The Role of MicroRNA-181a in Myocardial Fibrosis Following Myocardial Infarction in a Rat Model

Abstract

BACKGROUND The role of miR-181a in the development of cardiac disease and in particular, myocardial fibrosis following myocardial infarction (MI) remains unknown. The aim of this study was to explore the role of miR-181a in myocardial fibrosis in a rat model of MI and the expression of TGF-β receptor III (TβRIII). MATERIAL AND METHODS Forty adult male Wistar rats were randomly divided into an MI model group (n=30) and a control group with (n=10). The rat MI model involved ligating the left anterior descending (LAD) coronary artery in the model group; the control group was treated with a sham operation. Cardiac function was assessed using cardiac ultrasound. Myocardial fibroblasts were extracted from the rat hearts and transfected with a miR-mimic or miR-inhibitor, and cell growth was measured using an MTT assay. The level of miR-181a expression was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blots. RESULTS miR-181a expression was significantly increased during the progression of MI (P<0.05). Over-expression of miR-181a was associated with increased deposition of extracellular matrix (ECM) components, collagen I and fibronectin. This effect was reversed with the use of a miR-181a inhibitor (P<0.05). Upregulation of miR-181a suppressed the expression of TGF-β receptor III (TβRIII) by binding with 3'-UTR. CONCLUSIONS In this rat model of MI, the findings were that miR-181a had a role in the progression of myocardial fibrosis. The findings require further studies to determine whether miR-181a might provide a novel therapeutic target to limit myocardial fibrosis following MI.

Figure 1

Expression of miR-181a and the fibrotic index in a rat model of myocardial infarction (MI model group and control group). (A) The expression of miR-181a mRNA is significantly upregulated in the MI group compared with the control group. (B, C) The change in fibrotic index showed a significant increase in the expression of collagen I and fibronectin mRNA. Data are shown as the mean ±SD. * P 0.05 (Student’s t-test).

Figure 2

Effect of miR-181a on cell proliferation detected by the MMT assay. (A) The expression of miR-181a in myocardial fibroblasts treated with miR-Control or the miR-181a (mimic) was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (B) The MMT assay for the proliferation of myocardial fibroblasts transduced, as in A. Data are shown as the mean ±SD. * P<0.05 and *** P<0.001. (Student’s t-test).

Figure 3

Expression of collagen I and fibronectin, the fibrosis index, in myocardial fibroblasts. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) detects the mRNA levels of collagen I and fibronectin in cells treated with miR-Control or the miR-181a (mimic). (B) Western blot measurements of the expression of collagen I and fibronectin in cells transduced, as in A. (C) The expression of miR-181a in cells treated with control-inhibitor and miR-181a inhibitor detected by qRT-PCR. (D, E) Western blots and qRT-PCR for the expression of collagen I and fibronectin in cells treated, as in C. Data are shown as the mean ±SD. * P<0.05 and *** P<0.001. (Student’s t-test).

Figure 4

Upregulation of miR-181a suppresses the expression of TGF-β receptor III (TβRIII) by binding with 3′-UTR. Using bioinformatics analysis, 3′ UTR of TGF-β receptor III (TβRIII) is shown to be highly conserved and to bind to miR-181a. The 3′-UTR binding sites are shown as follows: (A) Luciferase reporter assay shows that transfection of miR-181a significantly restricts the relative luciferase activity in myocardial fibroblasts. (B) There appear to be few differences in luciferase activities between the control group and normal cells containing miR-21 in the control group and PTEN mutation 3′-UTR group (* P<0.5). (C) After cells were transfected with miR-181a inhibitor, TGF-β receptor III (TβRIII) expression levels are significantly upregulated. These findings show that miR-181a suppresses the expression of TβRIII by binding with the 3′-UTR.