RNA干扰靶向抑制胃癌细胞株SGC7901 hTERT基因表达

背景与目的：胃癌是全人类常见恶性肿瘤之一。在我国，胃癌居各种癌症死亡之首，其总体五年生存率仅15％～20％。在目前尚无有效一级预防措施的情况下，积极探讨有效防治胃癌的方法成为必然趋势。本研究利用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对胃癌细胞增殖的抑制效应。方法：设计靶向hTERT基因的小干扰RNA，构建重组表达质粒pGenesil—shRNA—hTERT并导入人胃癌细胞系SGC7901细胞株，经G418筛选，建立稳定表达siRNA—hTERT的细胞株。采用real time RT—PCR、MTT和PCR—TRAP法同时检测pGenesil-shRNA—hTERT稳定抑制组和未处理SGC7901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pGenesil—shRNA—hTERT的SGC7901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，是潜在的肿瘤基因治疗新方法。

Inhibition of hTERT gene by siRNA in SGC7901 gastric-carcinoma cells lines

Background and purpose:Stomach cancer is one of the most common malignancy of human being.In our country,it is one of leading cancer death,5-yr survival rate for the patients is just 15%-20%.At present,we do not have effective first line prevent measure for the disease,it becomes more and more important to pursue a effective method to prevent and treat gastric cancer.In our in vitro study,the inhibition of gastric-carcinoma cell SGC7901 proliferation by RNA interference could be observed.Methods:The stable screening-inhibition technique of RNAi was adopted to stably inhibit the expression of hTERT.Small hairpin interfering RNA(shRNA) targeting hTERT gene was designed,recombinant plasmid pGenesil-shRNA-hTERT was constructed,and the plasmid was transfected into gastric-carcinoma SGC7901 cells that clones were selected by G418 in order to establish gastric-carcinoma cell lines with stable expression of pGenesil-shRNA-hTERT.Real-time RT-PCR,MTT and PCR-TRAP were utilized to detect the alterations of hTERT mRNA expressions,telomerase activity and cell proliferation.Results:The effectiveness of RNAi could be observed in gastric-carcinoma SGC7901 cells with stable expression of pGenesil-shRNA-hTERT,in these cell lines,the expression of hTERT mRNA was obviously down-regulated.Meanwhile,the telomerase activity was significantly decreased.gastric-carcinoma SGC7901 cell proliferation was significantly inhibited in pGenesil-shRNA-hTERT group compared to negative control group.Conclusions:RNAi may continually and stably suppress hTERT mRNA expression and cell proliferation,which is a potential new approach for gene therapy of neoplasm.