Effect, Mechanism And Regulation Of C-SKI In Cardiac Fibrosis

Objective: To investigate the effect and mechanism of c-SKI in cardiac fibrosis: 1)in vitro,cardiac fibroblasts and coronary endothelial cells are cultured to establish two cell model of cardiac fibrosis and establish a model of rat cardiac fibrosis in vivo.To observe the effect on endothelial mesenchymal transition,cardiac fibrosis by lentivirus-mediated c-SKI overexpression or silencing and the effect on TGF?/Smad signaling pathway;2)To screen the microRNAs which can target c-SKI by bioinformatics analysis,and observe their effects on endothelial-mesenchymal transition and myocardial fibrosis.Methods: Part?: In vitro,cardiac fibroblasts and coronary endothelial cells were induced by different concentrations of TGF?1,western blot test the markers of myocardial fibrosis and endothelial-mesenchymal transition.To establish the rat model of myocardial fibrosis,use echocardiography,HE and Masson staining to assess.The expression of endogenous c-SKI was tested by Western blot in different fibrosis models.After c-SKI lentiviral vector was transfected,the effectiveness was tested by Western blot.The effect of c-SKI on cell proliferation was observed by MTT assay and clone formation assay.Part ? : In vitro,to observe the effect of lentiviral-mediated c-SKI overexpression on extracellular matrix and endothelialmesenchymal transition.In vivo,Echocardiography,HE staining,Masson staining and immunohistochemistry were used to detect cardiac parameters,heart tissue morphological change,extracellular matrix synthesis and endothelial-mesenchymal transition after c-SKI lentiviral vector was transfected.The downstream protein expression of TGF?/Smad signaling pathway were detected by Western blot.Part?: Through online software and bioinformatics analysis,screening to target MicroRNAs of c-SKI3'UTR,to verify their binding and regulatory relationships by using dual luciferase assays.To establish the mimics and inhibitor of screened microRNA,use the MTT assay and clone formation assay to test the cell proliferation.The expression of extracellular matrix markers and endothelial mesenchymal transition markers were determined by Western blot.Results: Part ? :(1)After 48 hours of induction with different concentration gradient TGF?1,the expression of collagen and ?-SMA in fibroblasts was significantly increased,the effect of TGF?1 at 10ng/ml was the most significant(P<0.01);the expression of vimentin and collagen were significantly up-regulated(P<0.01).the expression of E-cad was significantly down-regulated(P<0.01),the effect of TGF?1 at 5ng/ml was the most significant(P<0.01).The expression of endogenous c-SKI in the cells was decreased in a time and dose-dependent manner(P<0.01).(2)The results of echocardiography showed that the HR,HW,LVIDd,LVIDs,LVM,HMI,LVMI of C57 significantly increased(P<0.01),EF%,FS% significantly decreased(P<0.01).HE and Masson staining showed that the model group cardiac tissue arrangement disordered,collagen fiber increased.Compared with the control group,CVF% of the model group significantly decreased(3.85±0.61% vs 32.54±3.12%,P<0.01).The expression of endogenous c-SKI in myocardium of mice was decreased with the time of modeling.(3)Lentiviral c-SKI vector was transfected into fibroblasts,coronary endothelial cells and myocardium respectively.Western blot showed that lentivirus c-SKI vector could be transfected effectively.MTT assay and clone formation test showed,compared with the LV-NC group,overexpression of c-SKI could significantly inhibit the proliferation of fibroblasts(2.58±0.56 vs 2.04±0.48,P<0.01)and coronary endothelial cells(1.97±0.38 vs1.69±0.32,P<0.01).Part?:(1)In vitro,overexpression of c-SKI,compared with the LV-NC group,the expression of type I collagen and ?-SMA protein in fibroblasts was decreased(P<0.01),and the expression of vimentin in coronary endothelial cells was decreased,CD31 protein expression increased(P<0.01).(2)The results of echocardiography showed that compared with the LV-NC group,the HR,HW,LVIDd,LVIDs,LVM,HMI,LVMI significantly decreased(P<0.05),EF%,FS% significantly increased(P<0.01).HE and Masson staining showed that C57 myocardial fibrous tissue hyperplasia symptoms were significantly reduced,collagen fibers decreased,compared with the LV-NC group,the CVF% of LV-SKI group significantly decreased(33.61±2.61% vs 25.71±2.28%,P<0.01).Immunohistochemistry showed type?and type III collagen significantly decreased(3.11±0.19 vs 2.43±0.26?3.84±0.26 vs 2.61±0.24,P < 0.01).Western blot showed the expression of CD31 protein was up-regulated(P<0.01),the expression of ?-SMA protein was down-regulated in mouse myocardium(P<0.01).(3)After overexpression of c-SKI,compared with the LV-NC group,the expression of the phosphorylation of Smad2/Smad3 significantly decreased(P<0.01)in coronary endothelial cells and in C57.Part?:(1)By bioinformatics analysis and dual luciferase reporter,the miR-155 and mi R17 could target binding and inhibit the expression of c-SKI 909-915 and 971-977 sites respectively.Compared with the NC-inhibitor group,after transfection of mi R-155 and miR-17 mimics,the expression of c-SKI protein was significantly decreased by Western blot(P<0.01).(2)MTT and clone formation test results showed that after transfection of mi R-155 and mi R-17 inhibitors,compared with the TGF?1 group,fibroblasts(3.37±0.79 vs 2.83±0.54/2.99±0.38)and coronary endothelial cells(3.28±0.49 vs 2.68±0.38/2.86±0.35)was significantly decreased(P<0.01).(3)Western blot showed that the levels of collagen I and ?-SMA in fibroblasts were significantly decreased(P<0.01)after transfection of miR-155 and miR-17 inhibitors;the expression of vimentin in coronary endothelial cells was decreased(P <0.01),and the expression of cadherin were increased after transfection of miR-155 inhibitor(P<0.01).After transfection of miR-17 inhibitor,vimentin and cadherin did not change significantly(P>0.05).Conclusion: 1)Using fibroblast proliferation or endothelial-mesenchymal transformation method,can effectively establish the cell model of the myocardial fibrosis.In different fibrosis model,the expression of c-SKI is reduced in dose-and time-dependent way,and the overexpression of c-SKI can significantly inhibit fibroblasts and coronary endothelial cell proliferation;2)Overexpression of c-SKI can inhibit extracellular matrix synthesis and endothelial-mesenchymal transition,the mechanism is through inhibition Smad2,Smad3 protein phosphorylation,inhibit of TGF?/Smad signaling pathway,thereby inhibiting myocardial fibrosis;3)By screening and validation,mi R-155 and miR17 can target binding to inhibit the expression of c-SKI,and then regulate myocardial fibroblasts,coronary endothelial cell proliferation and extracellular matrix deposition,inhibition of miR-155 also inhibited endothelial-mesenchymal transition,TGF?1/miR-155/mi R17-5p/c-SKI forms a negative feedback pathway that plays an important role in the regulation of myocardial fibrosis.