RNAi沉默hTERT基因诱导大肠癌SW480细胞凋亡  

RNA interference-based hTERT gene silence induces apoptosis of colorectal cancer SW480 cells

作　　者：蔡艳玲[1] 罗小玲[1] 葛连英[2] 刘爱群[2] 谢裕安[1] 

机构地区：[1]广西医科大学附属肿瘤医院生物治疗科,广西壮族自治区南宁530021 [2]广西医科大学附属肿瘤医院内镜室,广西壮族自治区南宁530021

出　　处：《中国肿瘤生物治疗杂志》2011年第1期46-50,共5页Chinese Journal of Cancer Biotherapy

基　　金：广西自然科学基金资助项目( No. 2010GXNSFA013238);广西卫生厅重点课题( No. Z200971)~~

摘　　要：目的:探讨RNA干扰人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)的表达对大肠癌细胞SW480凋亡的影响。方法:构建携带hTERT小发夹干扰RNA(small hairpin RNA,shRNA)的重组表达载体pGPU6/GFP/Neo-hTERT-shRNA(简称hTERT-shRNA质粒),脂质体法转染SW480细胞,RT-PCR法检测不同转染时间点SW480细胞中hTERTmRNA的表达。TRAP-PCR-ELISA法检测转染后48h SW480细胞的端粒酶活性,透射电镜观察转染后48h SW480细胞超微结构。结果:hTERT-shRNA质粒转染48h时,hTERT-shRNA组SW480细胞hTERT mRNA表达的抑制率显著高于空白组、脂质体组、NC-shRNA组(75.0%vs39.2%、33.3%、28.0%,P<0.05)。hTERT-shRNA转染组SW480细胞端粒酶活性显著低于空白组、脂质体组、NC-shRNA组(2.242±0.285vs2.756±0.089、2.693±0.225、2.691±0.120,P<0.05)。hTERT-shRNA质粒转染后的SW480细胞体积明显缩小、细胞核固缩、染色质不均匀地沿核膜下聚集、空泡形成增多,出现典型的凋亡形态。结论:RNAi可有效沉默SW480细胞中hTERT的表达,降低SW480细胞端粒酶活性,诱导SW480细胞凋亡。Objective：To investigate the effect of RNA-mediated interference of hTERT （human telomerase reverse transcriptase） expression on the apoptosis of colorectal cancer cell line SW480. Methods：Small hairpin RNA （shRNA） targeting hTERT was synthesized and recombinant plasmid pGPU6/GFP/Neo-hTERT-shRNA （named hTERT-shRNA plasmid） containing hTERT-shRNA was constructed. SW480 cells were transfected with hTERT-shRNA plasmid by liposome method,and the expression of hTERT mRNA in SW480 cells was detected by RT-PCR analysis at different time points. The telomerase activity of SW480 cells was examined by TRAP-PCR-ELISA analysis. The ultrastructure of SW480 cells was examined by TEM （transparent electron microscope） 48 h after hTERT-shRNA transfection. Results：The inhibitory rate of hTERT mRNA expression in SW480 cells of hTERT-shRNA group was significantly higher than those of blank group,liposome group,and NC-shRNA group （75.0% vs 39.2%,33.3%,28.0%,P〈0.05）. Telomerase activity in SW480 cells of hTERT-shRNA group was significantly decreased compared with those of blank group,liposome group,and NC-shRNA group （2.242±0.285 vs 2.756±0.089,2.693±0.225,2.691±0.120,P〈0.05）. SW480 cells in hTERT-shRNA group showed smaller cell size,nuclear condensation,uneven aggregation of chromatin along the nuclear membrane,and increased vacuolization. Conclusion：RNA interference can effectively silence hTERT expression,reduce telomerase activity,and induce apoptosis of SW480 cells.

关 键 词：RNA干扰 大肠癌 HTERT 端粒酶 凋亡 

分 类 号：R735.34[医药卫生—肿瘤] R730.54[医药卫生—临床医学]