沉默lncRNA XIST对阿霉素诱导的扩张型心肌病的影响及其机制

Effect and mechanism of silencing lncRNA XIST on dilated cardiomyopathy induced by adriamycin

目的 探究长链非编码RNA X-无活性特异性转录物(lncRNA XIST)对阿霉素诱导的扩张型心肌病(DCM)大鼠心肌纤维化和心肌细胞凋亡的影响及其机制。方法 采用阿霉素诱导法建立DCM大鼠模型,将DCM大鼠分为模型组、sh-NC组和sh-XIST组,每组10只;另取10只健康SD大鼠设为对照组。采用超声影像系统检测各组大鼠心脏功能指数左室收缩末期内径(LVESD)、左室舒张末期内径(LVEDD)和左室射血分数(LVEF),采用苏木素伊红染色和Masson三色染色检测大鼠心肌组织病理学变化,TUNEL检测大鼠心肌细胞凋亡水平。采用阿霉素诱导H9c2细胞系构建DCM细胞模型,将DCM细胞分为模型组、sh-NC组、sh-XIST组、sh-XIST+miR-NC组和sh-XIST+miR-149-5p inhibitor组,未经阿霉素处理的H9c2细胞作为对照组。采用双荧光素酶报告试验分析XIST、miR-149-5p和血小板源性生长因子C(PDGFC)的靶向关系。采用流式细胞术检测心肌细胞凋亡水平。采用RT-qPCR检测大鼠心肌组织和H9c2细胞中XIST、miR-149-5p和PDGFC表达水平。采用Western blot检测大鼠心肌组织和H9c2细胞中PDGFC、α-SMA、collagenⅠ和TGF-β1表达水平。结果 (1)与sh-NC组比较,sh-XIST组大鼠心肌组织中XIST水平及PDGFC的mRNA和蛋白水平均降低(P<0.01),LVESD、LVEDD、TUNEL阳性细胞率以及α-SMA、collagenⅠ和TGF-β1蛋白水平均降低(P<0.05),miR-149-5p水平和LVEF水平均升高(P<0.01);心肌细胞坏死和组织纤维化均减少。(2)与sh-NC组比较,sh-XIST组H9c2细胞凋亡率、XIST水平、PDGFC mRNA和蛋白水平以及α-SMA、collagenⅠ和TGF-β1蛋白水平均降低(P<0.05),miR-149-5p表达水平升高(P<0.05)。与sh-XIST+miR-NC组比较,sh-XIST+miR-149-5p inhibitor组H9c2细胞凋亡率、PDGFC mRNA和蛋白水平以及α-SMA、collagenⅠ和TGF-β1蛋白水平均升高(P<0.05),miR-149-5p表达水平降低(P<0.05)。结论 沉默lncRNA XIST通过上调miR-149-5p表达和抑制PDGFC表达,进而抑制阿霉素诱导的DCM大鼠心肌纤维化和心肌细胞凋亡水平,从而改善DCM的发展。

Objective To investigate the effect and mechanism of long non-coding RNA X-inactive specific transcript(lncRNA XIST)on myocardial fibrosis and apoptosis in doxorubicin-induced dilated cardiomyopathy(DCM)rats.Methods The DCM rat model was established by adriamycin induction method.The DCM rats were divided into model group,sh-NC group and sh-XIST group,with 10 rats in each group,and another 10 healthy SD rats were selected as control group.Left ventricular end-systolic diameter(LVESD),left ventricular end-diastolic diameter(LVEDD)and left ventricular ejection fraction(LVEF)were measured by ultrasound imaging system.The histopathological changes of myocardium were detected by HE staining and Masson trichromatic staining,and the apoptosis of myocardium was detected by TUNEL.The H9c2 cells were treated with adriamycin to induce DCM cell model.DCM cells were divided into model group,sh-NC group,sh-XIST group,sh-XIST+miR-NC group and sh-XIST+miR-149-5p inhibitor group,and H9c2 cells without adriamycin treatment were treated as control group.The targeting relationships between XIST,miR-149-5p and PDGFC were analyzed by dual luciferase reporting assay.The apoptosis level of cardiomyocytes was detected by flow cytometry.The expression levels of XIST,miR-149-5p and PDGFC in rat myocardial tissue and H9c2 cells were detected by RT-qPCR.The expression levels of PDGFC,α-SMA,collagenⅠand TGF-β1 in rat myocardial tissue and H9c2 cells were detected by Western blot.Results①Compared with sh-NC group,the levels of XIST,and PDGFC mRNA and protein in myocardial tissue of rats were decreased in sh-XIST group(P<0.01),LVESD,LVEDD,TUNEL positive cell rate,and the protein levels ofα-SMA,collagenⅠand TGF-β1 were decreased(P<0.05),while the level of miR-149-5p and LVEF were increased(P<0.01).Compared with sh-NC group,both myocardial cell necrosis and tissue fibrosis were reduced in sh-XIST group.②Compared with sh-NC group,the apoptosis rate of H9c2 cells,and the levels of XIST,PDGFC mRNA and protein,α-SMA,collagenⅠand TGF-β1 protein in H9c2 cells were decreased in sh-XIST group(P<0.05),while the level of miR-149-5p was increased(P<0.05).Compared with sh-XIST+miR-NC group,the apoptosis rate of H9c2 cells,and the levels of PDGFC mRNA and protein,α-SMA,collagenⅠand TGF-β1 proteins in H9c2 cells were increased in sh-XIST+miR-149-5p inhibitor group(P<0.05),while the level of miR-149-5p was decreased(P<0.05).Conclusion Silencing lncRNA XIST can inhibit the adriamycin-induced myocardial fibrosis and the apoptosis in DCM rats by upregulating the expression of miR-149-5p and inhibiting the expression of PDGFC,thereby improving the development of DCM.