miR-106a-5p靶向GAB1影响人心肌细胞增殖和凋亡的机制研究  

Mechanism of miR-106a-5p Affecting the Proliferation and Apoptosis of Human Cardiomyoblast Cells via Targeting GAB1

作　　者：张曼 王鹏 ZHANG Man;WANG Peng(Department of Intensive Care Unit,Wuhan Red Cross Hospital,Wuhan,430015,China;Department of Pharmacy,Wuhan Sixth Hospital,Wuhan,430019,China)

机构地区：[1]武汉市红十字会医院重症医学科,武汉市430015 [2]武汉市第六医院药剂科,武汉市430019

出　　处：《医学分子生物学杂志》2021年第4期280-285,共6页Journal of Medical Molecular Biology

摘　　要：目的探究心力衰竭患者血浆中miR-106a-5p的表达水平及其影响人心肌细胞增殖和凋亡的潜在机制.方法利用RT-qPCR检测45例心力衰竭患者和健康志愿者血浆中miR-106a-5p的表达水平;利用Lipofectamine 2000转染miR-106a-5p模拟物及模拟物对照至人心肌细胞系,RT-qPCR验证转染效率后,分别使用CCK-8和流式细胞术检测各组转染细胞的增殖和凋亡水平;生物信息学结合双荧光素酶报告基因实验验证miR-106a-5p与接头蛋白GAB1的靶向关系;RT-qPCR和Western印迹检测转染miR-106a-5p对GAB1 mRNA和蛋白表达水平的影响;最后,将miR-106a-5p模拟物和GAB1的质粒共转染至人心肌细胞中,利用CCK-8和流式细胞术验证GAB1对miR-106a-5p诱导的心肌细胞增殖、凋亡的回复作用.结果心力衰竭患者血浆中的miR-106a-5p表达水平显著高于健康志愿者(t=48.54,P<0.001);CCK-8结果显示转染miR-106a-5p模拟物组人心肌细胞的A450值显著低于模拟物对照组和空白对照组(P<0.05);流式细胞仪结果显示过表达miR-106a-5p的人心肌细胞的凋亡率显著高于模拟物对照组和空白对照组(P<0.05);生物信息学分析结合双荧光素酶报告基因实验证实miR-106a-5p可结合到GAB1的3′-非翻译区(3′-UTR);RT-qPCR和Western印迹检测发现,过表达miR-106a-5p可显著抑制GAB1 mRNA和蛋白的表达水平;回复实验结果表明,过表达GAB1可逆转miR-106a-5p所致的心肌细胞增殖和凋亡能力改变.结论miR-106a-5p在心力衰竭患者中显著上调,其可通过靶向调控GAB1蛋白的表达抑制人心肌细胞增殖和诱导人心肌细胞凋亡,从而参与心力衰竭的病理过程.Objective To investigate the level of miR-106a-5p in plasma of patients with heart failure and the potential mechanism of its effect on the proliferation and apoptosis of human cardiomyoblast cells.Methods RT-qPCR was used to detect the level of miR-106a-5p in plasma of 45 patients with heart failure and 45 healthy volunteers.Lipofectamine 2000 was employed to transfect miR-106a-5p mimics and mimics control to human cardiomyoblast cells.After the transfection efE-ciency was verified by RT-qPCR,the proliferation and apoptosis level of transfected cells in each group were respectively detected by CCK-8 and flow cytometry.The targeting relationship between miR-106a-5p and adaptor protein GABI was verified by bioinformatics combined with dual luciferase reporter gene assay.Furthermore,the effects of miR-106a-5p transfection on GABI mRNA and protein levels were detected by RT-qPCR and Western blotting,respectively.Finally,miR-106a-5p mimics and GABI plasmid were co-transfected into human cardiomyocytes,and the recovery effects of GABI on miR-106a-5p-induced proliferation and apoptosis of human cardiomyoblast cell were verified by CCK-8 and flow cytometry,respectively.Results The level of miR-106a-5p was significantly higher in heart failure patients than in healthy volunteers(t=48.54,P<0.001).CCK-8 results showed that the A_(450)value was lower in human cardiomyoblast cells transfected with miR-106a-5p mimics than in mimics control group and blank control group(P<0.05).Flow cytometry results showed that the apoptosis rate of human cardiomyoblast cells with overexpression of miR-106a-5p was significantly higher than that of mimics control group and blank control group(P<0.05).Bioin­formatics combined with dual luciferase reporter assay confirmed that miR-106a-5p could bind to the 3r-untranslated region(3'-UTR)of GABI.Further RT-qPCR and Western blotting showed that overexpres­sion of miR-106a-5p could significandy inhibit the levels of GABI mRNA and protein.Furthermore,overexpression of GABI could reverse the prolife

关 键 词：心力衰竭 miR-106a-5p GAB1 增殖 凋亡 

分 类 号：R541.61[医药卫生—心血管疾病]