MicroRNA-101a inhibits cardiac fibrosis induced by hypoxia via targeting TGFβRI on cardiac fibroblasts.

Hypoxia is a basic pathological challenge that is associated with numerous cardiovascular disorders including aberrant cardiac remodeling. Transforming growth factor beta (TGF-β) signaling pathway plays a pivotal role in mediating cardiac fibroblast (CF) function and cardiac fibrosis. Recent data suggested that microRNA-101a (miR-101a) exerted anti-fibrotic effects in post-infarct cardiac remodeling and improved cardiac function. This study aimed to investigate the potential relationship between hypoxia, miR-101a and TGF-β signaling pathway in CFs. Two weeks following coronary artery occlusion in rats, the expression levels of both TGFβ1 and TGFβRI were increased, but the expression of miR-101a was decreased at the site of the infarct and along its border. Cultured rat neonatal CFs treated with hypoxia were characterized by the up-regulation of TGFβ1 and TGFβRI and the down-regulation of miR-101a. Delivery of miR-101a mimics significantly suppressed the expression of TGFβRI and p-Smad 3, CF differentiation and collagen content of CFs. These anti-fibrotic effects were abrogated by co-transfection with AMO-miR-101a, an antisense inhibitor of miR-101a. The repression of TGFβRI, a target of miR-101a, was validated by luciferase reporter assays targeting the 3'UTR of TGFβRI. Additionally, we found that overexpression of miR-101a reversed the improved migration ability of CFs and further reduced CF proliferation caused by hypoxia. Our study illustrates that miR-101a exerts anti-fibrotic effects by targeting TGFβRI, suggesting that miR-101a plays a multi-faceted role in modulating TGF-β signaling pathway and cardiac fibrosis.

Zhao X ，Wang K ，Liao Y ，Zeng Q ，Li Y ，Hu F ，Liu Y ，Meng K ，Qian C ，Zhang Q ，Guan H ，Feng K ，Zhou Y ，Du Y ，Chen Z ... -  《-》

MicroRNA-101 protects cardiac fibroblasts from hypoxia-induced apoptosis via inhibition of the TGF-β signaling pathway.

Cardiac fibroblasts (CFs) are the most numerous cells in the heart and are recognized primarily for their ability to maintain both the structural integrity and the physiological functions of the heart. The transforming growth factor beta (TGF-β) signaling pathway is reportedly involved in the modulation of CF functions, including apoptosis. Recent studies have indicated that microRNA-101 (miR-101) attenuates the TGF-β signaling pathway, either by inhibiting the expression of TGFβ1 or by targeting transforming growth factor-β receptor type I (TGFβRI). The present study aimed to determine whether miR-101 protects CFs from hypoxia-induced apoptosis and to investigate the mechanisms underlying its protective effects. The CCK-8 test, electron microscopy and TUNEL assay results demonstrated that miR-101a/b significantly inhibited hypoxia-induced CF apoptosis. The results of Western blotting, quantitative RT-PCR and immunofluorescence assays indicated that miR-101a dramatically inhibited the hypoxia-induced up-regulation of both TGFβRI and p-Smad 3 but not TGFβ1 in CFs. Additionally, miR-101a significantly reversed the hypoxia-induced up-regulation of Bax and Caspase-3, the down-regulation of Bcl-2 and the activation of Caspase-3 in CFs. Moreover, miR-101a markedly inhibited the intracellular Ca(2+) ([Ca(2+)]i) overload caused by hypoxia. Taken together, our results suggest that miR-101a protects CFs against hypoxia-induced apoptosis by inhibiting the TGF-β signaling pathway, which may be a potential therapeutic target for heart injury.

Zhao X ，Wang K ，Hu F ，Qian C ，Guan H ，Feng K ，Zhou Y ，Chen Z ... -  《-》

MicroRNA-101a suppresses fibrotic programming in isolated cardiac fibroblasts and in vivo fibrosis following trans-aortic constriction.

MiR-101a is reported to reduce post-infarction myocardial fibrosis through targeting c-FOS and TGFbr1. However the actions of miR-101a within the TGF-β signaling pathway are largely unknown. We demonstrate the mechanisms underlying mutual inhibition between miR-101a and TGF-β signaling and explore the therapeutic potential of miR-101a in suppressing pressure overload-induced cardiac fibrosis. The effects of miR-101a on fibroblast proliferation, myofibroblast transdifferentiation, collagen synthesis, apoptosis, and autophagy were assessed in isolated rat cardiac fibroblasts (cFB). MiR-101a effects upon cFB TGF-β signaling were assessed by qPCR, Western blotting, 3'UTR luciferase reporter assay and promoter activity assessments. Rats subjected to transverse aortic constriction (TAC) were treated with PBS or 1 mg/kg of a miR-101a mimic i.v. at 1, 3, and 7-day post-surgery. Left ventricular (LV) function (echocardiography; LV catheterization) and LV fibrosis (picro-sirius red staining, qPCR and WB) were assessed at 2-day and 2 & 10-week post-surgery. MiR-101a inhibited cFB proliferation by inducing cell apoptosis rather than through cell cycle inhibition; and reduced collagen synthesis by downregulation of collagen gene expression and increased autophagy. MiR-101a inhibited TGF-β signaling pathways by directly targeting TGFbr1, reducing Smad3 phosphorylation and inhibiting Tab3 promoter activity. Conversely TGF-β inhibited promoter activity for both miR-101a and b. In vivo endogenous miR-101a expression was downregulated 2-day post-TAC returning to baseline by 14-day. MiR-101a mimic treatment inhibited myocardial TGF-β signaling and collagen gene up-regulation at 2-day post-TAC. MiR-101a reduced fibrosis, improved +dP/dt and lowered end diastolic pressure (EDP) at 2-week post-TAC. Treatment also attenuated adverse LV remodeling and preserved cardiac ejection fraction at 10-week post-TAC. MiR-101a and TGF-β are mutually inhibitory and co-direct the activation, proliferation, and collagen synthesis of cFBs. The predominance of TGF-β signaling over reduced miR-101a expression early post-TAC is associated with cardiac fibrosis and dysfunction. Treatment with miR-101a, introduced early after imposition of abnormal pressure loading, inhibits TGF-β signaling, reduces cardiac fibrosis and preserves LV function.

Zhou Y ，Shiok TC ，Richards AM ，Wang P ... -  《-》

MiR-22 may Suppress Fibrogenesis by Targeting TGFβR I in Cardiac Fibroblasts.

Cardiac fibrosis after myocardial infarction (MI) has been identified as a key factor in the development of heart failure, but the mechanisms undelying cardiac fibrosis remained unknown. microRNAs (miRNAs) are novel mechanisms leading to fibrotic diseases, including cardiac fibrosis. Previous studies revealed that miR-22 might be a potential target. However, the roles and mechanisms of miR-22 in cardiac fibrosis remained ill defined. The present study thus addressed the impact of miR-22 in cardiac fibrosis. After seven days following coronary artery occlusion in mice, tissues used for histology were collected and processed for Masson's Trichrome staining. In addition, cardiac fibroblasts were transfected with mimics and inhibitors of miR-22 using Lipofectamin 2000, and luciferase activity was measured in cell lysates using a luciferase assay kit. Western blotting was used to detect the expression of collagen1, α-SMA and TGFβRI proteins levels, and real time-PCR was employed to measure the Col1α1, Col3α1, miR-22 and TGFβRI mRNA levels. In this study, we found that miR-22 was dynamically downregulated following MI induced by permanent ligation of the left anterior descending coronary artery for 7 days, an effect paralleled by significant collagen deposition. Inhibition of miR-22 with AMO-22 resulted in increased expression of Col1α1, Col3α1 and fibrogenesis in cultured cardiac fibroblasts. Conversely, overexpression of miR-22 in cultured cardiac fibroblasts significantly abrogated angiotensin II-induced collagen formation and fibrogenesis. Furthermore, we found that TGFβRI is a direct target for miR-22, and downregulation of TGFβR may have mediated the antifibrotic effect of miR-22. Our data clearly demonstrate that miR-22 acts as a novel negative regulator of angiotensin II-induced cardiac fibrosis by suppressing the expression of TGFβRI in the heart and may represent a new potential therapeutic target for treating cardiac fibrosis.

Hong Y ，Cao H ，Wang Q ，Ye J ，Sui L ，Feng J ，Cai X ，Song H ，Zhang X ，Chen X ... -  《-》

MicroRNA-328, a Potential Anti-Fibrotic Target in Cardiac Interstitial Fibrosis.

Deregulated myocardial fibrosis is associated with a wide spectrum of cardiac conditions, being considered one of the major causes for heart disease. Our study was designed to investigate the role of microRNA-328 (miR-328) in regulating cardiac fibrosis. We induced cardiac fibrosis following MI by occlusion of the left coronary artery in C57BL/6 mice. Real-time PCR was employed to evaluate the level of miR-328. Masson's Trichrome stain was used to evaluate the development of fibrosis. Luciferase activity assay was performed to confirm the miRNA's binding site in the TGFβRIII gene. Western blot analysis was used to examine TGFβRIII, p-smad2/3 and TGF-β1 at protein level. In this study, we found that miR-328 was significantly upregulated in the border zone of infarcted myocardium of wild type (WT) mice; TGFβRIII was downregulated whereas TGF-β1 was upregulated along with increased cardiac fibrosis. And miR-328 stimulated TGF-β1 signaling and promoted collagen production in cultured fibroblasts. We further found that the pro-fibrotic effect of miR-328 was mediated by targeting TGFβRIII. Additionally, cardiac fibrosis was significantly reduced in infarcted heart when treated with miR-328 antisense. These data suggest that miR-328 is a potent pro-fibrotic miRNA and an important determinant of cardiac fibrosis in diseased heart.

Du W ，Liang H ，Gao X ，Li X ，Zhang Y ，Pan Z ，Li C ，Wang Y ，Liu Y ，Yuan W ，Ma N ，Chu W ，Shan H ，Lu Y ... -  《-》