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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 investigate the inhibitory effect of RNAi on human telomerase reverse transcriptase (hTERT) gene in human colon cancer SW480 cells. METHODS: Four small interfering RNA (siRNA) sequences targeted to hTERT gene were chemically synthesized and transfected into SW480 cells using cationic liposomes as siRNA1 ~ 4, siRNA negative control and blank control group. The mRNA and protein levels of hTERT in colon cancer cells were detected by RT-PCR and Western blot respectively. The cell growth and proliferation were detected by MTS assay. Results: The relative expression levels of hTERT mRNA in siRNA1 ~ 4 groups were 0.54 ± 0.18,0.56 ± 0.19,0.46 ± 0.10 and 0.84 ± 0.18 respectively, and the relative activities of telomerase were 0.57 ± 0.18,0.56 ± 0.19,0.09 ± 0.08 and 0.70 ± 0.19, and the relative expression levels of hTERT protein in siRNA1 ~ 4 groups were 0.41 ± 0.18,0.45 ± 0.17,0.08 ± 0.07 and 0.82 ± 0.16, respectively. Among them, hTERT mRNA expression, telomerase activity and protein expression in siR-NA1 ~ 3 group The difference with the control group was statistically significant, P <0.05. The average cell growth inhibition rates of SW480 cells at 42 h after transfection were 42.14%, 39.12%, 48.05% and 35.85%, respectively. The results showed that different siRNA sequences had different transfection efficiency on SW480, in which siRNA3 group could significantly down-regulate hTERT mRNA and protein expression, inhibit telomerase activity and promote apoptosis of SW480 cells, compared with the blank control group, there were statistically significant differences Significance, P <0.01. Conclusion: RNAi targeting hTERT can significantly inhibit the proliferation of SW480, hTERT can be used as a target of gene therapy of colon cancer.