Haihai Liang

A novel reciprocal loop between microRNA-21 and TGFβRIII is involved in cardiac fibrosis

Cardiac fibrosis is characterized by aberrant proliferation of cardiac fibroblasts and exaggerated deposition of extracellular matrix (ECM) in the myocardial interstitial, and ultimately impairs cardiac function. It is still controversial whether microRNA-21 (miR-21) participates in the process of cardiac fibrosis. Our previous study confirmed that transforming growth factor beta receptor III (TGFβRIII) is a negative regulator of TGF-β pathway. Here, we aimed to decipher the relationship between miR-21 and TGFβRIII in the pathogenic process of myocardial fibrosis. We found that TGF-β1 and miR-21 were up-regulated, whereas TGFβRIII was down-regulated in the border zone of mouse hearts in response to myocardial infarction. After transfection of miR-21 into cardiac fibroblasts, TGFβRIII expression was markedly reduced and collagen content was increased. And, luciferase results confirmed that TGFβRIII was a target of miR-21. It suggests that up-regulation of miR-21 could increase the collagen content and at least in part through inhibiting TGFβRIII. Conversely, we also confirmed that overexpression of TGFβRIII could inhibit the expression of miR-21 and reduce collagen production in fibroblasts. Further studies showed that overexpression of TGFβRIII could also deactivate TGF-β1 pathway by decreasing the expression of TGF-β1 and phosphorylated-Smad3 (p-Smad3). TGF-β1 has been proven as a positive regulator of miR-21. Taken together, we found a novel reciprocal loop between miR-21 and TGFβRIII in cardiac fibrosis caused by myocardial infarction in mice, and targeting this pathway could be a new strategy for the prevention and treatment of myocardial remodeling.

Matrine inhibits breast cancer growth via miR-21/PTEN/Akt pathway in MCF-7 cells.

Background: Matrine is one of the major alkaloids extracted from Sophora flavescens and has been used clinically for breast cancer with notable therapeutic efficacy in China. However, the mechanisms are still largely unknown. Methods: Cell viability was analyzed by MTT assay. After MCF-7 cells were treated with matrine for 48h, apoptosis was detected by flow cytometry, TUNEL assay and transmission electron microscopy, and the cell cycle distribution was also analyzed by flow cytometry. Further, the expression of PTEN, pAkt, Akt, pBad, Bad, p21/WAF1/CIP1 , and p27/KIP1 was determined by Western blot. Changes of miR-21 level were quantified by real-time RT-PCR. After miR-21 was transfected in MCF-7 cells, PTEN protein level was measured by Western blot. Results: Matrine inhibited MCF-7 cell growth in a concentration-and time-dependent manner, by inducing apoptosis and cell cycle arrest at G1/S phase. Matrine up-regulated PTEN by downregulating miR-21 which in turn dephosphorylated Akt, resulting in accumulation of Bad, p21/WAF1/CIP1 and p27/KIP1. Conclusion: Our study unraveled, for the first time, the ability of matrine to suppress breast cancer growth and elucidated the miR-21/PTEN/Akt pathway as a signaling mechanism for the anti-cancer action of matrine. Our findings also reinforce the notion that miRNAs can act as mediators of the therapeutic efficacy of natural medicines.

microRNA-124 Regulates Cardiomyocyte Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells Via Targeting STAT3 Signaling†‡§

Accumulating evidence demonstrated that bone marrow‐derived mesenchymal stem cells (BMSCs) may transdifferentiate into cardiomyocytes and replace apoptotic myocardium so as to improve functions of damaged hearts. However, little information is known about molecular mechanisms underlying myogenic conversion of BMSCs. microRNAs as endogenous noncoding small molecules function to inhibit protein translation post‐transcriptionally by binding to complementary sequences of targeted mRNAs. Here, we reported that miR‐124 was remarkably downregulated during cardiomyocyte differentiation of BMSCs induced by coculture with cardiomyocytes. Forced expression of miR‐124 led to a significant downregulation of cardiac‐specific markers—ANP, TNT, and α‐MHC proteins as well as reduction of cardiac potassium channel currents in cocultured BMSCs. On the contrary, the inhibition of endogenous miR‐124 with its antisense oligonucleotide AMO‐124 obviously reversed the changes of ANP, TNT, and α‐MHC proteins and increased cardiac potassium channel currents. Further study revealed that miR‐124 targeted the 3′UTR of STAT3 gene so as to suppress the expression of STAT3 protein but did not affect its mRNA level. STAT3 inhibitors AG490, WP1066, and S3I‐201 were shown to attenuate the augmented expression of ANP, TNT, α‐MHC, GATA‐4 proteins, and mRNAs in cocultured BMSCs with AMO‐124 transfection. Moreover, GATA‐4 siRNA reduced the expression of ANP, TNT, α‐MHC, and GATA‐4 proteins but did not impact STAT3 protein in cocultured BMSCs, indicating GATA‐4 serves as an effector of STAT3. In summary, we found that miR‐124 regulated myogenic differentiation of BMSCs via targeting STAT3 mRNA, which provides new insights into molecular mechanisms of cardiomyogenesis of BMSCs. STEM CELLS2012;30:1746–1755

The Antifibrotic Effects and Mechanisms of MicroRNA-26a Action in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and high-lethality fibrotic lung disease characterized by excessive fibroblast proliferation, extracellular matrix accumulation, and, ultimately, loss of lung function. Although dysregulation of some microRNAs (miRs) has been shown to play important roles in the pathophysiological processes of IPF, the role of miRs in fibrotic lung diseases is not well understood. In this study, we found downregulation of miR-26a in the lungs of mice with experimental pulmonary fibrosis and in IPF, which resulted in posttranscriptional derepression of connective tissue growth factor (CTGF), and induced collagen production. More importantly, inhibition of miR-26a in the lungs caused pulmonary fibrosis in vivo, whereas overexpression of miR-26a repressed transforming growth factor (TGF)-β1-induced fibrogenesis in MRC-5 cells and attenuated experimental pulmonary fibrosis in mice. Our study showed that miR-26a was downregulated by TGF-β1-mediated phosphorylation of Smad3. Moreover, miR-26a inhibited the nuclear translocation of p-Smad3 through directly targeting Smad4, which determines the nuclear translocation of p-Smad2/Smad3. Taken together, our experiments demonstrated the antifibrotic effects of miR-26a in fibrotic lung diseases and suggested a new strategy for the prevention and treatment of IPF using miR-26a. The current study also uncovered a novel positive feedback loop between miR-26a and p-Smad3, which is involved in pulmonary fibrosis.

Integrated analyses identify the involvement of microRNA-26a in epithelial-mesenchymal transition during idiopathic pulmonary fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and highly lethal fibrotic lung disease with poor treatment and unknown etiology. Emerging evidence suggests that epithelial–mesenchymal transition (EMT) has an important role in repair and scar formation following epithelial injury during pulmonary fibrosis. Although some miRNAs have been shown to be dysregulated in the pathophysiological processes of IPF, limited studies have payed attention on the participation of miRNAs in EMT in lung fibrosis. In our study, we identified and constructed a regulation network of differentially expressed IPF miRNAs and EMT genes. Additionally, we found the downregulation of miR-26a in mice with experimental pulmonary fibrosis. Further studies showed that miR-26a regulated HMGA2, which is a key factor in the process of EMT and had the maximum number of regulating miRNAs in the regulation network. More importantly, inhibition of miR-26a resulted in lung epithelial cells transforming into myofibroblasts in vitro and in vivo, whereas forced expression of miR-26a alleviated TGF-β1- and BLM-induced EMT in A549 cells and in mice, respectively. Taken together, our study deciphered the essential role of miR-26a in the pathogenesis of EMT in pulmonary fibrosis, and suggests that miR-26a may be a potential therapeutic target for IPF.

Upregulation of microRNA-1 and microRNA-133 contributes to arsenic-induced cardiac electrical remodeling.

BACKGROUND A large body of evidence showed that arsenic trioxide (As2O3), a front-line drug for the treatment of acute promyelocytic leukemia, induced abnormal cardiac QT prolongation, which hampers its clinical use. The molecular mechanisms for this cardiotoxicity remained unclear. This study aimed to elucidate whether microRNAs (miRs) participate in As2O3-induced QT prolongation. METHODS A guinea pig model of As2O3-induced QT prolongation was established by intravenous injection with As2O3. Real-time PCR and Western blot were employed to determine the expression alterations of miRs and mRNAs, and their corresponding proteins. RESULTS The QT interval and QRS complex were significantly prolonged in a dose-dependent fashion after 7-day administration of As2O3. As2O3 induced a significant upregulation of the muscle-specific miR-1 and miR-133, as well as their transactivator serum response factor. As2O3 depressed the protein levels of ether-a-go-go related gene (ERG) and Kir2.1, the K(+) channel subunits responsible for delayed rectifier K(+) current IKr and inward rectifier K(+) current IK1, respectively. In vivo transfer of miR-133 by direct intramuscular injection prolonged QTc interval and increased mortality rate, along with depression of ERG protein and IKr in guinea pig hearts. Similarly, forced expression of miR-1 widened QTc interval and QRS complex and increased mortality rate, accompanied by downregulation of Kir2.1 protein and IK1. Application of antisense inhibitors to knockdown miR-1 and miR-133 abolished the cardiac electrical disorders caused by As2O3. CONCLUSIONS Deregulation of miR-133 and miR-1 underlies As2O3-induced cardiac electrical disorders and these miRs may serve as potential therapeutic targets for the handling of As2O3 cardiotoxicity.

MicroRNA-328 as a regulator of cardiac hypertrophy

Cardiac hypertrophy is a primary predictor of progressive heart disease that often results in heart failure. Growing evidence has demonstrated that microRNAs (miRNAs) play a critical role in regulating cardiac hypertrophy. This study was designed to evaluate the effect of miR-328 on cardiac hypertrophy and the potential molecular mechanisms. We found that transgenic overexpression of miR-328 in the heart induced cardiac hypertrophy in mice, which was accompanied by reduced SERCA2a level increased intracellular calcium concentration and calcineurin protein level, and enhanced NFATc3 nuclear translocation. However, normalization of miR-328 level by its antisense chemically modified with locked nucleic acid (LNA-antimiR-328) reversed the changes. Forced expression of miR-328 resulted in cardiomyocyte hypertrophy in cultured neonatal rat ventricular cells, which was accompanied by downregulation of SERCA2a expression and activation of the calcineurin/NFATc3 signaling pathway. These changes were abolished by LNA-antimiR-328. We validated the SERCA2a as a direct target for miR-328. MiR-328 expression was upregulated in cardiomyocyte treated with isoproterenol (ISO) to induce hypertrophy; while knockdown of miR-328 attenuated the hypertrophic responses. The level of miR-328 was significantly elevated in a mouse model of hypertrophy by thoracic aortic banding (TAC). Consistently, SERCA2a was downregulated, whereas calcineurin were upregulated, and NFATc3 nuclear translocation was enhanced. In contrast, hypertrophy in these mice was significantly alleviated when treated with miR-328 antisense. MiR-328 promotes cardiac hypertrophy by targeting SERCA2a. Our study therefore uncovered a novel molecular mechanism for cardiac hypertrophy and indicated miR-328 as a potential therapeutic target for this cardiac condition.

Arsenic-induced interstitial myocardial fibrosis reveals a new insight into drug-induced long QT syndrome.

AIMS Arsenic trioxide (ATO), an effective therapeutic agent for acute promyelocytic leukaemia, can cause sudden cardiac death due to long QT syndrome (LQTS). The present study was designed to determine whether ATO could induce cardiac fibrosis and explore whether cardiac fibroblasts (CFs) are involved in the development of LQTS by ATO. METHODS AND RESULTS ATO treatment of guinea pigs caused substantial interstitial myocardial fibrosis and LQTS, which was accompanied by an increase in transforming growth factor β1(TGF-β1) secretion and a decrease in ether-à-go-go-related gene (HERG) and inward rectifying potassium channel (I(K1)) subunit Kir2.1 protein levels. ATO promoted collagen production and TGF-β1 expression and secretion in cultured CFs. Whole-cell patch clamp and western blotting showed that treatment with TGF-β1 markedly reduced HERG and I(K1) current densities and downregulated HERG and Kir2.1 protein expression in HEK293 cells stably transfected with the human recombinant HERG channel and in cardiomyocytes (CMs). These changes were completely reversed by treatment with the protein kinase A (PKA) antagonist, H89. CM and CF co-cultures showed that ATO significantly increased TGF-β1 levels in the culture medium, whereas markedly reduced HERG and Kir2.1 protein levels were observed in CMs compared with ATO-treated CMs not co-cultured with CFs. Finally, in vivo administration of LY364947, a pharmacological antagonist of TGF-β signalling, dramatically prevented interstitial fibrosis and LQTS and abolished aberrant expression of TGF-β1, HERG, and Kir2.1 in ATO-treated guinea pigs. CONCLUSION ATO-induced TGF-β1 secretion from CFs aggravates QT prolongation, suggesting that modulation of TGF-β signalling may provide a novel strategy for the treatment of drug-induced LQTS.

Calcineurin suppresses AMPK-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress.

Calcineurin signalling plays a critical role in the pathogenesis of many cardiovascular diseases. Calcineurin has been proven to affect a series of signalling pathways and to exert a proapoptotic effect in cardiomyocytes. However, whether it is able to regulate autophagy remains largely unknown. Here, we report that prolonged oxidative stress-induced activation of calcineurin contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signalling and inhibits autophagy in cardiomyocytes. Primary cardiomyocytes exhibited rapid formation of autophagosomes, microtubule-associated protein 1 light chain 3 (LC3) expression and phosphorylation of AMPK in response to hydrogen peroxide (H2O2) treatment. However, prolonged (12 h) H2O2 treatment attenuated these effects and was accompanied by a significant increase in calcineurin activity and apoptosis. Inhibition of calcineurin by FK506 restored AMPK function and LC3 expression, and decreased the extent of apoptosis caused by prolonged oxidative stress. In contrast, overexpression of the constitutively active form of calcineurin markedly attenuated the increase in LC3 induced by short-term (3 h) H2O2 treatment and sensitised cells to apoptosis. In addition, FK506 failed to induce autophagy and alleviate apoptosis in cardiomyocytes expressing a kinase-dead K45R AMPK mutant. Furthermore, inhibition of autophagy by 3-methylanine (3-MA) or by knockdown of the essential autophagy-related gene ATG7 abrogated the protective effect of FK506. These findings suggest a novel role of calcineurin in suppressing adaptive autophagy during oxidative stress by downregulating the AMPK signalling pathway. The results also provide insight into how altered calcineurin and autophagic signalling is integrated to control cell survival during oxidative stress and may guide strategies to prevent cardiac oxidative damage.

By targeting Stat3 microRNA-17-5p promotes cardiomyocyte apoptosis in response to ischemia followed by reperfusion.

Background: Several studies have confirmed the role of microRNAs in regulating ischemia/reperfusion-induced cardiac injury (I/R-I). MiR-17-5p has been regarded as an oncomiR in the development of cancer. However, its potential role in cardiomyocytes has not been exploited. The aim of this study is to investigate the role of miR-17-5p in I/R-I and the underlying mechanism through targeting Stat3, a key surviving factor in cardiomyocytes. Methods: MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay was used to detect the cell viability. ELISA and TUNEL were performed to measure apoptosis of neonatal rat ventricular cardiomyocytes (NRVCs). Infarct area was estimated by TTC (triphenyltetrazolium chloride) and Evans blue staining. Western blot analysis was employed to detect the Stat3 and p-Stat3 levels and real-time RT-PCR was used to quantify miR-17-5p level. Results: The miR-17-5p level was significantly up-regulated in I/R-I mice and in NRVCs under oxidative stress. Overexpression of miR-17-5p aggravated cardiomyocyte injury with reduced cell viability and enhanced apoptotic cell death induced by H2O2, whereas inhibition of miR-17-5p by its antisense AMO-17-5p abrogated the deleterious changes. Moreover, the locked nucleic acid-modified antisense (LNA-anti-miR-17-5p) markedly decreased the infarct area and apoptosis induced by I/R-I in mice. Furthermore, overexpression of miR-17-5p diminished the p-Stat3 level in response to H2O2. The results from Western blot analysis and luciferase reporter gene assay confirmed Stat3 as a target gene for miR-17-5p. Conclusion: Upregulation of miR-17-5p promotes apoptosis induced by oxidative stress via targeting Stat3, accounting partially for I/R-I.