SiRNA稳定抑制肝癌细胞hTERT基因的表达
【关键词】 肝肿瘤Stable inhibition of hTERT gene by siRNA in hepatocarcinoma cells
【Abstract】 AIM: To elucidate the timeefficiency relation of stable inhibition of hepatocarcinoma cell proliferation by RNA interference in vitro. METHODS: The stable screeninginhibition technique of RNAi was adopted to suppress the expression of hTERT stably. After small hairpin RNA (shRNA) targeting hTERT gene was designed, recombinant vector pGenesilshRNAhTERT was constructed and transfected into hepatocarcinoma SMMC7721 cells, which were stably selected by G418 to establish hepatocarcinoma cell lines stablely expressing pGenesilshRNAhTERT. Realtime RTPCR, MTT and PCRTRAP were utilized to detect the alterations of hTERT mRNA expressions, telomerase activity and cell proliferation. RESULTS: The effectiveness of RNAi existed continually and stably in hepatocarcinoma SMMC7721 cells expressing stably pGenesilshRNAhTERT. In the cell lines expressing stably pGenesilshRNAhTERT, hTERT mRNA was obviously suppressed, and the telomerase activities were significantly decreased. Hepatocarcinoma SMMC7721 cell proliferation significantly inhibited in pGenesilshRNAhTERT group compared to that in negative control group. CONCLUSION: RNAi may continually and stably suppress hTERT mRNA expression and carcinoma cell proliferation, which is a potential new approach for antitumor gene therapy.
【Keywords】 RNA interference; liver neoplasms; telomerase
【摘要】 目的： 利用RNA干扰稳定筛选抑制技术，抑制人端粒酶逆转录酶（hTERT）基因表达，探讨靶向hTERT基因RNAi与抑制肝癌细胞增殖的时效关系. 方法： 设计靶向hTERT基因的小干扰RNA，构建重组表达质粒pGenesilshRNAhTERT并导入肝癌SMMC7721细胞株，经G418筛选，建立稳定表达siRNAhTERT的细胞株. 采用realtime RTPCR、MTT和PCRTRAP法同时检测pGenesilshRNAhTERT稳定抑制组和未处理SMMC7721细胞组hTERT基因表达、端粒酶活性及细胞增殖变化. 结果： 在稳定表达pGenesilshRNAhTERT的SMMC7721细胞株中，RNAi效力持续、稳定存在，hTERT mRNA表达、端粒酶活性明显降低，瘤细胞增殖被抑制. 结论： RNA干扰能持续、稳定地抑制靶基因hTERT mRNA表达及肿瘤细胞增殖，是有潜力的基因治疗肿瘤新方法.
【关键词】 RNA干扰；肝肿瘤；端粒，末端转移酶
0引言
端粒酶的异常表达与恶性肿瘤的发生发展和预后密切相关［1］， hTERT基因在肝癌中的表达阳性率为89.5%［2］. 研究表明hTERT的高表达能通过多分化刺激来抑制细胞凋亡，导致细胞永生化［3］. 我们依据RNA干扰（RNA interference, RNAi）原理，使用短发夹样RNA（small hairpin RNA, shRNA）设计的RNAiDNA载体方法［4］，以hTERT基因为靶点，构建稳定表达小干扰RNA(small interfering RNA, siRNA) 的'肝癌SMMC7721细胞株，从体外研究siRNA稳定、特异诱导hTERT基因转录后沉默，逆转癌细胞恶性表型的能力，探讨RNAi基因治疗恶性肿瘤的时效性.
1材料和方法
1.1材料
肝癌SMMC7721细胞株购自上海细胞生物研究所. 质粒pGenesil1购自武汉晶赛生物工程技术有限公司. 限制性内切酶BamHⅠ和HindⅢ购自Roche公司,总RNA提取试剂、转染试剂LipofectamineTM2000等分别购自Takara和Invitrogen公司；端粒酶TRAPHyb Kit购自华美生物工程公司. Taqman探针和引物由Takara公司合成，hTERT及内参PO的探针和引物序列参照文献［4］. siRNAhTERT转录模板由上海博亚公司合成.
1.2方法