Roles And Mechanisms Of P16 Methylation In Atorvastatin-induced Inhibition Of VSMCs Proliferation And Migration

Part ?.Effects of p16 on atorvastatin-induced inhibition of VSMCs proliferation and migrationObjective: To study the effects of atorvastatin on the proliferation,migration and apoptosis of vascular smooth muscle cells(VSMCs),and the role of p16 in this process.Methods: Rat aortic VSMCs were primary cultured and identified by immunofluorescence.VSMCs were treated with different concentrations of atorvastatin(0,0.5,1.0,and 2.0 ?M).The effect of atorvastatin on VSMCs proliferation was determined by CCK-8.Transwell chamber was used to detect the effect of atorvastatin on VSMCs migration.Annexin V-PI assay was used to detect the effect of atorvastatin on apoptosis of VSMCs.The effects of atorvastatin on the expression of p16 and p53 were detected by q PCR and Western blot.Silencing p16 gene with short hairpin RNA(sh RNA)and observing the effect of p16 on the proliferation and migration of VSMCs.Results:(1)Compared with the control group,atorvastatin inhibited the proliferation and migration of VSMCs in a dose-dependent manner(P<0.05).(2)Compared with the control group,atorvastatin promoted the apoptosis of VSMCs in a dose-dependent manner and increased expression of p53 protein.(3)Compared with the control group,atorvastatin up-regulated the expression of p16 m RNA and protein in a dose-and time-dependent manner(P<0.05).(4)The inhibitory effects of atorvastatin on VSMCs proliferation and migration were partially released when p16 was knocked down(P<0.05).Conclusion: Atorvastatin affects the proliferation,migration and apoptosis of VSMCs by regulating the expression of p16.Part ?.DNA methylation mediates the regulation of p16 expression by atorvastatin and the underlying mechanismsObjective: To study the role of DNA methylation in the regulation of p16 expression by atorvastatin,and the underlying mechanisms.Methods: VSMCs were treated with different concentrations of atorvastatin(0,0.5,1.0,2.0 ?M)and 5-Azacytidine(AZA)(0,0.5,1.0 ?M).Expressions of DNA methyltransferases(DNMTs)(DNMT1,DNMT3 a,DNMT3b)and p16 were detected by q-PCR and Western blot.The methylation status of Cp G island in p16 promoter region was detected by bisulfite pyrosequencing analysis.DNMT1 overexpression and silencing cell model were established,and the expression of p16 was detected by q-PCR and Western blot.Results:(1)Compared with the control group,the expression of DNMT1 in AZA and atorvastatin group was significantly down-regulated,while the expression of DNMT3 a and DNMT3 b was not changed.(2)The expression of p16 m RNA and p16 protein was up-regulated in AZA group(P<0.05).(3)Compared with the control group(20.0%),the methylation level of Cp G island in p16 promoter region was significantly reduced in AZA(10.9%)and atorvastatin(7.3%)group.(4)Compared with control group,overexpressed DNMT1 inhibited p16 expression,while the expression of p16 was elevated when DNMT1 was knocked down(P<0.05).(5)Compared with the control group,atorvastatin increased the p16 expression.The repression of DNMT1 could alleviate the atorvastatin-induced p16 expression and overexpressed DNMT1 enhanced it(P<0.05).Conclusion: Atorvastatin affects the methylation of p16 promoter region through down-regulation of DNMT1,which in turn regulates p16 expression.Part ?.The MAPK pathway is involved in atorvastatin-induced inhibition of VSMCs proliferation and migrationObjective: To study the effect of mitogen-activated protein kinases(MAPK)pathway on the expression of DNMT1 and p16,and the effect on the proliferation and migration of VSMCs.Methods: VSMCs were treated with different concentrations of atorvastatin(0,0.5,1.0,and 2.0 ?M).Western blot was used to detect the expression of JNK,ERK,p38-MAPK and their phosphorylated forms.VSMCs were treated with atorvastatin(1.0 ?M)combined with MAPKs inhibitors(p-JNK inhibitor SP600125,p-ERK inhibitor AZD6244 and p-p38 inhibitor SB203580),and Western blot was used to detect the expression of p-JNK,p-ERK,p-p38,DNMT1 and p16.CCK-8 assay and Transwell chamber were used to detect proliferation and migration of VSMCs.Results:(1)Compared with the control group,the levels of JNK,ERK and p38-MAPK protein in atorvastatin group did not change significantly,while the levels of p-JNK,p-ERK and p-p38 protein increased significantly.(2)Compared with atorvastatin group,atorvastatin combined with p-JNK inhibitor/p-ERK inhibitor/p-p38 inhibitor group up-regulated DNMT1 expression and down-regulated p16 expression.(3)Atorvastatin combined with p-JNK inhibitor/p-ERK inhibitor/p-p38 inhibitor group increased proliferation and migration of VSMCs compared to atorvastatin group(P<0.05).Conclusion: The MAPK pathway affected atorvastatin-induced inhibition of cell proliferation and migration via modulating DNMT1/p16 expression in VSMCs.Part ?.Effect of atorvastatin on neointima formation after balloon injury in rat carotid arteryObjective: To study the effects of atorvastatin on intimal hyperplasia and p16 expression in rat carotid artery after balloon injury.Methods: SD male rats were divided into three groups:(1)sham group,(2)balloon injury group,(3)balloon injury+atorvastatin group.HE staining was used to observe vessel wall morphology and intimal hyperplasia.q-PCR and Western blot were used to detect the p16 expression in the injured arteries.Bisulfite pyrosequencing analysis was used to detect the methylation level of Cp G island in p16 promoter region.Results:(1)In the Sham group,the structure of the carotid wall was clear and complete,no neointimal hyperplasia was seen,and the medial VSMCs were well-arranged.In the balloon injury group,the vessel wall was thickened and the lumen was narrowed,and neointima containing a large number of VSMCs was observed.Neointimal hyperplasia was significantly reduced in the balloon injury+atorvastatin group.(2)Compared with the sham group,the levels of p16 m RNA and protein expression in the balloon injury group were significantly downregulated.Compared with the balloon injury group,the levels of p16 m RNA and protein expression in the balloon injury+atorvastatin group were increased(P<0.05).(3)Compared with the sham group,the methylation level of Cp G islands in the p16 promoter region was elevated in the balloon injury group.Compared with the balloon injury group,the methylation level of the Cp G island in the p16 promoter region was reduced in the balloon injury+atorvastatin group.Conclusion: Atorvastatin effectively inhibits neointimal hyperplasia after carotid balloon injury in rat carotid artery,and promotes p16 expression by decreasing the methylation level of Cp G island in p16 promoter region.