小干扰RNA抑制人端粒酶逆转录酶基因对胃癌细胞增殖的影响

Effects of inhibiting human telomerase reverse transcriptase gene by small interfering RNA interference on proliferation of gastric cancer SGC7901 cells

目的 应用小干扰RNA（siRNA）技术特异性抑制人端粒酶逆转录酶（hTERT）基因在胃癌SGC7901细胞中的表达,观察其对细胞增殖的影响.方法 hTERT基因的RNA干扰表达重组体用脂质体介导方法转染SGC7901细胞.荧光定量聚合酶链反应（PCR）检测hTERT基因表达.TRAP-PCR法检测端粒酶活性、噻唑蓝（MTT）比色法检测细胞增殖.结果 SGC7901细胞转染前后hTERT基因的表达强度分别为：转染pU6组平均表达量为6.5×105拷贝/μg RNA;转染pU6-hTERT-siRNA Ⅰ组平均表达量为4.1 × 104拷贝/μg RNA;转染pU6-hTERT-siRNAⅡ组平均表达量为5.4×104拷贝/μg RNA.转染pU6组与其余两组P值〈0.01;转染pU6-hTERT-siRNA Ⅰ组和转染pU6-hTERT-siRNAⅡ组P值〉0.05.端粒酶活性下降,细胞生长速度明显减慢.结论 小干扰RNA能有效抑制SGC7901细胞中hTERT基因表达,hTERT基因表达下调影响SGC7901细胞增殖.

Objective To investigate the effect of inhibiting human telomerase reverse transcriptase （hTERT） gene by small interfering RNA （siRNA） interference on the proliferation of gastric cancer SGC7901 cells.Methods The recombinant plasmids containing hTERT target sequences were transfected into SGC7901 cells by LipofectamineTM 2000.The expression of hTERT was detected by FQ-polymerase chain reaction （PCR） quantitatively.The activity of telomerase was tested by TRAP-PCR.The proliferation activity of SGC7901 cells was determined using methylthiazol tetrazolium （MTr） assay.Results After transfection of recombinant plasmids containing hTERT target sequences,the mean hTERT mRNA expression was 6.5 × 105 copy/μg RNA in pU6 group,4.1×104 copy/μg RNA in pU6-hTERT-siRNA Ⅰ group and 5.4 × 104 copy/μg RNA in pU6-hTERT-siRNA Ⅱ group,respectively.The expression of hTERT gene was specifically inhibited by pU6-hTERT-siRNAs in SGC7901 cells （P 〈0.01 ）,but there was no difference between the pU6-hTERT-siRNA Ⅰ and Ⅱ groups （P 〉 0.05）.The proliferation of gastric cancer SGC7901 cells was significantly reduced.Conclusion hTERT expression in SGC7901 cells can be inhibited significantly with siRNA and the down-regulation of hTERT expression can cause the reduction of proliferation of SGC7901 cells.