siRNA技术沉默SOCS3对缺氧复氧心肌细胞增殖、凋亡的影响以及作用机制

Effect of SOCS3 on proliferation and apoptosis of hypoxic reoxygenation myocardial cells and its mechanism of action by siRNA

目的:研究小干扰RNA(siRNA)靶向沉默细胞因子信号转导抑制因子3(SOCS3)基因对心肌细胞增殖、凋亡的影响以及可能作用机制。方法:采用脂质体Lipofectamine 2000转染大鼠H9C2心肌细胞,分为对照组、缺氧/复氧组、缺氧/复氧+siRNA NC组、缺氧/复氧+siRNA SOCS3组;免疫印迹试验(Western blot)检测细胞的转染效果;噻唑蓝(MTT)观察细胞的增殖活性,观察细胞中乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平的变化;膜联蛋白V-FITC(Annexin V-FITC)/碘化丙啶(PI)染色法检测细胞凋亡率,Western blot检测核因子-κB(NF-κB)、细胞周期蛋白D1(Cyclin D1)、c-Myc蛋白水平。结果:与对照组相比,缺氧/复氧组心肌细胞SOCS3蛋白水平显著增加(P<0. 05),细胞的增殖活性、SOD显著下降(P<0. 05),LDH、MDA、凋亡率显著升高(P<0. 05);靶向沉默缺氧复氧心肌细胞SOCS3基因表达较缺氧/复氧组细胞的增殖活性和SOD显著增加(P<0. 05),LDH、MDA、凋亡率显著降低(P<0. 05);缺氧/复氧干预心肌细胞显著抑制NF-κB、Cyclin D1、cMyc蛋白表达(P<0. 05),下调SOCS3基因表达显著增加NF-κB、Cyclin D1、c-Myc蛋白表达(P<0. 05)。结论:沉默SOCS3表达促进缺氧复氧心肌细胞增殖,抑制其凋亡,增强机体氧自由基的清除能力,其作用机制与NF-κB信号通路的激活有关。

Objective:To investigate the relationship between Suppressor of cytokine signaling 3(SOCS3)and cardiomyocyte proliferation and apoptosis and its possible mechanism by Small interference RNA(siRNA).Methods:RNAi was transfected into H9C2 cells to silence the expression of SOCS3 gene by lipofectamine 2000,and the experiment were divided into control group,hypoxia/reoxygenation group,hypoxia/reoxygenation+siRNA NC group,hypoxia/reoxygenation+siRNA SOCS3 group.The transfected efficiency of cells was detected by Western blot.The proliferation activity of cells was observed by MTT assay and the apoptotic rate was analyzed by Annexin V-FITC/PI staining.The activities of Lactate dehydrogenase(LDH),superoxide dismutase(SOD),Maleic Dialdehyde(MDA)were observed.The protein levels of nuclear factor-κB(NF-κB),cyclin D1 and c-Myc were examined by Western blot.Results:Compared with the control group,the SOCS3 protein level in cardiomyocytes of hypoxia/reoxygenation group was significantly increased(P<0.05);Hypoxia/reoxygenation had the significantly effects on inhibiting cell proliferation activity and SOD level(P<0.05),promoted cell apoptosis,and increased the levels of LDH,MDA(P<0.05).Compared with hypoxia/reoxygenation group,silencing SOCS3 expression in hypoxia-reoxygenation cardiomyocytes could promoted cell proliferation activity and SOD level(P<0.05),inhibited cell apoptosis,and decreased the levels of LDH,MDA(P<0.05).The protein levels of NF-κB,Cyclin D1 and c-Myc decreased significantly in hypoxia-reoxygenation cardiomyocytes.Down-regulation of SOCS3 gene expression significantly increased the expression of NF-κB,Cyclin D1 and c-Myc protein in H9C2 cells of hypoxia/reoxygenation(P<0.05).Conclusion:Silencing SOCS3 expression can promote the proliferation of hypoxia-reoxygenation cardiomyocytes,inhibit cells apoptosis,and enhance the ability of scavenging oxygen free radicals.Its mechanism is related to the activation of NF-κB signaling pathway.