The Regulatory Mechanism Of Long Noncoding RNA PUNISHER On Proliferation And Apoptosis Of Vascular Smooth Muscle Cell

Objective Explore the relationship of long noncoding RNA PUNISHER with atherosclerosis and its effects on the proliferation and apoptosis of vascular smooth muscle cell(VSMC).Methods Twelve healthy male 4 week old apolipoprotein E(Apo E)knockout mice(ApoE-/-mice),produce atherosclerosis model by high fat fed(model group);12 healthy C57BL/6J mice were fed with basal diet as control group;human vascular smooth muscle cells(HA-VSMCs)were divided in small interfering-PUNISHER group(siPUNISHER)and noncoding interfering group(NC)by their different small interfering RNA.Aortic arch tissue sample of the atherosclerosis model and control mice were used to determine the PUNISHER expression using fluorescence quantitative PCR method.Small interference RNA of PUNISHER were used to downregulate the expression of PUNISHER in vascular smooth muscle cell.Moreover,CCK 8,scratch-wound experiment,Mitochondrial staining and flow cytometry method were used to detect the regulatory role of PUNISHER in vascular smooth muscle cell proliferation migration mitochondrial division and apoptosis by respectively.Finally western blot was used to determine the expression levels of B-cell leukemia-lymphoma-2(Bcl-2),cysteinylaspartae specific proteinase 3(caspase3),caspase8 P53 and OPA1.Results(1)The levels of triacylglycerol(TG),total cholesterol(TC),low density lipoprotein-cholesterol(LDL-C),high density lipoprotein-cholesterol(HDL-C)along with Hematoxulin and Eosin(HE)staining proved that model building success.The fluorescence quantitative PCR results showed that the PUNISHER expression is higher(t=9.597,p<0.05)in model mice than in control group.(2)According to the CCK-8 assay,the knockdown of PUNISHER group showed significantly lower proliferation level than the control group(t36=2.638 p < 0.05,t48=2.554 p < 0.05).Scratch-wound experiments siPUNISHER cells migration has been suppressed(t=3.136 p<0.05).(3)Flow cytometry showed that silencing of PUNISHER lncRNA significantly increased VSMC apoptosis cell number compared to control group(x2=4.245 p<0.05).(4)the knockdown of PUNISHER group showed significantly high percent of divided mitochondrion(5)Western blot results showed a significant downregulation of expression of Bcl-2 caspase3 30 kD p53(tbcl-2=9.546 p<0.05,tcapase3 30kD=5.647 p<0.05,tp53=5.42 p<0.05)and a upregulation of expression of caspase3 17 k D and OPA1(tcaspase3 17kD=4.892 p < 0.05,tOPA1=12.258,p < 0.05)upon silencing of PUNISHER lncRNA in VSMC.Conclusion Silence long noncoding RNA PUNISHER can accelerate vascular smooth muscle mitochondrial division and cell apoptosis.