人端粒酶逆转录酶基因siRNA质粒的构建及其对不同p53状态卵巢癌细胞生长和凋亡的影响  

Construction of siRNA plasmid for human telomerase reverse transcriptase gene and its effect on growth and apoptosis of ovarian cancer cells in presence or absence of p53

作　　者：罗祎[1] 姚珍薇[1] 杨雅莹[2] 易永芬[2] 

机构地区：[1]重庆医科大学附属第一医院妇产科,重庆400016 [2]重庆医科大学分子医学与肿瘤研究中心病理学教研室,重庆400016

出　　处：《中国生物制品学杂志》2013年第6期780-786,791,共8页Chinese Journal of Biologicals

摘　　要：目的构建人端粒酶逆转录酶(Human telomerase reverse transcriptase,hTERT)基因小干扰RNA(Small interferingRNA,siRNA)质粒,并探讨其对不同p53状态卵巢癌细胞SKOV3(缺p53)和OVCAR3(含p53)生长和凋亡的影响。方法设计并合成针对hTERT基因的siRNA,与表达的质粒pGenesil-1连接,构建重组表达质粒pG1、pG2和pGCR(阴性对照质粒),分别转染卵巢癌细胞SKOV3和OVCAR3,并设未转染细胞对照组和转染空载体对照组。转染后48 h,荧光显微镜观察并计算转染效率;实时荧光定量PCR法检测转染细胞中hTERT基因mRNA的表达;Western blot检测转染细胞中hTERT、p53和p21蛋白的表达;端粒重复序列扩增(Telomeric repeat amplification portocol,TRAP)法检测转染细胞端粒酶的活性;CCK-8法检测细胞生长情况;流式细胞仪和AnnexinⅤ-PE/7-AAD双染法分别检测细胞周期和细胞凋亡的情况。结果已成功构建了靶向hTERT siRNA重组质粒pG1、pG2和阴性对照质粒pGCR,具有较高的转染效率,与pGCR转染组相比,pG1和pG2转染组hTERT基因和蛋白表达(P<0.05)及端粒酶活性均明显降低,pG1和pG2转染组均可抑制两种细胞的生长,且对OVCAR3细胞的抑制作用强于SKOV3细胞;pG1和pG2转染组G0-G1期细胞比例明显下降,S期细胞比例明显增多(P<0.05);在OVCAR3细胞中,hTERT表达受抑后均出现了明显的凋亡,同时伴随p53和p21蛋白表达的增加,在缺乏p53基因的SKOV3细胞中并未导致明显的凋亡,其p21蛋白表达明显低于pGCR转染组(P<0.05)。结论构建的靶向hTERT siRNA重组表达质粒在体外能有效和特异地沉默卵巢癌细胞hTERT基因的表达,降低端粒酶活性,并引起肿瘤细胞的生长抑制和凋亡,其机制可能与抑癌基因p53及p21上调有关。Objective To construct the siRNA plasmid for human telomerase reverse transcriptase（hTERT）gene and investigate its effect on the growth and apoptosis of ovarian cancer cells in presence or absence of p53.Methods The siRNA targeting to hTERT gene was designed and synthesized,and inserted into expression vector pGenesil-1.Ovarian cancer SKOV3（p53 null）and OVCAR3（with p53）cells were transfected with the constructed recombinant plasmids pG1,pG2 and pGCR（negative control）separately,using the untransfected cells and empty vector-transfected cells as controls,and observed by fluorescent microscopy 48 h later,based on which the transfection efficiencies were calculated.The expression of hTERT at mRNA level in transfected cells was determined by PCR,while those of hTERT,p53 and p2a at protein level by Western blot.The telomerase activity in transfected cells was determined by TRAP method.The cell growth was observed by CCK-8 method.The cell cycle and cell apoptosis were determined by flow cytometry and Annexin Ⅴ-PE/7-AAD staining respectively.Results The siRNA plasmids pG1 and pG2 targeting to hTERT as well as negative control plasmid pGCR were constructed successfully,which showed high transfection efficiencies.As compared with those transfected with pGCR,the expression of hTERT at mRNA and protein levels（both P ＆lt; 0.05） as well as telomerase activity in the cells transfected with pG1 and pG2 decreased significantly.Both pG1 and pG2 inhibited the growth of SKOV3 and OVCAR3 cells,while the inhibiting effect on the latter was superior to that on the former.The percentages of cells at G 0-G 1 phases in pG1 and pG2 transfection groups decreased significantly,while those at S phase increased significantly（P ＆lt; 0.05）;obvious apoptosis was observed in OVCAR3 cells after the expression of hTERT was inhibited;however,no obvious apoptosis was observed in the SKOV3 cells with the increased expressions of p53 and p21 proteins,while the expression level of p21 protein was significantly lower than tho

关 键 词：人端粒酶逆转录酶 RNA干扰 卵巢癌 细胞增殖 细胞凋亡 

分 类 号：R737.31[医药卫生—肿瘤] Q782[医药卫生—临床医学]