摘要
目的:探讨microRNA-181a(miR-181a)对缺氧/复氧诱导下大鼠心肌细胞株H9c2凋亡的作用。方法:通过生物信息学软件筛选出可调控Bcl-2表达的miRNAs。建立H9c2细胞缺氧/复氧模型,通过Western blot检测Bcl-2蛋白表达变化,荧光定量PCR检测miR-181a表达变化。进一步构建Bcl-2的荧光素酶报告载体,双荧光素酶报告基因系统检测荧光素酶的表达变化。采用体外合成的大鼠miR-181a的模拟物(mimic)和抑制剂(inhibitor),用脂质体转染培养的H9c2细胞48h后,缺氧/复氧处理细胞,实验分为4组:正常对照组(CTL)、缺氧/复氧组(H/R)、缺氧复氧加miR-181a的模拟物组(mimic)、缺氧复氧加miR-181a的抑制剂组(inhibitor)。TUNEL法检测各组细胞凋亡;Western blot分析各组凋亡关键蛋白cleaved caspase-3的表达量的变化。结果:生物学软件预测miR-181a可调控Bcl-2蛋白的表达,在缺氧/复氧模型中Bcl-2蛋白的表达明显下调,而miR-181a的表达上升近4倍。成功构建双荧光酶素报告载体以及突变体,实验组的报告荧光比对照组明显下降,而突变体的荧光强度下降不明显。与H/R组相比,inhibitor组cleaved caspase-3蛋白、凋亡率下降,mimic组却拮抗了上述变化。结论:miR-181a能够增加氧化应激条件下H9c2心肌细胞损伤,促进H9c2心肌细胞凋亡,miR-181a通过调控Bcl-2蛋白的表达,在H/R介导的凋亡途径中扮演了关键性的作用。
Objective：To investigate the effects of microRNA-181a（miR-181a）on hypoxia/reoxygenation（H/R）-induced apoptosis in H9c2 cardiomyocytes in order to understand the molecular mechanisms.Method：Current bioinformatics were performed to select miRNAs for possible interactions with Bcl-2.The expression changes of miR-181 aand Bcl-2after H/R treatment were determined by real-time PCR and Western blot,respectively.Dual luciferase assay combined with mutation and immunoblotting was used to screen and verify the bioinformatically predicted miRNAs.H9c2 cells were transfected with miR-181 amimics or miR-181 ainhibitor and cultured 48 h.After H/R treatment,H9c2 cells were divided into the following 4groups：control,H/R,mimic,inhibitor groups.The changes of apoptosis in H9c2 cells were quantitatively assayed by TUNEL method.The expression of cleaved caspase-3in H9c2 cells were detected by Western blotting.Reslut：miR-181 awas predicted as negative regulator of Bcl-2by commonly utilized miR target prediction algorithms.After H/R treatment,miR-181 aexpression were increased compared with normal group,whereas Bcl-2protein expressions decreased（P〈0.05）.Compared with H/R group,the apoptosis rates and cleaved caspase-3expression were significantly decreased（P〈0.05）.However,upregulation of miR-181 aexacerbated these changes.Conclusion：The inhibition of miR-181 aconfers cardiac protection against oxidative stress-induced H9c2 cell apoptosis through the direct target Bcl-2expression.
出处
《临床心血管病杂志》
CAS
CSCD
北大核心
2015年第6期664-669,共6页
Journal of Clinical Cardiology
作者简介
通信作者：马建群，E-mail：mjianqun@163．com
