载体介导的siRNA对SGC-7901胃癌细胞中hPOT1表达的抑制作用

Suppression of the expression of hPOT1 gene in human gastric cancer cell line SGC-7901 with vector-based RNA interference technique

目的构建人端粒保护蛋白(hPOT1)基因的短链干扰核糖核酸(siRNA)表达载体,观察其在胃癌细胞中抑制hPOT1基因表达的作用,为进一步研究hPOT1在胃癌细胞生长增殖中的作用打下基础。方法设计并化学合成64nt编码hPOT1siRNA基因片段的寡核苷酸,退火成双链后将其连接到pSUPER质粒的H1RNA启动子下游,构建psiRNAhPOT1重组质粒。经酶切、测序鉴定后,用脂质体介导的基因转染方法,转入SGC7901胃癌细胞,用半定量RTPCR检测hPOT1表达水平的变化。结果测序结果表明重组质粒psiRNAhPOT164个碱基成功插入到预定位置,并且序列完全一致。hPOT1siRNA表达载体psiRNAhPOT1转入胃癌细胞后,可明显抑制SGC7901细胞hPOT1的表达。结论构建的hPOT1基因的siRNA表达载体psiRNAhPOT1重组质粒能显著抑制hPOT1基因在SGC7901胃癌细胞中的表达。

Objective To construct the expression vector of siRNA targeting the human protection of telomeres （hPOT1） gene for the observation of the inhibitory effect of the vector on hPOT1 gene expression in gastric cancer cell line SGC-7901, and to offer a ground work for further study on the roles of hPOT1 in growth and proliferation of gastric cancer cells. Methods 64 base-pair oligonucleotide for small interfering RNA expression targeting hPOT1 gene was designed and chemically synthesized. After annealing, double oligonucleotides were inserted into the downstream of HI RNA promoter of pSUPER to recombine psiRNA-hPOT1 plasmids, and transiently transfected into gastric cancer cell line SGC-7901 with liposome-mediated transfection after restriction enzyme and sequencing analysis. Semi-quantitative RT-PCR was used to detect the expression levels of hPOT1 gene. Result Recombinant psiRNA-hPOT1 vector was confirmed by se- quencing analysis. The results demonstrated that 64nt had been inserted into the expected site. Furthermore, the inserted sequence was exactly correct. The recombinant psiRNA-hPOT1 vector could significantly suppress the hPOT1 expression in gastric cancer cell line SGC- 7901. Condusion The constructed siRNA expression vector of hPOT1 gene psiRNA-hPOT1 recombinant plasmid can block the expression of hPOT1 gene in gastric cancer cell line SGC-7901.