MicroRNA-133b-5p通过靶向调控Fas基因影响H9c2心肌细胞凋亡  

Effects of microRNA-133b-5p on cell apoptosis by targeting Fas gene in myocardial H9c2 cells

作　　者：程洁[1] 何淑芳[1] 韩正怡 朱海娟[1] 张野[1] 

机构地区：[1]安徽医科大学第二附属医院麻醉科,合肥230601

出　　处：《安徽医科大学学报》2016年第2期189-194,共6页Acta Universitatis Medicinalis Anhui

基　　金：安徽高校省级自然科学研究重大项目(编号:KJ2014ZD16);安徽省科技厅年度重点项目(编号:1301043030)

摘　　要：目的研究microRNA(miR)-133b-5p在H9c2心肌细胞凋亡中发挥的作用及其靶向调控Fas基因的机制。方法取对数生长期大鼠H9c2胚胎心肌细胞,分为空白对照组、miR-133b-5p抑制剂组、抑制剂阴性对照组。分别转染48 h后收集细胞,实时荧光定量PCR(qRT-PCR)检测miR-133b-5p表达,微板法检测细胞培养液中乳酸脱氢酶(LDH)活性,Annexin V-FITC联合PI双染流式细胞术检测细胞凋亡。双荧光素酶报告基因系统验证miR-133b-5p与Fas mRNA的3'UTR区的靶向结合。最后运用qRT-PCR和Westernblot分别检测各组Fas mRNA与Fas蛋白表达水平。结果miR-133b-5p抑制剂组细胞内miR-133b-5p表达水平下调至空白对照组的33%(P<0.05);LDH活性升高,细胞凋亡率增加,均明显高于空白对照组与抑制剂阴性对照组(P<0.05)。双荧光素酶报告基因证明Fas是miR-133b-5p的一个靶基因;miR-133b-5p inhibitor显著上调Fas mRNA和Fas蛋白水平(P<0.05)。结论利用化学合成miR-133b-5p inhibitor抑制大鼠H9c2心肌细胞中miR-133b-5p表达,可诱导心肌细胞损伤和凋亡,其机制可能与调控靶基因Fas表达有关。Objective To investigate the effects of miR-133b-5p on cell apoptosis in H9c2 cells and the mechanisms of which targeting Fas gene. Methods H9c2 cells in logarithmic phase were divided into the CON group, the miR-133b-5p inhibitor group, and the miR inhibitor-NC group. The cells in different groups were collected after 48 h transfection to examine miR-133b-5p level by qRT-PCR. The activity of lactate dehydrogenase（LDH） in the culture medium was measured by colorimetric kit. Cell apoptosis was determined by flow cytometry after Annexin V/PI double staining. Dual luciferase reporter assay was performed to confirm the direct regulation of miR-133b-5p on the 3′UTR of target gene Fas. Finally, the levels of Fas mRNA and protein were detected by qRT-PCR and Western blot, respectively. Results The level of miR-133b-5p in miR-133b-5p inhibitor group was reduced to 33% as compared with the CON group （P 〈 0. 05）. Inhibition of miR-133b-5p led to elevated LDH activity and increased apoptosis rate as compared with the CON group or miR inhibitor-NC group （P 〈 0. 05 ）. Dual luciferase reporter assay results confirmed that Fas was a target gene of miR-133b-5p. In addition, miR-133b-5p inhibitor obviously up-regulated the levels of Fas mRNA and Fas protein （P 〈 0. 05 ）. Conclusion The chemically synthesized miR-133b-5p inhibitor could effectively inhibit miR-133b-5p expression in H9c2 cells, leading to cell injury and apoptosis, and its mechanism might involve the regulation on Fas expression.

关 键 词：心肌细胞 微小RNAS 乳酸脱氢酶 凋亡 FAS 

分 类 号：R363[医药卫生—病理学]