microRNA-423-5p调节PI3K/AKT通路在大鼠心力衰竭进展中的作用探究
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摘要
目的探究microRNA-423-5p(miR-423-5p)调节磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/AKT)通路在心力衰竭进展中的作用。方法构建大鼠心力衰竭模型,同时用AngⅡ处理(100 n M,24 h)大鼠心肌细胞H9C2构建心力衰竭体外模型。H9C2细胞转染miR-423-5p的抑制剂和模拟剂实现miR-423-5p的敲除和过表达;LY294002用来抑制PI3K/AKT通路的激活。RT-PCR技术检测miR-423-5p的表达; Western blot检测凋亡相关蛋白Bcl-2、Bax、casapse-3/9及PI3K、AKT总蛋白及磷酸化蛋白的表达;流式细胞术检测细胞凋亡。结果 miR-423-5p的表达水平在模型组大鼠心肌组织和AngⅡ处理的H9C2细胞中升高;敲除miR-423-5p可抑制由AngⅡ造成的H9C2细胞凋亡,并促进了p-PI3K及p-AKT的表达,但敲除miR-423-5p的以上作用在PI3K/AKT通路被抑制后全部削弱。结论敲除miR-423-5p可通过激活PI3K/AKT通路抑制心肌细胞凋亡,进而缓解心力衰竭的恶性进展。
Objective To investigate the effect and mechanism of microRNA-423-5 p( miR-423-5 p) in heart failure through regulation of the PI3 K/AKT signaling pathway. Methods Heart failure models were constructed in vivo with Rat and in vitro through treatment rat cardiac myocyte H9 C2 cells with AngⅡ( 100 nM,24 h). MiR-423-5 p-inhibitors and miR-423-5 p-mimics were used to down-regulate and up-regulate miR-423-5 p expression in H9 C2 cells via cell transfection; LY294002,an inhibitor of PI3 K/AKT signaling pathway was used to repress the activation of PI3 K/AKT signaling. RT-PCR was used to detect miR-423-5 p expression; western blot was performed to analyze the protein levels of apoptosis-related proteins such as Bcl-2,Bax,caspase-3/9 and the levels of total and phosphorylated PI3 K and AKT protein; flow cytometry was used to test cell apoptosis. Results The level of miR-423-5 p was elevated in model rat myocyte tissues and H9 C2 cells treated with AngⅡ. Knockdown of miR-423-5 p inhibited the apoptosis of H9 C2 cells induced by AngⅡ treatment and promoted the expression of p-PI3 K and p-AKT,whereas the above effects of miR-423-5 p down-regulation were all weakened after the inhibition of PI3 K/AKT pathway. Conclusion Knockdown of miR-423-4 p inhibits myocardial cell apoptosis through activating PI3 K/AKT signaling,repressing the progression of heart failure.
Objective To investigate the effect and mechanism of microRNA-423-5 p( miR-423-5 p) in heart failure through regulation of the PI3 K/AKT signaling pathway. Methods Heart failure models were constructed in vivo with Rat and in vitro through treatment rat cardiac myocyte H9 C2 cells with AngⅡ( 100 nM,24 h). MiR-423-5 p-inhibitors and miR-423-5 p-mimics were used to down-regulate and up-regulate miR-423-5 p expression in H9 C2 cells via cell transfection; LY294002,an inhibitor of PI3 K/AKT signaling pathway was used to repress the activation of PI3 K/AKT signaling. RT-PCR was used to detect miR-423-5 p expression; western blot was performed to analyze the protein levels of apoptosis-related proteins such as Bcl-2,Bax,caspase-3/9 and the levels of total and phosphorylated PI3 K and AKT protein; flow cytometry was used to test cell apoptosis. Results The level of miR-423-5 p was elevated in model rat myocyte tissues and H9 C2 cells treated with AngⅡ. Knockdown of miR-423-5 p inhibited the apoptosis of H9 C2 cells induced by AngⅡ treatment and promoted the expression of p-PI3 K and p-AKT,whereas the above effects of miR-423-5 p down-regulation were all weakened after the inhibition of PI3 K/AKT pathway. Conclusion Knockdown of miR-423-4 p inhibits myocardial cell apoptosis through activating PI3 K/AKT signaling,repressing the progression of heart failure.
引文
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[12]Wencker D,Chandra M,Nguyen K,et al.A mechanistic role for cardiac myocyte apoptosis in heart failure[J].J Clin Invest,2003,111(10):1497-1503.
[13]魏玲,李慧萍,魏伟,等.慢性心衰大鼠心肌细胞凋亡、心肌纤维化及其相关性研究[J].西南国防医药,2018,28(1):35-38.
[14]Zhao H,Qi G,Han Y,et al.20-Hydroxyeicosatetraenoic Acid Is a Key Mediator of Angiotensin II-induced Apoptosis in Cardiac Myocytes[J].J Cardiovasc Pharmacol,2015,66(1):86-95.
[15]范亮亮,马立宁,彭元亮,等.PI3K/AKT信号通路与心力衰竭[J].生命科学研究,2015,19(1):85-90.
[16]Chen BC,Shibu MA,Kuo CH,et al.E4BP4 inhibits AngⅡ-induced apoptosis in H9c2 cardiomyoblasts by activating the PI3K-Akt pathway and promoting calcium uptake[J].Exp Cell Res,2018,363(2):227-234.
[2]秦晓毅,卢新政.2010年NICE慢性心力衰竭诊治指南更新的解读[J].心血管病学进展,2011,32(4):490-492.
[3]Watson CJ,Gupta SK,O'Connell E,et al.MicroRNA signatures differentiate preserved from reduced ejection fraction heart failure[J].Eur JHeart Fail,2015,17(4):405-415.
[4]Li H,Fan J,Yin Z,et al.Identification of cardiac-related circulating microRNA profile in human chronic heart failure[J].Oncotarget,2016,7(1):33-45.
[5]Tijsen AJ,Creemers EE,Moerland PD,et al.MiR423-5p As a Circulating Biomarker for Heart Failure[J].Circ Res,2010,106(6):1035-1039.
[6]王仁俊,温美玲,未晓巍,等.MicroRNA-214在慢性心衰大鼠心肌细胞钙调控中的作用与机制研究[J].吉林师范大学学报(自然科学版),2016,37(1):112-118.
[7]尚佳.MicroRNA-10a-5p在大鼠心衰发展中的作用和机制研究[D].银川:宁夏医科大学,2017.
[8]杨阳,罗义,黄建楷,等.心力衰竭患者循环miR-423-5p的表达及其与左室肥厚的相关性[J].中国循环杂志,2014,29(z1):18-19.
[9]姜蕴珊,谈红,李晓燕,等.培哚普利对慢性心力衰竭患者血浆miR-423-5p的调控及对心功能的影响[J].山东大学学报(医学版),2016,54(8):55-59.
[10]陈立强,韩曦,赵勇,等.GHRP-6对心衰模型大鼠心肌细胞PI3K/Akt/Caspase-9信号通路调节作用[J].中国民康医学,2015,27(14):120.
[11]Abbate A,Bussani R,Amin MS,et al.Acute myocardial infarction and heart failure:role of apoptosis[J].Int J Biochem Cell Biol,2006,38(11):1834-1840.
[12]Wencker D,Chandra M,Nguyen K,et al.A mechanistic role for cardiac myocyte apoptosis in heart failure[J].J Clin Invest,2003,111(10):1497-1503.
[13]魏玲,李慧萍,魏伟,等.慢性心衰大鼠心肌细胞凋亡、心肌纤维化及其相关性研究[J].西南国防医药,2018,28(1):35-38.
[14]Zhao H,Qi G,Han Y,et al.20-Hydroxyeicosatetraenoic Acid Is a Key Mediator of Angiotensin II-induced Apoptosis in Cardiac Myocytes[J].J Cardiovasc Pharmacol,2015,66(1):86-95.
[15]范亮亮,马立宁,彭元亮,等.PI3K/AKT信号通路与心力衰竭[J].生命科学研究,2015,19(1):85-90.
[16]Chen BC,Shibu MA,Kuo CH,et al.E4BP4 inhibits AngⅡ-induced apoptosis in H9c2 cardiomyoblasts by activating the PI3K-Akt pathway and promoting calcium uptake[J].Exp Cell Res,2018,363(2):227-234.