端粒酶逆转录酶小干扰RNA对结肠癌SW480细胞体外抑制作用的观察

Anti-cancer effect of siRNA targeting human telomerase reverse transcriptase in SW480 cells

目的:探讨RNA干扰(RNAi)对结肠癌SW480细胞人端粒酶逆转录酶(hTERT)基因的抑制效应。方法:化学合成靶向于hTERT基因的4个小分子干扰RNA(siRNA)序列,用阳离子脂质体为载体转染SW480细胞,分为siRNA1～4、siRNA阴性对照和空白对照6组。通过RT-PCR和蛋白质印迹法分别检测结肠癌细胞转染后细胞hTERT mRNA和蛋白水平,并用MTS法检测细胞生长增殖。结果:siRNA1～4组hTERT mRNA相对表达量分别为0.54±0.18、0.56±0.19、0.46±0.10和0.84±0.18,端粒酶相对活性分别为0.57±0.18、0.56±0.19、0.09±0.08和0.70±0.19,siRNA1～4组hTERT蛋白相对表达量分别为0.41±0.18、0.45±0.17、0.08±0.07和0.82±0.16,其中siR-NA1～3组的hTERT mRNA表达量、端粒酶活性和蛋白表达量与空白对照组的差异有统计学意义,P<0.05。SW480细胞在转染后48h时的细胞增长平均抑制率分别为42.14%、39.12%、48.05%和35.85%。提示不同siRNA序列对SW480的转染效率不同,其中siRNA3组能显著下调hTERT mRNA和蛋白的表达水平,并能抑制端粒酶活性和促进SW480细胞凋亡,与空白对照组比较均差异有统计学意义,P<0.01。结论:靶向hTERT的RNAi能明显抑制SW480的增殖,hTERT可作为结肠癌基因治疗的靶点。

OBJECTIVE：To explore the inhibitory effect of RNA interference （RNAi） targeting human telomerase reverse transcriptase （hTERT） gene of colon carcinoma SW480 cells. METHODS： Four small interfering RNAs （siRNAs） targeting hTERT gene were synthesized by chemistry, and siRNAs were transfected into SW480 cells by cationic liposome. And SW480 cells were divided into six groups： blank group （untransfected SW480 cells group）, siRNA-NC group（negative control group）, siRNA 1-- 4 group（RNA interference group）. The hTERT mRNA, hTERT protein and telomerase activity were detected by RT PCR,Western blot and TRAP process, respectively. The proliferation of the SW480 cell was analyzed by MTS kits. RESULTS： The hTERT mRNA relative expression of siRNA 1--4 groups after transfection were as follow： 0.54±0.18,0.56±0.19,0.46±0.10,0.84± 0.18. The telomerase relative activity of siRNA 1- 4 groups were 0. 57±0.18,0. 56±0. 19,0.09±0. 08,0.70±0.19, respectively. The relative expression level of hTERT protein of siRNA 1- 4 groups were 0.41±0.18,0.45±0.17,0.08±0. 07,0.82±0.16, respectively. SW480 cells average inhibition rate were 42. 14%, 39.12% ,48.05% ,35.85% ,respectively. In contrast to the blank groups, the relative expression level of the hTERT mRNA and pro- tein and telomerase activity of siRNA-3 group was declined （P〈 0. 05）,in addition,the difference of them in siRNA 3 group vs the blank were significant （P〈0.01）. CONCLUSIONS： RNAi can in- hibit the proliferation of the cancer cells. The hTERT may be a target point of gene therapy for Colon carcinoma.