RNAi抑制iASPP基因表达对大肠癌细胞凋亡的影响及机制

Effect of Inhibiting of iASPP Gene Expression by RNA Interference on Apoptosis of Colorectal Cancer Cells

[目的]探讨RNAi抑制iASPP基因表达对大肠癌细胞凋亡的影响。[方法]参照脂质体LipofectamineTM2000转染说明转染i ASPP的siRNA (iASPP-siRNA组)进入人大肠癌LoVo细胞株,并设置阴性对照组(转染无义的siRNA序列)和空白对照组(仅加入脂质体),Western bloting检测转染48h的3组细胞中iASPP的蛋白表达;CCK8法于转染的24h、48h、72h检测细胞活力;流式细胞术检测转染48h的细胞凋亡率;Western blotting检测Ki-67、含半胱氨酸的天冬氨酸蛋白水解酶3 (Caspase3)、STAT3和磷酸化的信号转导与转录因子3(pSTAT3)的蛋白表达。[结果] i ASPP-siRNA组(0.108±0.013)LoVo细胞株iASPP的蛋白表达显著低于空白对照组(0.389±0.036)(P<0.05);i ASPP-siRNA组细胞在24h (0.267±0.034)、48h(0.495±0.067)和72h(0.682±0.068)的活力细胞均显著低于空白对照组(0.385±0.042、0.688±0.074、0.977±0.097)(P<0.05),在48h的细胞凋亡率(11.18%±0.78%)显著高于空白对照组(2.13%±0.45%)(P<0.05)。Ki-67(0.126±0.018)和p-STAT3(0.110±0.012)的蛋白表达水平显著低于空白对照组(0.365±0.045、0.169±0.018),Caspase3(0.197±0.020)表达水平高于空白对照组(0.086±0.009)(P<0.05),3组间STAT3蛋白表达水平差异无统计学意义(P>0.05)。[结论]通过RNAi抑制iASPP基因表达可降低大肠癌细胞增殖,诱导细胞凋亡,机制可能是下调STAT3信号,对细胞增殖的影响是下调Ki-67表达,凋亡的影响是上调Caspase3表达。

[Objective]To investigate the effect of inhibiting iASPP gene expression by RNA interference on apoptosis of colorectal cancer cells.[Methods]Human colorectal cancer LoVo cells were transfected with i ASPP siRNA(iASPP-siRNA group),nonsense sequence siRNA(negative control group)or liposome alone(blank control group)for 24,48 and 72 h,respectively.The expression of iASPP protein in three groups was detected by Western blotting;cell viability was detected by CCK8 assay,cell apoptosis was detected by flow cytometry;the expression of Ki-67,Caspase3,STAT3 and p-STAT3 proteins was detected by Western blotting.[Results]The expression of i ASPP protein in iASPP-siRNA group(0.108±0.013)was significantly lower than that in blank control group(0.389±0.036)(P<0.05).The cell activity in iASPP-siRNA group at 24 h(0.267±0.034),48 h(0.495±0.067)and 72 h(0.682±0.068)were significantly decreased compared with the control group(0.385±0.042,0.688±0.074,0.977±0.097).Cell apoptosis rate(11.18%±0.78%)increased significantly at 48 h compared with the blank control group(2.13%±0.45%)%.The expression of Ki-67(0.126±0.018)and p-STAT3 protein(0.110±0.012)was significantly decreased compared with the control group(0.365±0.045,0.169±0.018),and the Caspase3 protein expression(0.197±0.020)was significantly increased compared with the control group(0.086±0.009)(all P<0.05).There was no significant difference in protein expression of STAT3 among three groups(P>0.05).[Conclusion]Inhibition of i ASPP expression by RNA interference can reduce the proliferation of colorectal cancer cells and induce cell apoptosis,which is associated with the down-regulation of p-STAT3 and Ki-67 expression,and up-regulation of Caspase3 expression.