LncRNA MIAT靶向调节miR-532-3p对心力衰竭小鼠心肌细胞凋亡的影响

Influence of LncRNA MIAT on cardiomyocyte apoptosis in mice with heart failure by targeting miR-532-3p

目的:探讨长链非编码RNA心肌梗死相关转录本(lncRNA-MIAT)靶向调节miR-532-3p对心力衰竭(HF)小鼠心肌细胞凋亡的影响。方法:建立HF小鼠模型,将小鼠分为Control组、Sham组、HF组、sh-NC组、sh-MIAT组、sh-MIAT+anti-NC组、sh-MIAT+anti-miR-532-3p组,每组10只,除Control组、Sham组、HF组尾静脉注射生理盐水外,其余各组尾静脉注射相应的质粒。心脏彩超检查小鼠心脏功能,HE染色和Masson染色观察心肌组织病理变化,TUNEL法检测心肌细胞凋亡,实时荧光定量PCR(RT-qPCR)检测心肌组织LncRNA MIAT、miR-532-3p表达;Western blot检测心肌组织半胱氨酸蛋白酶-3(Caspase-3)、Ⅰ型胶原蛋白(collagenⅠ)和Ⅲ型胶原蛋白(collagenⅢ)表达;双荧光素酶报告基因检测LncRNA MIAT与miR-532-3p的相互关系。结果:造模后HF组小鼠左心室射血分数(LVEF)<60%,提示造模成功;与Sham组比较,HF组小鼠心功能显著降低,心肌组织损伤加重,心肌纤维化及心肌细胞凋亡增加,LncRNA MIAT、Caspase-3、collagenⅠ和collagenⅢ蛋白表达水平显著升高(P<0.05);与sh-NC组比较,sh-MIAT组小鼠心功能改善,心肌组织损伤减轻、心肌纤维化及心肌细胞凋亡减少,LncRNA MIAT、Caspase-3、collagenⅠ和collagenⅢ蛋白表达水平显著降低(P<0.05);与sh-MIAT组比较,sh-MIAT+anti-miR-532-3p组小鼠心功能降低,心肌组织病理损伤加重,心肌纤维化及心肌细胞凋亡率增加,Caspase-3、collagenⅠ和collagenⅢ蛋白表达水平显著升高(P<0.05);双荧光素酶报告基因实验结果显示,LncRNA MIAT靶向负调控miR-532-3p表达。结论:HF小鼠心肌组织LncRNA MIAT表达水平升高,抑制LncRNA MIAT表达可通过靶向上调miR-532-3p改善HF引起的小鼠心肌损伤与心肌细胞凋亡。

Objective:To investigate the influence of long non-coding RNA myocardial infarction-associated transcript(lncRNA-MIAT)targeting miR-532-3p on cardiomyocyte apoptosis in heart failure(HF)mice.Methods:The mice model of HF was established and divided into Control group,Sham group,HF group,sh-NC group,sh-MIAT group,sh-MIAT+anti-NC group and sh-MIAT+anti-miR-532-3p group,10 animals per group.The Control group,Sham group and HF group were injected with normal saline through the tail vein,the other groups were injected with corresponding plasmids through the tail vein.Cardiac function of mice was examined by cardiac color ultrasound,pathological changes of myocardial tissue were observed by HE staining and Masson staining,apoptosis of myocardial cells was detected by TUNEL method,expression of LncRNA MIAT and miR-532-3p in myocardial tissue were detected by Real-time fluorescence quantitative PCR(RT-qPCR).The expressions of Caspase-3,collagenⅠand collagenⅢin myocardial tissue were detected by Western blot.The relationship between LncRNA MIAT and miR-532-3p was detected by dual luciferase reporter assay.Results:After modeling,the left ventricular ejection fraction(LVEF)of mice in the HF group was less than 60%,indicating that the modeling was successful;compared with the Sham group,the cardiac function of the mice in the HF group was obviously decreased,the myocardial tissue damage was aggravated,myocardial fibrosis and myocardial cell apoptosis were increased,and the protein expression levels of LncRNA MIAT,Caspase-3,collagenⅠand collagenⅢwere obviously increased(P<0.05);compared with the sh-NC group,the sh-MIAT group had improved cardiac function,reduced myocardial tissue damage,myocardial fibrosis and cardiomyocyte apoptosis were decreased,and obviously decreased the protein expression levels of LncRNA MIAT,Caspase-3,collagenⅠand collagenⅢ(P<0.05);compared with sh-MIAT group,the cardiac function of mice in sh-MIAT+anti-miR-532-3p group was decreased,myocardial tissue pathological injury was aggravated,myocardial fibrosis and myocardial cell apoptosis rate were increased,and the protein expression levels of Caspase-3,collagenⅠand collagenⅢwere significantly increased(P<0.05);the results of the dual-luciferase reporter gene assay showed that LncRNA MIAT targeted and negatively regulated the expression of miR-532-3p.Conclusion:The expression level of LncRNA MIAT in the myocardial tissue of HF mice is increased,and inhibiting the expression of LncRNA MIAT can improve myocardial injury and myocardial cell apoptosis in mice caused by HF by targeting up-regulation of miR-532-3p expression.