LncRNA Terc在心肌纤维化过程中的功能研究

Functional Study of lncRNA Terc during Myocardial Fibrosis

目的:探讨lncRNA端粒酶RNA组分(Terc)在心肌纤维化(MF)过程中的作用。方法:使用不同浓度的TGF-β1诱导心肌成纤维细胞(CFs)转分化,通过免疫荧光染色、western blot检测α-SMA、Vimentin、Collagen I、Collagen III蛋白的表达水平,qRT-PCR检测lncRNA Terc表达水平。过表达和敲减Terc后,通过western blot、CCK-8和流式细胞术观察模型细胞的胞外基质产生、细胞增殖、凋亡和Smads信号传导情况。皮下注射异丙肾上腺素(ISO)构建小鼠心肌纤维化模型,并使用Terc敲低慢病毒干预,其后多普勒超声仪检测小鼠的心脏射血分数(EF)和左室短轴缩短率(FS),称量小鼠心脏湿重,HE、Masson染色检测小鼠心脏的病理改变,IHC检测α-SMA、Vimentin蛋白的表达水平,qRT-PCR检测lncRNA Terc表达水平。结果:TGF-β1处理增加CFs的α-SMA、Vimentin、Collagen I、Collagen III的蛋白表达以及Terc水平;过表达Terc促进α-SMA、Vimentin、Collagen I、Collagen III的蛋白表达以及Smad2/3的磷酸化水平,同时还可促进CFs的增殖、抑制CFs的凋亡;敲减Terc则起相反的作用;动物模型中,ISO可抑制EF和FS,增加心脏湿重,加重心肌的病理损伤,而敲减Terc可有效缓解上述过程。结论:LncRNA Terc可通过促进Smads信号传导,加速心肌成纤维细胞转分化和心肌纤维化进展。

Objective:To investigate the role of telomerase RNA component(lncRNA Terc)in myocardial fibrosis(MF).Methods:TGF-β1 was used to induce cardiac fibroblasts(CFS)fibrosis cell model.The protein expression level was detected by immunofluorescence staining,Western blot and IHC staining,meanwhile the expression level of TERC was detected by qRT-PCR;CCK-8 and flow cytometry were used to detect cell proliferation and apoptosis,respectively;the myocardial fibrosis model of mice was established by subcutaneous injection of isoproterenol(ISO).The ejection fraction(EF)and left ventricular short axis shortening(FS)of the mice were measured by Doppler ultrasound.The wet weight of the mouse heart was also weighed.The pathological changes of the mouse heart were detected by HE and Masson staining;the expression levels ofα-SMA and Vimentin detected by IHC;the expression level of TERC was detected by qRT-PCR.Results:TGF-β1 treated CFs have a higher expression ofα-SMA,vimentin,collagen I and collagen III and m RNA level of Terc.Overexpression of TERC promoted the expression ofα-SMA,vimentin,collagen I and collagen III and the phosphorylation level of Smad2/3,and also promoted the proliferation of CFs and inhibit the apoptosis of CFs.Knockdown of Terc had the opposite effect.In animal models,ISO can inhibit EF and FS,increase cardiac wet weight,and aggravate the pathological damage of myocardium;while Terc knockdown with shRNA can effectively alleviate the above process.Conclusion:TERC in CFs promotes the phosphorylation of Smad2/3 after myocardial fibrosis,which aggravates the pathological changes of myocardial fibrosis.