Abstract：
Cardiac fibrosis is a commonly seen pathophysiological process in various cardiovascular disorders, such as coronary heart disorder, hypertension, and cardiomyopathy. Cardiac fibroblast trans-differentiation into myofibroblasts (MFs) is a key link in myocardial fibrosis. LncRNA PVT1 participates in fibrotic diseases in multiple organs; however, its role and mechanism in cardiac fibrosis remain largely unknown. Human cardiac fibroblasts (HCFs) were stimulated with TGF-β1 to induce myofibroblast; Immunofluorescent staining, Immunoblotting, and fluorescence in situ hybridization were used to detect the myofibroblasts phenotypes and lnc PVT1 expression. Cell biological phenotypes induced by lnc PVT1 knockdown or overexpression were detected by CCK-8, flow cytometry, and Immunoblotting. A mouse model of myocardial fibrosis was induced using isoproterenol (ISO), and the cardiac functions were examined by echocardiography measurements, cardiac tissues by H&E, and Masson trichrome staining. In this study, TGF-β1 induced HCF transformation into myofibroblasts, as manifested as significantly increased levels of α-SMA, vimentin, collagen I, and collagen III; the expression level of lnc PVT1 expression showed to be significantly increased by TGF-β1 stimulation. The protein levels of TGF-β1, TGFBR1, and TGFBR2 were also decreased by lnc PVT1 knockdown. Under TGF-β1 stimulation, lnc PVT1 knockdown decreased FN1, α-SMA, collagen I, and collagen III protein contents, inhibited HCF cell viability and enhanced cell apoptosis, and inhibited Smad2/3 phosphorylation. Lnc PVT1 positively regulated MYC expression with or without TGF-β1 stimulation; MYC overexpression in TGF-β1-stimulated HCFs significantly attenuated the effects of lnc PVT1 knockdown on HCF proliferation and trans-differentiation to MFs. In the ISO-induced myocardial fibrosis model, lnc PVT1 knockdown partially reduced fibrotic area, improved cardiac functions, and decreased the levels of fibrotic markers. In addition, lnc PVT1 knockdown decreased MYC and CDK4 levels but increased E-cadherin in mice heart tissues. lnc PVT1 is up-regulated in cardiac fibrosis and TGF-β1-stimulated HCFs. Lnc PVT1 knockdown partially ameliorates TGF-β1-induced HCF activation and trans-differentiation into MFs in vitro and ISO-induced myocardial fibrosis in vivo, potentially through interacting with MYC and up-regulating MYC.
摘要：
心脏纤维化是各种心血管疾病中常见的病理生理过程。比如冠心病，高血压,和心肌病。心肌成纤维细胞转分化为肌成纤维细胞（MFs）是心肌纤维化的关键环节。LncRNAPVT1参与多个器官的纤维化疾病;然而,其在心脏纤维化中的作用和机制尚不清楚。用TGF-β1刺激人心肌成纤维细胞（HCFs）诱导成肌纤维细胞；免疫荧光染色，免疫印迹,用荧光原位杂交技术检测肌成纤维细胞表型和lncPVT1表达。CCK-8、流式细胞术检测PVT1敲低或过表达诱导的细胞生物学表型,和免疫印迹。使用异丙肾上腺素（ISO）诱导心肌纤维化的小鼠模型，心脏功能通过超声心动图检查，心脏组织由H&E，和Masson三色染色.在这项研究中,TGF-β1诱导HCF转化为肌成纤维细胞，表现为α-SMA水平显著增加，波形蛋白,胶原蛋白I，和胶原蛋白III；通过TGF-β1刺激，lncPVT1的表达水平显着增加。lncPVT1敲低也降低了TGF-β1，TGFBR1和TGFBR2的蛋白水平。在TGF-β1刺激下，lncPVT1敲低降低FN1、α-SMA、胶原蛋白I，和胶原蛋白III含量，抑制HCF细胞活力，增强细胞凋亡，并抑制Smad2/3磷酸化。在有或没有TGF-β1刺激的情况下，LncPVT1正调节MYC表达；在TGF-β1刺激的HCF中MYC过表达显着减弱了lncPVT1敲低对HCF增殖和转分化为MF的影响。在ISO诱导的心肌纤维化模型中，lncPVT1敲低部分减少纤维化面积，改善心脏功能，并降低纤维化标志物的水平。此外,lncPVT1敲低降低了小鼠心脏组织中的MYC和CDK4水平，但增加了E-cadherin。lncPVT1在心脏纤维化和TGF-β1刺激的HCFs中上调。LncPVT1敲低部分改善TGF-β1诱导的HCF活化和转分化为MF的体外和ISO诱导的心肌纤维化，可能通过与MYC相互作用和上调MYC。
