摘要
目的:探讨miR-499a-5p靶向细胞周期蛋白依赖性激酶抑制剂1A(CDKN1A)抑制血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞肥大的作用机制。方法:体外培养大鼠心肌细胞H9c2,并将H9c2细胞随机分为正常对照(Con)组、细胞肥大模型(AngⅡ)组、转染对照(AngⅡ+miR-NC)组和转染(AngⅡ+miR-499a-5p)组。采用Image J软件测量单细胞表面积,实时荧光定量聚合酶链式反应(qRT-PCR)分析各组细胞中miR-499a-5p的表达水平,细胞计数试剂盒(CCK-8)检测细胞活性,流式细胞术检测细胞凋亡率,蛋白免疫印迹法(Western Blot)检测CDKN1A、cleaved Caspase-3以及心肌肥大标志蛋白心钠肽(ANP)、脑钠肽(BNP)和β-肌球蛋白重链(β-MHC)的表达水平,双荧光素酶报告基因实验验证miR-499a-5p和CDKN1A的靶向作用关系。结果:与Con组比较,AngⅡ组H9c2细胞表面积、凋亡率、CDKN1A、Cleaved Caspase-3、ANP、BNP和β-MHC蛋白表达水平明显升高(P<0.05),而细胞活力和miR-499a-5p蛋白表达水平明显降低(P<0.05)。双荧光素酶报告基因实验证实,CDKN1A是miR-499a-5p的靶基因。与AngⅡ组和AngⅡ+miR-NC组比较,AngⅡ+miR-499a-5p组H9c2细胞的表面积、凋亡率、CDKN1A、Cleaved Caspase-3、ANP、BNP和β-MHC的蛋白表达水平明显降低(P<0.05),而细胞活力和miR-499a-5p的表达水平明显升高(P<0.05)。结论:AngⅡ诱导的H9c2细胞中miR-499a-5p的表达明显降低,过表达miR-499a-5p能够减轻AngⅡ诱导的心肌细胞肥大,其作用机制可能与其靶向调控CDKN1A基因的表达有关。
Objective:To explore the mechanism of miR-499a-5p targeting cyclin-dependent kinase inhibitor 1A(CDKN1A)in inhibiting angiotensinⅡ(AngⅡ)induced cardiomyocyte hypertrophy.Methods:Rat cardiomyocytes H9c2 were cultured in vitro and randomly divided into normal control group(Con),cell hypertrophy model(AngⅡ)group,transfection control(AngⅡ+miR-NC)group and transfection(AngⅡ+miR-499a-5p)group.The surface area of single cells was measured by Image J software.The expression level of miR-499a-5p in each group was analyzed by real-time quantitative fluorescent polymerase chain reaction(qRT-PCR).The cell activity was detected by CCK-8.The cell apoptosis rate was detected by flow cytometry.Expression levels of CDKN1A,cleaved Caspase-3,cardiac natriuretic peptide(ANP),brain natriuretic peptide(BNP),andβ-myosin heavy chain(β-MHC),were detected by Western Blot.Dual luciferase reporter assay verified the targeting relationship between miR-499a-5p and CDKN1A.Results:Compared with the Con group,the H9c2 cell surface area,apoptosis rate,CDKN1A,cleaved Caspase-3,ANP,BNP,andβ-MHC expression levels in AngⅡgroup were significantly increased(P<0.05),while the cell activity and miR-499a-5p expression levels were significantly decreased(P<0.05).Double luciferase reporter assay confirmed that CDKN1A was the target gene of miR-499a-5p.Compared with AngⅡgroup and AngⅡ+miR-NC group,H9c2 cell surface area,apoptosis rate,CDKN1A,cleaved Caspase-3,ANP,BNP,andβ-MHC expression levels in AngⅡ+miR-499a-5p group were significantly decreased(P<0.05).The cell activity and the expression level of miR-499a-5p were significantly increased(P<0.05).Conclusion:The expression of miR-499a-5p in H9c2 cells induced by AngⅡwas significantly decreased,and overexpression of miR-499a-5p could reduce the cardiomyocyte hypertrophy induced by AngⅡ,which may be related to its targeted regulation of CDKN1A expression.
作者
牟寒霜
黄震
郭婧
MOU Hanshuang;HUANG Zhen;GUO Jing(Baoji Central Hospital,Baoji 721000,Shaanxi,China)
出处
《中西医结合心脑血管病杂志》
2023年第7期1218-1223,共6页
Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease
