RNA干扰MBP-1基因对胃癌细胞SGC-7901增殖影响

Effects of RNAi of MBP-1 gene on proliferation of gastric cancer SGC-7901 cell line

目的探讨MBP-1(c-myc promter binding protein 1,MBP-1)基因表达沉默对胃癌细胞株SGC-7901细胞增殖影响。方法实验分3组:空白对照组(未转染胃癌细胞)、阴性对照组(转染错义序列)和干扰组(转染MBP-1shRNA)。设计2条针对MBP-1基因的小干扰RNA片段及1条阴性对照siRNA,并构建入pSIREN-retroQ质粒。将构建的重组pSIREN-retroQ质粒通过Lipofectamine 2000脂质体转染胃癌SGC-7901细胞,嘌呤霉素筛选稳转株细胞。Real time PCR和Western blot分别检测MBP-1表达。MTT法对MBP-1干扰后SGC-7901细胞增殖进行检测。结果通过PCR扩增阳性克隆及测序,说明已成功构建MBP-1干扰及对照重组pSIREN-retroQ质粒。通过Lipofectamine 2000脂质体将重组质粒转染胃癌SGC-7901细胞,并通过嘌呤霉素筛选2周,说明已成功构建MBP-1干扰及对照SGC-7901稳转株细胞。Real time PCR检测,干扰组MBP-1mRNA相对表达量与空白对照组相比显著下调(P<0.05)。Western blot检测MBP-1蛋白表达,干扰组MBP-1表达量与空白对照组相比也都显著下调。MTT法检测结果表明,MBP-1干扰组细胞在48、72、96和120h增殖能力比空白对照组都有显著的升高(P<0.05)。结论下调MBP-1基因表达能明显促进胃癌细胞SGC-7901的增殖,从而为胃癌基因治疗提供了新靶点。

Objective To investigate the effects of c-myc promoter binding protein（MBP-1）gene expression silencing on the pro-liferation in vitro in human gastric cancer cell line SGC-7901.Methods The cells divided into three groups：blank control group （cells without transfecting gastric cancer cell）,negative control group（cells transfecting missense sequence）and experimental group （cells transfecting MBP-1 shRNA）.Two MBP-1 shRNA sequences and one negative control shRNA sequence were designed,syn-thesized and cloned into pSIREN-retroQ plasma.Then the recombinant plasmids were constructed and transfected into human gas-tric cancer SGC-7901 cells by Lipofectamine 2000.After selection of cells with puromycin for two weeks,stable cell line SGC-7901 expressing gene of MBP-1 specific shRNA was obtained.The expressions of MBP-1 mRNA and protein in SGC-7901 were deter-mined by the real time PCR and Western blot,respectively.The effects of altered expression of MBP-1 on the cell proliferation were measured by MTT cell proliferation assay.Results PCR and sequencing indicated that the recombinant plasmids pSIREN-retroQ was constructed.Then the recombinant plasmids were transfected into human gastric cancer SGC-7901 cells by Lipofectamine 2000. After selection of cells with puromycin for two weeks,stable cell line SGC-7901 expressing gene of MBP-1 specific shRNA was ob-tained.The relative expression level MBP-1 mRNA in the MBP-1 siRNA transfection group was significantly decreased compared with the blank control group（P 〈0.05）.Compared with the blank group,the expression levels of MBP-1 protein in the experimental group also significantly decreased.The proliferation abilities of SGC-7901 cells at 48,72,96,120 h after MBP-1 siRNA transfection were significantly increased compared with the blank control group （P 〈 0.05 ）.Conclusion Down-regulating the expression of MBP-1 can obviously promote the proliferation of human gastric cancer cell line SGC-7901.MBP-1 gene may become the new target of gene therapy for gastric cancer.