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目的:构建靶向人大肠癌细胞端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)基因的RNAi载体,探讨其对人大肠癌SW480细胞增殖的影响。方法:设计3条靶向hTERT的shRNA序列和阴性对照序列,分别克隆入pGPU6/GFP/Neo载体,构建RNAi载体pGPU6-hTERT-1、2、3和阴性对照载体pGPU6-hTERT-NC,转染SW480细胞。RT-PCR检测各组载体对hTERT mRNA表达的影响,MTT法检测下调hTERT mRNA表达对SW480细胞增殖的影响。结果:成功构建3个携带hTERT mRNA序列的重组载体,3种RNAi载体均能明显抑制SW480细胞hTERT mRNA的表达,pGPU6-hTERT-3组SW480细胞hTERT mRNA表达水平较空白对照组下调最为显著(0.347±0.028 vs 0.513±0.032,P<0.01)。转染3种RNAi载体均能明显抑制SW480细胞增殖,pGPU6-hTERT-3组细胞增殖抑制率较空白对照组、脂质体对照组和pGPU6-hTERT-NC组升高最为显著[(50.08±0.43)%vs(4.11±0.39)%、(3.88±0.35)%、(3.38±0.35)%,P<0.05]。结论:转染RNAi载体pGPU6-hTERT-3能够抑制SW480细胞的增殖,其机制可能与降低hTERT基因的表达从而抑制端粒酶活性有关。
OBJECTIVE: To construct RNAi vector targeting human telomerase reverse transcriptase (hTERT) gene in human colorectal cancer cells and investigate its effect on the proliferation of human colorectal cancer SW480 cells. METHODS: Three hTERT-targeting shRNA and negative control sequences were designed and cloned into pGPU6 / GFP / Neo vector respectively. RNAi vector pGPU6-hTERT-1,2,3 and negative control vector pGPU6-hTERT-NC were constructed and transfected SW480 cells. The expression of hTERT mRNA in each group was detected by RT-PCR. The effect of hTERT mRNA expression on the proliferation of SW480 cells was detected by MTT assay. Results: Three recombinant vectors carrying hTERT mRNA sequence and three RNAi vectors were successfully constructed. The expression of hTERT mRNA in SW480 cells was significantly inhibited (p <0.05), and the expression of hTERT mRNA in SW480 cells in pGPU6-hTERT-3 group was significantly lower than that in control group ± 0.028 vs 0.513 ± 0.032, P <0.01). Transfection of three kinds of RNAi vectors significantly inhibited the proliferation of SW480 cells, and the inhibition rate of pGPU6-hTERT-3 cells was the highest than that of blank control group, liposome control group and pGPU6-hTERT-NC group [(50.08 ± 0.43 )% vs (4.11 ± 0.39)%, (3.88 ± 0.35)%, (3.38 ± 0.35)%, P <0.05]. CONCLUSION: Transfection of RNAi vector pGPU6-hTERT-3 can inhibit the proliferation of SW480 cells. The mechanism may be related to the decrease of hTERT gene expression and the inhibition of telomerase activity.