长链非编码RNA punisher对血管平滑肌细胞增殖和凋亡的影响及机制

The regulatory mechanism of long noncoding RNA punisher on proliferation and apoptosis of vascular smooth muscle cell

目的探究长链非编码RNA(long noncoding RNA,lncRNA)punisher与动脉粥样硬化的关系及其对血管平滑肌细胞增殖和凋亡的影响及机制。方法 12只健康纯种雄性8周龄载脂蛋白E(apolipoprotein E,Apo E)基因敲除小鼠(Apo E-/-小鼠),通过高脂饲喂制作动脉粥样硬化模型小鼠(模型组);12只健康C57BL/6J小鼠饲以基础饲料为对照组;人体血管平滑肌细胞系根据转染情况分为小干扰-punisher组和阴性对照(negative control,NC)组。通过荧光定量聚合酶链反应测定模型组与对照组小鼠主动脉弓的punisher表达。通过合成punisher的小干扰RNA作为反向对照抑制punisher在人血管平滑肌细胞中的表达,应用实时荧光定量聚合酶链反应验证punisher表达情况;分别采用细胞计数试剂盒(cell counting kit-8,CCK-8)、划痕实验及流式细胞术检测抑制punisher表达对人血管平滑肌细胞增殖、迁移和凋亡的影响;蛋白印迹法检测B淋巴细胞瘤-2(B-cell leukemia-lymphoma-2,Bcl-2)、半胱氨酸的天冬氨酸蛋白水解酶3(cysteinyl aspartate specific proteinase 3,caspase3)、caspase8、p-p38、p53凋亡相关蛋白的表达。结果 1血脂4项及血管苏木精-伊红染色证明造模成功。荧光定量聚合酶链反应显示,动脉粥样硬化模型组小鼠血管punisher表达高于对照组(P<0.05);2CCK-8检测显示小干扰-punisher组的光密度值在转染36 h之后低于NC组(t36=2.683、P<0.05,t48=2.554、P<0.05);划痕实验显示小干扰-punisher组细胞迁移低于NC组(t=3.136、P<0.05);3流式细胞术显示,小干扰-punisher组中凋亡细胞数占比多于NC组(22.6%和11.7%,χ2=4.245、P<0.05);4蛋白印迹法显示小干扰-punisher组Bcl-2、caspase3-30×103、p53蛋白表达水平明显低于NC组(t分别为9.546、5.647、5.42,P<0.05),caspase3-17×103蛋白表达水平高于NC组(t=4.892、P<0.05)。结论沉默lncRNA punisher可以促进血管平滑肌细胞的凋亡并抑制其增殖。

Objective Explore the relationship and mechanism of long noncoding RNA( lncRNA) punisher with atherosclerosis( AS) and its effects on the proliferation and apoptosis of vascular smooth muscle cell( VSMC). Methods Twelve healthy male 8 week old apolipoprotein E( Apo E)knockout mice( Apo E- /-mice),produced the model of atherosclerosis in mice by high fat fed( model group); 12 healthy C57BL/6J mice were fed with basal diet as control group; hunman vascular smooth muscle cell were divided in small interfering-punisher group and negative control( NC)group by their different transfection. Aortic arch tissue sample of the AS model and control mice were used to determine the punisher expression using fluorescence quantitative polymerase chain reaction( PCR) method. Small interference RNA of punisher were used to downregulate the expression of punisher in vascular smooth muscle cell. Moreover,cell counting kit-8( CCK-8),scratch-wound experiment and flow cytometry method were used to detect the regulatory role of punisher in vascular smooth muscle cell proliferation migration and apoptosis respectively. Finally Western Blot was used to determine the expression levels of B-cell leukemia-lymphoma-2( Bcl-2). Results(1) The levels of triacylglycerol( TG),total cholesterol( TC),low density lipoprotein-cholesterol( LDL-C) and high density lipoprotein-cholesterol( HDL-C) along with Hematoxylin 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( P>0. 05) lower proliferation level than the NC group( t36= 2. 683,P<0. 05; t48= 2. 554,P<0. 05). Scratch-wound experiments si-punisher cells migration has been supressed( t = 3. 136,P < 0. 05).(3) Flow cytometry showed that silencing of punisher lncRNA significantly increased VSMC apoptosis cell number compared to NC group( 22. 6% and 11. 7%; χ2= 4. 245,P<0. 05).(4)Western Blot results showed a significant down regulation of expression of Bcl-2,caspase3-30 × 103 and p53( t respectively 9. 546,5. 647,5. 42; P<0. 05) and a significant upregulation of expression of caspase 3-17×103( t= 4. 892,P<0. 05) upon silencing of punisher lncRNA in VSMC. Conclusion Silence lncRNA punisher can increasing vascular smooth muscle cell apoptosis and downregulation of cell proliferation.