miR-522-3p通过靶向结合PTEN促进心肌肥厚
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摘要
目的:探讨miR-522-3p对心肌肥厚的影响及机制。方法:运用生物信息学分析GEO数据库中心力衰竭病人和正常对照人中miRNA的表达差异,采用实时定量聚合酶连反应(Real-time PCR)检测主动脉弓缩窄模型(TAC)小鼠和假手术(sham)小鼠心肌组织中差异表达miRNA miR-522-3p和磷酸酶及张力蛋白同源基因(PTEN)的mRNA表达;选取HEK293T细胞分别转染(miR-522-3p)类似物(miR-522-3p mimic)及类似物对照剂(mimic control)48 h,然后进行荧光报告基因实验;选取H9C2细胞分别转染miR-522-3p类似物(miR-522-3p mimic)及类似物对照剂(mimic control)48 h,利用Real-time PCR与Western blot法检测PTEN-AKT-mTOR通路相关基因的表达;将H9C2细胞分为对照组(不做处理)、AngII组(加用AngII干预细胞24 h)、miR-522-3p mimic+AngII组(转染miR-522-3p mimic 24 h后,加用AngII干预24 h)及mimic control+AngII组(转染mimic control 24 h后,加用AngII干预24 h),利用Real-time PCR与Western blot法检测心房利钠肽(ANP)、脑利钠肽(BNP)、β型肌球蛋白重链(βMHC)的表达。结果:芯片数据显示(GSE61741),在缺血性心力衰竭和非缺血性心力衰竭病人中,血液循环miR-522-3p表达明显增加,差异有统计学意义(P<0.0001);TAC小鼠中,miR-522-3p表达升高,PTEN表达下降,miR-522-3p的表达与PTEN负相关,差异有统计学意义(P<0.01);MiR-522-3p可调控PTEN/AKT-mTOR通路,过表达miR-522-3p明显使ANP、BNP、βMHC表达升高,差异有统计学意义(P<0.05)。结论:上调miR-522-3p可通过靶向结合PTEN使心肌肥厚加重,为治疗心力衰竭提供了一个新靶标。
Objective: To investigate the effect of miR-522-3 p on cardiac hypertrophy and its action mode. Methods: Differential miRNA expression between patients with heart failure and normal controls from GEO database was analyzed using bioinformatics methods. Real-time quantitative polymerase chain reaction(qPCR) was used to detect expression levels of miR-522-3 p and phosphatase and tensin homolog(PTEN). HEK293 T cells were transfected with miR-522-3 p analog(miR-522-3 p mimic) and mimic control for 48 h, followed by fluorescent reporter gene experiments; H9 C2 cells were transfected with miR-522-3 p analog(miR-522-3 p mimic) and analog control(mimic control) for 48 h, and qPCR and Western blot were used to detect the expression of PTEN-AKT-mTOR pathway-related genes. H9 C2 cells were divided into control group(no treatment), AngII group(with AngII intervention for 24 h), miR-522-3 p mimic+AngII group(after transfection of miR-522-3 p mimic for 24 h, plus AngII for 24 h) and mimic control+AngII group(after 24 h transfection with mimic control, add AngII for 24 h). The expression of atrial natriuretic peptide(ANP), brain natriuretic peptide(BNP) and β-type myosin heavy chain(βMHC) were measured using qPCR and Western blot. Results: The analysis of the chip data(GSE61741) showed that the expression of miR-522-3 p in blood circulation was significantly increased in patients with ischemic heart failure and non-ischemic heart failure(P<0.000 1). In TAC mice, the expression of miR-522-3 p was increased, but PTEN expression was decreased. In addition, the expression levels of miR-522-3 p were negatively correlated with PTEN expression levels(P<0.01). miR-522-3 p could regulate PTEN/AKT-mTOR. Pathway. Overexpression of miR-522-3 p significantly increased the expression of ANP, BNP and βMHC(P<0.05). Conclusion: Up-regulation of miR-522-3 p can increase cardiac hypertrophy by targeting PTEN. Our study provides a new target for the treatment of heart failure.
Objective: To investigate the effect of miR-522-3 p on cardiac hypertrophy and its action mode. Methods: Differential miRNA expression between patients with heart failure and normal controls from GEO database was analyzed using bioinformatics methods. Real-time quantitative polymerase chain reaction(qPCR) was used to detect expression levels of miR-522-3 p and phosphatase and tensin homolog(PTEN). HEK293 T cells were transfected with miR-522-3 p analog(miR-522-3 p mimic) and mimic control for 48 h, followed by fluorescent reporter gene experiments; H9 C2 cells were transfected with miR-522-3 p analog(miR-522-3 p mimic) and analog control(mimic control) for 48 h, and qPCR and Western blot were used to detect the expression of PTEN-AKT-mTOR pathway-related genes. H9 C2 cells were divided into control group(no treatment), AngII group(with AngII intervention for 24 h), miR-522-3 p mimic+AngII group(after transfection of miR-522-3 p mimic for 24 h, plus AngII for 24 h) and mimic control+AngII group(after 24 h transfection with mimic control, add AngII for 24 h). The expression of atrial natriuretic peptide(ANP), brain natriuretic peptide(BNP) and β-type myosin heavy chain(βMHC) were measured using qPCR and Western blot. Results: The analysis of the chip data(GSE61741) showed that the expression of miR-522-3 p in blood circulation was significantly increased in patients with ischemic heart failure and non-ischemic heart failure(P<0.000 1). In TAC mice, the expression of miR-522-3 p was increased, but PTEN expression was decreased. In addition, the expression levels of miR-522-3 p were negatively correlated with PTEN expression levels(P<0.01). miR-522-3 p could regulate PTEN/AKT-mTOR. Pathway. Overexpression of miR-522-3 p significantly increased the expression of ANP, BNP and βMHC(P<0.05). Conclusion: Up-regulation of miR-522-3 p can increase cardiac hypertrophy by targeting PTEN. Our study provides a new target for the treatment of heart failure.
引文
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[20]OUDIT G Y,PENNINGER J M.Cardiac regulation by phosphoinositide 3-kinases and PTEN [J].Cardiovasc Res,2009,82(2):250-260.
[21]MAILLET M,VAN BERLO J H,MOLKENTIN J D.Molecular basis of physiological heart growth:fundamental concepts and new players [J].Nat Rev Mol Cell Biol,2013,14(1):38-48.
[2] HEINEKE,KEMPF T,KRAFT T,et al.Downregulation of cytoskeletal muscle LIM protein by nitric oxide:impact on cardiac myocyte hypertrophy [J].Circulation,2003,107(10):1424-1432.
[3] WILCOX J E,FONAROW G C,ARDEHALI H,et al.“Targeting the heart” in heart failure:myocardial recovery in heart failure with reduced ejection fraction [J].JACC Heart Fail,2015,3(9):661-669.
[4] JESSUP M,BROZENA S.Heart failure [J].N Engl J Med,2003,348(20):2007-2018.
[5] LAI E C.Micro RNAs are complementary to 3’UTR sequence motifs that mediate negative post-transcriptional regulation [J].Nat Genet,2002,30(4):363-364.
[6] BARTEL DP.MicroRNAs:genomics,biogenesis,mechanism,and function [J].Cell,2004,116(2):281-297.
[7] THUM T,GROSS C,FIEDLER J,et al.MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts [J].Nature,2008 456(7224):980-984.
[8] LI R C,TAO J,GUO Y B,et al.In vivo suppression of microRNA-24 prevents the transition toward decompensated hypertrophy in aortic-constricted mice [J].Circ Res,2013,112(4):601-605.
[9] YANCY C W,JESSUP M,BOZKURT B,et al.2013 ACCF/AHA guideline for the management of heart failure:Executive summary:A report of the American college of cardiology foundation/american heart association task force on practice guidelines [J].Circulation,2013,128(16):1810-1852.
[10]WANG B,NIE J,WU J,et al.AMPKα2 protects against the development of heart failure by enhancing mitophagy via PINK1 phosphorylation [J].Circ Res,2018,122(5):712-729.
[11]DRUMMOND GA,SQUIRE IB.The cardiac insufficiency bisoprolol study II [J].Lancet,1999,353(9161):1361.
[12]SHARPE N.Benefit of beta-blockers for heart failure:proven in 1999 [J].Lancet,1999,353(9169):1988-1989.
[13]PACKER M,COATS A J,FOWLER M B,et al.Effect of carvedilol on survival in severe chronic heart failure [J].N Engl J Med,2001,344(22):1651-1658.
[14]PONIKOWSKI P,ANKER S D,ALHABIB K F,et al.Heart failure:preventing disease and death worldwide[J].ESC Heart Failure,2014,1(1):4-25.
[15]MCMURRAY J V,PACKER M,DESAI A S,et al.Angiotensin-neprilysin inhibition versus enalapril in heart failure[J].N Engl J Med,2014,371(11):993-1004.
[16]RICHARDSON K,LOUIE-GAO Q,ARNETT D K,et al.The PLIN4 variant rs8887 modulates obesity related phenotypes in humans through creation of a novel miR-522 seed site [J].PLoS One,2011,6(4):e17944.
[17]ZHANG S,ZHANG H,ZHU J,et al.MiR-522 contributes to cell proliferation of human glioblastoma cells by suppressing PHLPP1 expression [J].Biomed Pharmacother,2015,70:164-169.
[18]ZHANG T,HU Y,JU J,et al.Downregulation of miR-522 suppresses proliferation and metastasis of non-small cell lung cancer cells by directly targeting DENN/MADD domain containing 2D [J].Sci Rep,2016,6:19346.
[19]SHI Y H,QI B B,LIU X B,et al.Upregulation of miR-522 is associated with poor outcome of hepatocellular carcinoma [J].Eur Rev Med Pharmacol Sci,2016,20(15):3194-3198.
[20]OUDIT G Y,PENNINGER J M.Cardiac regulation by phosphoinositide 3-kinases and PTEN [J].Cardiovasc Res,2009,82(2):250-260.
[21]MAILLET M,VAN BERLO J H,MOLKENTIN J D.Molecular basis of physiological heart growth:fundamental concepts and new players [J].Nat Rev Mol Cell Biol,2013,14(1):38-48.