The Smad3-miR-29b/miR-29c axis mediates the protective effect of macrophage migration inhibitory factor against cardiac fibrosis.

Although macrophage migration inhibitory factor (MIF) is known to have antioxidant property, the role of MIF in cardiac fibrosis has not been well understood. We found that MIF was markedly increased in angiotension II (Ang-II)-infused mouse myocardium. Myocardial function was impaired and cardiac fibrosis was aggravated in Mif-knockout (Mif-KO) mice. Functionally, overexpression of MIF and MIF protein could inhibit the expression of fibrosis-associated collagen (Col) 1a1, COL3A1 and α-SMA, and Smad3 activation in mouse cardiac fibroblasts (CFs). Consistently, MIF deficiency could exacerbate the expression of COL1A1, COL3A1 and α-SMA, and Smad3 activation in Ang-II-treated CFs. Interestingly, microRNA-29b-3p (miR-29b-3p) and microRNA-29c-3p (miR-29c-3p) were down-regulated in the myocardium of Ang-II-infused Mif-KO mice but upregulated in CFs with MIF overexpression or by treatment with MIF protein. MiR-29b-3p and miR-29c-3p could suppress the expression of COL1A1, COL3A1 and α-SMA in CFs through targeting the pro-fibrosis genes of transforming growth factor beta-2 (Tgfb2) and matrix metallopeptidase 2 (Mmp2). We further demonstrated that Mif inhibited reactive oxygen species (ROS) generation and Smad3 activation, and rescued the decrease of miR-29b-3p and miR-29c-3p in Ang-II-treated CFs. Smad3 inhibitors, SIS3 and Naringenin, and Smad3 siRNA could reverse the decrease of miR-29b-3p and miR-29c-3p in Ang-II-treated CFs. Taken together, our data demonstrated that the Smad3-miR-29b/miR-29c axis mediates the inhibitory effect of macrophage migration inhibitory factor on cardiac fibrosis.

Liang JN ，Zou X ，Fang XH ，Xu JD ，Xiao Z ，Zhu JN ，Li H ，Yang J ，Zeng N ，Yuan SJ ，Pan R ，Fu YH ，Zhang M ，Luo JF ，Wang S ，Shan ZX ... -  《-》

MicroRNA-323a-3p Promotes Pressure Overload-Induced Cardiac Fibrosis by Targeting TIMP3.

Cardiac fibrosis is a major cause of diverse cardiovascular diseases. MicroRNAs have recently been proven a novel class of regulators of cardiac fibrosis. In this study, we sought to investigate the role of miR-323a-3p and its mechanisms in regulating cardiac fibrosis. The transverse aortic constriction (TAC) mice model was induced and neonatal cardiac fibroblasts (CFs) were cultured. MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay was used to detect the cell viability. Echocardiography was used to evaluate cardiac function. Masson's Trichrome stain was used to evaluate the development of fibrosis. Luciferase activity assay was performed to confirm the miRNA's binding site. Real-time PCR and Western blot were used to evaluate the level of mRNA and protein. MiR-323a-3p was found up-regulated in myocardial tissues subjected to TAC and in CFs cultured with Angiotensin Ⅱ (Ang Ⅱ). Overexpression of miR-323a-3p significantly increased the mRNA levels of collagen Ⅰ, collagen Ⅲ, MMP2 and MMP9, while inhibition of miR-323a-3p prevented the proliferation, collagen production and the protein level of transforming growth factor (TGF-β) in rat neonatal CFs. Strikingly, injection of antagomiR-323a-3p elevated cardiac function and inhibited the expression of TGF-β in the TAC mice. TIMP3 was a direct target of miR-323a-3p, as the overexpression of miR-323a-3p decreased the protein and mRNA levels of TIMP3. In the CFs with pre-treatment of Ang Ⅱ, siRNA-TIMP abolished the effects of AMO-323a-3p on the inhibition of the proliferation of CFs, the down-regulation of collagen Ⅰ and collagen Ⅲ, and the expression of TGF-β. Our findings provide evidence that miR-323a-3p promotes cardiac fibrosis via miR-323a-3p-TIMP3-TGF-β pathway. miR-323a-3p may be a new marker for cardiac fibrosis progression and that inhibition of miR-323a-3p may be a promising therapeutic target for the treatment of cardiac fibrosis.

Zhang J ，Lang Y ，Guo L ，Pei Y ，Hao S ，Liang Z ，Su G ，Shu L ，Liu H ，Huang C ，Xu J ... -  《-》

microRNA-29b Mediates the Antifibrotic Effect of Tanshinone IIA in Postinfarct Cardiac Remodeling.

Yang F ，Li P ，Li H ，Shi Q ，Li S ，Zhao L ... -  《-》

Inhibition of circHIPK3 prevents angiotensin II-induced cardiac fibrosis by sponging miR-29b-3p.

Circular RNAs (circRNAs) are emerging as powerful regulators of cardiac development and disease. Nevertheless, detailed studies describing circRNA-mediated regulation of cardiac fibroblasts (CFs) biology and their role in cardiac fibrosis remain limited. PCR and Sanger sequencing were performed to identify the expression of circHIPK3 in CFs. Edu corporation assays, Transwell migration assays, and immunofluorescence staining assays were conducted to detect the function of circHIPK3 in CFs in vitro. Bioinformatics analysis, dual luciferase activity assays, RNA immunoprecipitation, and fluorescent in situ hybridization experiments were conducted to investigate the mechanism of circHIPK3-mediated cardiac fibrosis. Echocardiographic analysis, Sirius Red staining and immunofluorescence staining were performed to investigate the function of circHIPK3 in angiotensin II (Ang II) induced cardiac fibrosis in vivo. circHIPK3 expression markedly increased in CFs and heart tissues after the treatment of Ang II. circHIPK3 silencing attenuates CFs proliferation, migration and the upregulation of a-SMA expression levels induced by Ang II in vitro. circHIPK3 acted as a miR-29b-3p sponge and overexpression of circHIPK3 effectively reverses miR-29b-3p-induced inhibition of CFs proliferation and migration and alters the expression levels of miR-29b-3p targeting genes (a-SMA, COL1A1, COL3A1) in vitro. Combination of circHIPK3 silencing and miR-29b-3p overexpression had a stronger effect on cardiac fibrosis suppression in vivo than did circHIPK3 silencing or miR-29b-3p overexpression alone. Our data suggest that circHIPK3 serves as a miR-29b-3p sponge to regulate CF proliferation, migration and development of cardiac fibrosis, revealing a potential new target for the prevention of Ang II-induced cardiac fibrosis.

Ni H ，Li W ，Zhuge Y ，Xu S ，Wang Y ，Chen Y ，Shen G ，Wang F ... -  《-》

MicroRNA-223 Regulates Cardiac Fibrosis After Myocardial Infarction by Targeting RASA1.

Percutaneous coronary intervention reduces acute myocardial infarction (MI)-induced mortality to a great extent, but effective treatments for MI-induced cardiac fibrosis and heart failure are still lacking. MicroRNAs (miRNAs) play a variety of roles in cells and have thus been investigated extensively. MicroRNA-223 (miR-223) expression has been reported to be altered in post-MI heart failure in humans; however, the roles of miR-223 in MI remain unknown. Our study aimed to elucidate the roles of miR-223 in cardiac fibrosis. Cultured cardiac fibroblasts (CFs) were activated by TGF-β1 stimulation. Gain and loss of miR-223 and RAS p21 protein activator 1 (RASA1) knockdown in CFs were achieved by transfecting the cells with miR-223 mimics and inhibitors, as well as small interfering RNA-RASA1 (siRASA1), respectively. Quantitative real-time reverse transcriptase-polymerase chain reactions (qRT-PCR) was used to determine miR-223-3p and RASA1 expression levels, and Cell Counting Kit-8 (CCK-8), transwell migration and scratch assays were performed to assess CFs viability and migration, respectively. Western blotting was used to detect collagen I, collagen III, alpha-smooth muscle actin (a-SMA), RASA1, p-Akt/t-Akt, p-MEK1/2/t-MEK1/2, and p-ERK1/2/t-ERK1/2 protein expressions, and immunofluorescence assays were used to detect the expression of α-actin, vimentin and α-SMA. Luciferase assays were carried out to determine whether miR-223 binds to RASA1. Rat models of MI were established by the ligation of the left anterior descending (LAD) coronary artery. MiR-223 inhibition in vivo was achieved via intramyocardial injections of the miR-223 sponge carried by adeno-associated virus 9 (AAV9). The cardiac function was detected by echocardiography, and cardiac fibrosis was shown by Masson's trichrome staining. miR-223 was increased in CFs compared to cardiomypcytes, and TGF-β1 treatment increased miR-223 expression in CFs. The miR-223 mimics enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs, while the miR-223 inhibitors had contrasting effects and partially prevented the promoting effects of TGF-β1. qRT-PCR and western blotting revealed that miR-223 negatively regulated RASA1 expression, and the luciferase assays showed that miR-223 suppressed the luciferase activity of the RASA1 3' untranslated region (3'UTR), indicating that miR-223 binds directly to RASA1. Similar to transfection with the miR-223 mimics, RASA1 knockdown enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs. Moreover, RASA1 knockdown partially reversed the inhibitory effects of the miR-223 inhibitor on cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression, indicating that the effects of miR-223 in CFs are partially mediated by the regulation of RASA1 expression. Further exploration showed that miR-223 mimics and siRASA1 promoted MEK1/2, ERK1/2 and AKT phosphorylation, while the miR-223 inhibitors had contrasting effects. The in vivo experiments confirmed the results of the in vitro experiments and showed that miR-223 inhibition prevented cardiac functional deterioration and cardiac fibrosis. miR-223 enhanced cell proliferation, migration, and differentiation in CFs, thus mediated cardiac fibrosis after MI partially via the involvement of RASA1.

Liu X ，Xu Y ，Deng Y ，Li H ... -  《-》