靶向Plk1的siRNA对肝癌细胞凋亡的影响  

Small interfering RNA-mediated knockdown of polo-like kinase 1 promotes apoptosis in human hepatocellular carcinoma cell line BCL-7402

作　　者：孙威[1] 刘宝林[1] 陈爱山[1] 曹献馗[1] 苏琪[1] 

机构地区：[1]中国医科大学附属盛京医院结直肠,肛门病外科,辽宁省沈阳市110004

出　　处：《世界华人消化杂志》2011年第27期2822-2828,共7页World Chinese Journal of Digestology

摘　　要：目的:观察靶向作用于Plk1的siRNA对肝癌细胞系BCL-7402细胞中Plk1基因和p53基因表达及细胞凋亡的影响,探求靶向该基因的治疗在肝癌基因治疗中的可行性及效应.方法:设计合成两对靶向作用于Plk1基因的双链siRNA序列,分别命名为siRNA1和siRNA2,应用脂质体法将其转入BCL-7402细胞中.实验分为siRNA1、siRNA2、无关对照及空白对照4组.通过RT-PCR检测各组细胞中Plk1 mRNA表达的变化;Western blot检测各组细胞中Plk1和P53蛋白表达的变化;流式细胞仪检测细胞周期及细胞凋亡的改变;透射电镜观察细胞超微结构的变化.结果:转染后24h和48h,Plk1 mRNA相对水平siRNA1组分别较无关对照组和空白对照组下降了50%、51%和60%、62%,siRNA2组分别较无关对照组和空白对照组下降了42%、42%和54%、56%,siRNA1组、siRNA2组的Plk1 mRNA相对水平与无关对照组和空白对照组相比有统计学差异(P<0.01).Plk1蛋白表达的变化趋势与Plk1 mRNA的变化趋势相似,P53蛋白的表达随着Plk1表达的抑制而明显增加(P<0.01).转染后24h,转染组中G2/M期的细胞数量明显增加(P<0.01).转染后48h,转染组中出现大量的凋亡细胞,透射电镜下可见凋亡早期及晚期形态学改变.结论:靶向作用于Plk1的siRNA能够抑制肝癌细胞系BCL-7402细胞中Plk1基因的表达,同时增加p53基因的表达,促进转染细胞的凋亡,提示Plk1基因在对肝癌细胞的细胞周期及凋亡的调控中起重要作用.AIM： To investigate the effect of small interfering RNA （siRNA）-mediated Polo-like kinase 1 （Plkl） gene silencing on p53 expression and cell apoptosis in human hepatocellular carcinoma cell line BCL-7402, and to explore the feasibility of targeting the human Plkl gene as a therapeutic strategy for hepatocellular carcinoma.METHODS： Two siRNA sequences （siRNA1 and siRNA2） targeting the human Plkl gene were designed and synthesized. BCL-7402 cells were transfected with blank control, negative control, siRNA1 or siRNA2 via lipofection. After transfection, reverse transcription-polymerase chain reaction （RT- PCR） was used to examine the expression of Plkl mRNA, and Western blot was used to examine the expression of Plkl and P53proteins in transfected BCL-7402 cells. Cell cycle distribution and apoptosis of transfected cells were monitored by flow cytometry （FCM）. The ultrastructural changes of transfected BCL-7402 cells were observed by transmission electron microscopy （TEM）.RESULTS： BCL-7402 cells transfected with low doses of siRNAs targeting the Plkl gene showed greatly decreased levels of Plkl mRNA and protein. In the siRNA1 group, Plkl mRNA ex- pression was reduced by 51% and 62% and Plkl protein expression by 65% and 81% 24 and 48 h after transfection （all P 〈 0.01）. In the siRNA2 group, Plkl mRNA expression was reduced by 42% and 56% and Plkl protein expression by 51% and 65% 24 and 48 h after transfection （all P 〈 0.01）. P53 protein levels increased obviously with the decrease in Plkl protein levels （P 〈 0.01）. The percentage of cells at G2/M phase increased obviously 24 h after transfection （P 〈 0.01）. Apoptosis rate increased remarkably and apoptotic phenotypes could be seen by TEM. in cells 48 h after transfection.CONCLUSION： SiRNAs targeting the human Plkl gene remarkably inhibited Plkl expression, increased p53 gene expression, and promoted apoptosis, suggesting that the Plkl gene plays important roles in cell cycle control and apoptosis of BCL-7402

关 键 词：小干扰RNA 保罗样激酶1 肝脏肿瘤 BCL-7402细胞 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]