample t test. The experiment was repeated 3 times. *P<0.05 compared with the negative control (NC) group.

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  Figure 3. MicroRNA‐101 (miR‐101) overexpression or runt‐related transcription factor 1 (RUNX1) silencing led to improved cardiac function in rats with myocardial infarction (MI). The MI rats used for following measurements were injected with lentiviral vectors expressing empty vector, miR‐101 mimic, miR‐101 inhibitor, and/or small interfering RNA targeting RUNX1 (si‐RUNX1). A, The level of left ventricular end‐diastolic diameter (LVEDD) in sham‐operated and MI rats. B, The levels of left ventricular end‐systolic diameter (LVESD) in sham‐operated and MI rats. C, The level of left ventricular ejection fraction (LVEF) in sham‐operated and MI rats. D, The level of left ventricular fractional shortening (LVFS) in sham‐operated and MI rats. The data were expressed as mean±SD and analyzed using ANOVA (n=20 in each group). *P<0.05 compared with the sham‐operated rats; # P<0.05 compared with the MI rats injected with empty vector; & P<0.05 compared with the MI rats injected with both miR‐101 inhibitor and si‐RUNX1. NC indicates negative control.

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  Figure 4. Myocardial infarction (MI)–induced damage to cardiomyocytes was alleviated with the enhancement of microRNA‐101 (miR‐101) or depletion of runt‐related transcription factor 1 (RUNX1). The MI rats used for following assays were injected with lentiviral vectors expressing empty vector, miR‐101 mimic, miR‐101 inhibitor, and/or small interfering RNA targeting RUNX1 (si‐RUNX1). A, Representative images of cardiomyocytes in sham‐operated and MI rats observed by hematoxylin‐eosin staining (×400). B, The quantitative surface areas of cardiomyocytes in sham‐operated and MI rats. The data were expressed as mean±SD and analyzed using ANOVA (n=20 in each group). *P<0.05 compared with the sham‐operated rats. # P<0.05 compared with the MI rats injected with empty vector. & P<0.05 compared with the MI rats injected with both miR‐101 inhibitor and si‐RUNX1. NC indicates negative control.

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  Figure 5. Myocardial infarct size in rats with myocardial infarction (MI) was reduced in response to microRNA‐101 (miR‐101) elevation or runt‐related transcription factor 1 (RUNX1) depletion. The MI rats used for following assays were injected with lentiviral vectors expressing empty vector, miR‐101 mimic, miR‐101 inhibitor, and/or small interfering RNA targeting RUNX1 (si‐RUNX1). A, Representative images of myocardial infarct in sham‐operated and MI rats detected by triphenyltetrazolium chloride staining. B, The percentage of myocardial infarct area of sham‐operated and MI rats. The data were expressed as mean±SD and analyzed using ANOVA (n=10 in each group). *P<0.05 compared with the sham‐operated rats. # P<0.05 compared with the MI rats injected with empty vector. & P<0.05 compared with the MI rats injected with both miR‐101 inhibitor and si‐RUNX1. NC indicates negative control.

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  Figure 6. Myocardial fibrosis following myocardial infarction (MI) was ameliorated in response to microRNA‐101 (miR‐101) elevation or runt‐related transcription factor 1 (RUNX1) depletion. The MI rats used for following assays were injected with lentiviral vectors expressing empty vector, miR‐101 mimic, miR‐101 inhibitor, and/or small interfering RNA targeting RUNX1 (si‐RUNX1). A, Representative images showing myocardial fibrosis in myocardial tissues of sham‐operated and MI rats assessed by Masson staining (×400). B, The percentage of myocardial fibrosis area in sham‐operated and MI rats. The data were expressed as mean±SD and analyzed using ANOVA (n=20 in each group). *P<0.05 compared with the sham‐operated rats. # P<0.05 compared with the MI rats injected with empty vector. & P<0.05 compared with the MI rats injected with both miR‐101 inhibitor and si‐RUNX1. NC indicates negative control.

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  Figure 7. Cardiomyocyte apoptosis after myocardial infarction (MI) was found to be repressed with the elevation of microRNA‐101 (miR‐101) or depletion of runt‐related transcription factor 1 (RUNX1). The MI rats used for following assays were injected with lentiviral vectors expressing empty vector, miR‐101 mimic, miR‐101 inhibitor, and/or small interfering RNA targeting RUNX1 (si‐RUNX1). A, Representative images showing cell apoptosis in myocardial tissues of sham‐operated and MI rats detected by terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling staining (×400). B, The cell apoptotic rate in myocardial tissues. The data were expressed as mean±SD and analyzed using ANOVA (n=20 in each group). *P<0.05 compared with the sham‐operated rats. # P<0.05 compared with the MI rats injected with empty vector. & P<0.05 compared with the MI rats injected with both miR‐101 inhibitor and si‐RUNX1. AI indicates apoptotic index; NC indicates negative control.

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  Figure 8. Overexpression of microRNA‐101 (miR‐101) downregulated the expression of runt‐related transcription factor 1 (RUNX1), thus inactivating the transforming growth factor β1 (TGF‐β1)/Smad family member 2 (Smad2) signaling pathway and repressing cardiomyocyte apoptosis after myocardial infarction (MI). The MI rats used for following assays were injected with lentiviral vectors expressing empty vector, miR‐101 mimic, miR‐101 inhibitor, and/or small interfering RNA targeting RUNX1 (si‐RUNX1). A, Expression of miR‐101, RUNX1, regulated upon activation normal T‐cell expressed and secreted (RANTES), chemokine receptor 5 (CCR5), TGF‐β1, Smad2, B‐cell CLL/lymphoma 2 (Bcl‐2), and Bcl‐2–associated X protein (Bax) in myocardial tissues of sham‐operated and MI rats determined by reverse transcription quantitative polymerase chain reaction. B and C, Protein expression of RUNX1, RANTES, CCR5, TGF‐β1, Smad2, Bcl‐2, and Bax relative to GAPDH in myocardial tissues of sham‐operated and MI rats measured by western blot analysis. D, The positive rate of phosphorylated Smad2 in myocardial tissues of sham‐operated and MI rats detected by immunohistochemistry. The data were expressed as mean±SD and…

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  Figure 9. The regulatory mechanism of microRNA‐101 (miR‐101) in cardiac remodeling following myocardial infarction (MI). miR‐101 downregulated the expression of runt‐related transcription factor 1 (RUNX1) to inactivate the transforming growth factor β1 (TGF‐β1)/Smad family member 2 (Smad2) signaling pathway, thus inhibiting cardiomyocyte apoptosis, preventing cardiac dysfunction, and eventually alleviating cardiac remodeling following MI.