摘要
目的探讨miR-30a-5p通过调控组蛋白去乙酰化酶9(HDAC9)对缺氧复氧诱导的心肌细胞凋亡、氧化应激的影响及作用机制。方法本研究通过体外培养心肌细胞H9c2,建立缺氧复氧损伤细胞模型,细胞分组为:Control组、H/R组、H/R+miR-NC组、H/R+miR-30a-5p组、H/R+si-NC组、H/R+si-HDAC9组、H/R+miR-30a-5p+pcDNA组、H/R+miR-30a-5p+HDAC9组。qRT-PCR检测miR-30a-5p和HDAC9 mRNA表达;Western blot检测HDAC9、Bax和Bcl-2蛋白表达;流式细胞术检测细胞凋亡;ELISA检测超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量和乳酸脱氢酶(LDH)含量;双荧光素酶报告基因检测miR-30a-5p和HDAC9靶向关系。结果与Control组比较,H/R组的心肌细胞中miR-30a-5p表达、Bcl-2蛋白表达和SOD活性均显著降低(P<0.05),而细胞凋亡率、Bax蛋白表达、MDA含量、LDH含量HDAC9 mRNA和蛋白表达水平均显著增加(P<0.05);与H/R+miR-NC组比较,H/R+miR-30a-5p组的心肌细胞中miR-30a-5p表达、Bcl-2蛋白表达和SOD活性均显著增加(P<0.05)。与miR-NC组比较,miR-30a-5p组中HDAC93’UTR-wt荧光素酶活性显著降低(P<0.05),而HDAC93’UTR-mut荧光素酶活性没有任何变化。与anti-miR-N组比较,anti-miR-30a-5p组中miR-30a-5p表达显著降低(P<0.05)。与miR-NC组比较,miR-30a-5p组中HDAC9蛋白表达显著降低(P<0.05);与anti-miR-N组比较,anti-miR-30a-5p组中HDAC9蛋白表达显著增加(P<0.05)。与Control组比较,H/R组的心肌细胞中HDAC9蛋白表达、细胞凋亡率、Bax蛋白表达、MDA含量和LDH含量均显著增加(P<0.05);而Bcl-2蛋白表达和SOD活性均显著降低(P<0.05);与H/R+si-NC组比较,H/R+si-HDAC9组的心肌细胞中HDAC9蛋白表达、细胞凋亡率、Bax蛋白表达、MDA含量和LDH含量均显著降低(P<0.05),而Bcl-2蛋白表达和SOD活性均显著增加(P<0.05)。与H/R+miR-NC组比较,H/R+miR-30a-5p组的心肌细胞中HDAC9蛋白表达、细胞凋亡率、Bax蛋白表达、MDA含量和LDH含量均显著降低(P<0.05),而Bcl-2蛋白表达和SOD活性�
Objective To investigate the protective effects of miR-30a-5p on the oxidative stress injury of cardiomyocytes by regulating HDAC9 and its action mechanism.Methods The cardiomyocytes(H9c2)were cultured in vitro,and the hypoxia-reoxygenation(H/R)injury cell model was established.The experiment was divided into control group,H/R group,H/R+miR-NC group,H/R+miR-30a-5p group,H/R+si-NC group,H/R+si-HDAC9 group,H/R+miR-30a-5p+pcDNA group,H/R+miR-30a-5p+HDAC9 group.The expression levels of miR-30a-5p and HDAC9 mRNA were detected by qRT-PCR,and those of HDAC9,Bax and Bcl-2 protein were measured by Westen Blot,and cell apoptosis was detected by flow cytometry,and SOD activity and the levels of MDA and LDH were detected by ELISA.Moreover the dual luciferase reporter gene assay was used to analyze the targeting correlation between miR-30a-5p and HDAC9.Results Compared with control group,the expression of miR-30a-5p,Bcl-2 protein and SOD activity in cardiomyocytes of H/R group were significantly decreased(P<0.05),while the apoptosis rate,Bax protein expression,MDA content,LDH content and HDAC9 mRNA and protein expression were significantly increased(P<0.05);Compared with H/R+miR-NC group,the expression of miR-30a-5p,Bcl-2 protein expression and SOD activity in cardiomyocytes of H/R+miR-30a-5p group were significantly increased(P<0.05).Compared with mir-NC group,HDAC93’UTR-wt luciferase activity decreased significantly in miR-30a-5p group(P<0.05),while HDAC93’UTR-mut luciferase activity did not change.Compared with anti-miR-N group,the expression of miR-30a-5p in anti-miR-30a-5p group decreased significantly(P<0.05).Compared with miR-NC group,the expression of HDAC9 protein in miR-30a-5p group decreased significantly(P<0.05);Compared with anti-miR-n group,the expression of HDAC9 protein in anti-miR-30a-5p group was significantly increased(P<0.05).Compared with control group,HDAC9 protein expression,apoptosis rate,Bax protein expression,MDA content and LDH content in cardiomyocytes of H/R group were significantly increased(
作者
余敏
崔跃
黎鹏飞
谢丹
陈芬
YU Min;CUI Yue;LI Pengfei(Department of Cardiology,Wuhan Red Cross Hospital,Hubei,Wuhan 430015,China)
出处
《河北医药》
CAS
2022年第8期1125-1129,1135,共6页
Hebei Medical Journal
基金
国家自然科学基金项目(编号:81300196)。
