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目的:研究微小RNA-34a(microRNA-34a,miR-34a)在阿霉素诱导的心肌细胞凋亡中的作用及其作用靶基因。方法:建立阿霉素(doxorubicin,Dox)诱导的大鼠H9c2心肌细胞凋亡模型;TUNEL染色观察H9c2细胞凋亡;双萤光素酶报告实验检测miR-34a与潜在靶基因沉默信息调节因子1(silent information regulator 1,SIRT1)3’端非翻译区(3’-untranslated region,3’UTR)的结合作用;实时荧光定量PCR检测miR-34a和SIRT1 mRNA表达水平,Western blot检测SIRT1和凋亡相关蛋白表达水平。结果:阿霉素处理H9c2细胞之后,细胞发生凋亡,miR-34a的表达显著增强;双萤光素酶报告实验提示miR-34a与SIRT1 3’UTR可相互作用,并证实miR-34a可在转录后水平抑制SIRT1的表达,SIRT1蛋白水平在阿霉素处理的心肌细胞中显著下调;过表达miR-34a及沉默SIRT1均能一致性抑制Bcl-2表达,促进Bax和p66shc的表达,而过表达SIRT1能有效抑制阿霉素诱导的H9c2细胞凋亡。结论:SIRT1是miR-34a的靶基因,并介导了miR-34a在阿霉素诱导的心肌细胞凋亡中的作用。
AIM: To investigate the role of microRNA-34a (miR-34a) in doxorubicin-induced cardiomyocyte apoptosis and its target genes. Methods: Apoptosis model of rat H9c2 cardiomyocytes induced by doxorubicin (Dox) was established. TUNEL staining was used to observe the apoptosis of H9c2 cells. Dual luciferase reporter assay was used to detect miR-34a and potential target gene silencing information regulators 1 (3’-untranslated region, 3’UTR) of silent information regulator 1 (SIRT1). The expression of miR-34a and SIRT1 mRNA were detected by real-time fluorescence quantitative PCR. The levels of SIRT1 and apoptosis Related protein expression levels. Results: Adriamycin treatment of H9c2 cells, the cell apoptosis, miR-34a expression was significantly enhanced; dual luciferase reporter experiments suggest that miR-34a and SIRT1 3’UTR interaction and confirmed miR-34a in The post-transcriptional level inhibited the expression of SIRT1 and the SIRT1 protein level was significantly down-regulated in doxorubicin-treated cardiomyocytes. Overexpression of miR-34a and silencing SIRT1 both inhibited the expression of Bcl-2 and promoted the expression of Bax and p66shc SIRT1 expression can effectively inhibit doxorubicin-induced apoptosis in H9c2 cells. Conclusion: SIRT1 is the target gene of miR-34a and mediates the role of miR-34a in doxorubicin-induced cardiomyocyte apoptosis.