SiRNA Inhibite PCSK9 Gene And Effects On Proliferation And Apoptosis Of VSMC

Background Vascular smooth muscle cells are the main compo-nents of the artery wall and plays an important role in keeping the normal function of the Vascular.In atherosclerosis,vascular smooth muscl-ecell proliferation,apoptosis can affect by many factors such as stress,oxidized low density lipoprotein cholesterol,NO,reactive oxygen species and cytokines.Uncoordinated proliferation and apoptosis of the vascular smooth muscle cells not only contributed to the acceleration of the atherosclerosis plaques,but also affected the instability of atherosclerotic plaques and in-stent restenosis.Proprotein convertase subtilisin/kexin 9 is the third gene of familial hypercholesterolemia,mainly expressed in the liver,intestine,kidney and brain and can be detected in artery atherom-atous plaque,vascular smooth muscle cells,endothelial cells,mononuclear macrophages.Recent study found that PCSK9 not only play a key role in the dynamic balance of cholesterol,but also have important fanction in the inflammatory response,cell migration and apoptosis.It has being reported that using RNA interference to silence PCSK9 gene can inhibit apoptosis of THP-1 source macrophages and umbilical vein endothelial cells.But the effect on vascular smooth muscle cells proliferation and apoptosis have not been reported at home and abroad.Objective Using siRNA technique to silence PCSK9 gene,observe its effect on vascular smooth muscle cells proliferation and apoptosis,and explore the possible mechanisms.Metheodsl.Ascular smooth muscle cell culture and identification.2.Design 3 different PCSK9 gene targets siRNA sequences,The PCSK9 siRNAs gene were transfected into vascular smooth muscle cells by positive ion liposome Lipofectamine 2000.Transfection efficiency was assessed by fluorescence microscope assay.The most efficient siRNA was selected to transfected into vascular smooth muscle cells.3.SiRNA inhibite PCSK9 gene and the effects on vascular smooth muscle cells proliferation:Cells(3×104/well)were distributed in 96-well plates and subjected to 10ng/mlPlatelet-derived growth factor BB,Divi-ded into three groups:Blank group(without PDGF+Transfection reagents),Negtive transfection group(10 ng/ml PDGF BB+not transfection PCSK9siRNA),Positive transfection group(10 ng/ml PDGF-BB+transfection PCSK9 siRNA).Each group of six holes,inl2,24,36,48,60 hours,OD,value is measured by MTT method,then draw the proliferation curve.After 48 hours detect the expression of LRP-1.4.SiRNA inhibite PCSK9 gene and the effects on vascular smooth muscle cells apoptosis:Cells(3-5×105well)were distributed in 6-well plates and subjected to 100?g/ml ox-LDL.Divided into three groups:Blank group(without PDGF+not transfection PCSK9siRNA),Negtive tran-sfection group(10 ng/ml PDGF BB+Transfection reagents),the positive transfection group(10 ng/ml PDGF-BB+transfection PCSK9 siRNA),Each group of three holes,After 24 hours.Hoechst33342 dyeing,fluorescence microscope to observe the cell apoptosis morphology;Flow cytometry to detect morphology characters of apoptosis.Results1.80 nmol/L PCSK9siRNA can inhibite expression of PCSK9mRNA and protein in vascular smooth muscle cells effectively.2.After deal with 10 ng/ml PDGF-BB 12,24,36,48 h,compared with negative control group,test Group OD value significantly decreased,indicated that transfection of PCSK9 siRNA could effectively restrain vascular smooth muscle proliferation induced by PDGF(P<0.05),Western blot results show that the LRP-1 May play an important role.3.100?g/ml oxLDL treatment after 24 h,flow cytometry detection of apoptosis,found that the blank group of smooth muscle cell apoptosisrat-e was 7.48%;Negative control group was 19.40%;Test Group on was 4.70%.Conclusion1.PCSK9siRNA can effectively inhibit PCSK9mRNA and protein expression in vascular smooth muscle cells.2.PCSK9siRNA can effectively restrain proliferation of vascular sm-ooth muscle cells mediated by PDGF.3.PCSK9siRNA can effectively restrain apoptosis of vascular smooth muscle cells mediated by xo-LDL.