RNA干扰enolase-1基因治疗胃癌的实验研究

The Effect of RNA Interference of the Enolase-1 Gene on Gastric Carcinoma

目的:探讨RNA干扰enolase-1基因对人胃癌的治疗作用。方法:根据enolase-1mRNA序列构建表达enolase-1特异的小干扰RNA(small interfering RNA,siRNA)真核表达质粒pMSCVU6/ENO-1si1,pMSCVU6/ENO-1si2,pMSCVU6/ENO-1si3。种植人胃癌SGC-7901细胞于裸鼠皮下建立裸鼠肿瘤模型。将构建成功的三种siRNA质粒分别转染SGC-7901细胞株,pMSCVU6/ENO-1si2和pMSCVU6/ENO-1si3转染裸鼠瘤体内。MTT法检测SGC-7901细胞增殖状况;测量荷瘤裸鼠的肿瘤体积,计算抑瘤率;RT-PCR检测SGC-7901细胞enolase-1mRNA表达;分别以免疫组化SP法和Western blot检测体内外enolase-1蛋白表达。结果:SGC-7901细胞株及裸鼠瘤体均被所采用的质粒成功转染。SGC-7901细胞受pMSCVU6/ENO-1si2转染后,其生长活性受到明显抑制(P<0.01),enolase-1mRNA及蛋白表达显著降低(P<0.01)。体内抑瘤实验表明,pMSCVU6/ENO-1si2组平均瘤体积为(380±26)mm3,pMSCVU6/ENO-1si3组与空白对照组分别为(993±124)mm3和(1102±131)mm3;pMSCVU6/ENO-1si2组抑瘤率为65.52%,pMSCVU6/ENO-1si3组为9.89%;pMSCVU6/ENO-1si2组肿瘤体积明显小于pMSCVU6/ENO-1si3组和空白对照组,差异具有显著性(P<0.01);pMSCVU6/ENO-1si3组和空白对照组之间肿瘤体积无显著差异(P>0.05);pM-SCVU6/ENO-1si2组与pMSCVU6/ENO-1si3组和空白对照组相比,enolase-1蛋白表达减弱。结论:表达siRNA的enolase-1基因特异的真核表达质粒能成功转染体内、外胃癌细胞,其中pMSCVU6/ENO-1si2可有效抑制人胃癌细胞的生长。

Objective： To assess the effect of RNA interference of enolase-1 mRNA in the treatment of gastric carcinoma. Methods： Three types of plasmids were synthesized： pMSCVU6/ENO-1si1, pMSCVU6/ENO-1si2. and pMSCVU6/ENO-lsi3. The tumor model was established in nude mice with subcutaneous implantation of SGC-7901 gastric carcinoma cells. pMSCVU6/ENO-1si1. pMSCVU6/ENO-1si2 and pMSCVU6/ENO-1si3 were transfected into SGC-7901 cells, pMSCVU6/ ENO-1si2 and oMSCVU6/ENO-1si3 were transfected into the transplanted tumors. The proliferation of SGC-7901 cells was detecled by MTT assay. The growth inhibiting tale was ealeulaled. Enolase-1 mRNA expression in SGC-7901 cells was deteeled by RT-PCR. Western blot and immunohistochemistry were employed to confirm enolase-1 protein expression in vitro and in vivo. Results： Aider the SGC-7901 cells were transfected with the plasmids, the viahility of cells was inhibited by pMSCVU6/ENO-1si2 （P〈0.01）. pMSCVU6/ENO-1si2 decreased the level of enolase-1 mRNA and protein expression in SGC-7901 cells（P〈0.01）. In vivo experiments demonslrated that the tumor volume was 380±26mm^3 in the pMSCVU6/ENO-1si2 group, 993±124mm^3 in the pMSCVU6/ENO-1si3 group, and 1102±131 mm^3 in the control group. The growth inhibiting rate was 65.52% in the pMSCV U6/ENO-1si2 group and 9.89% in the pMSCVU6/ENO-1si3 group. The tumor volume in the pMSCV U6/ENO-1si2 group was smaller than that in the pMSCVU6/ENO-1si3 group and the control group（P〈0.01）. No significant difference was found in tumor volume between the pMSCVU6/ENO-1si3 group and the controll group （P〉0.05）. Enolase-1 protein expression was depressed in the pMSCVU6/ENO-1si2 group compared with that inthe pMSCVU6/ENO-1si3 group and the control group, pMSCVU6/ENO-1si2 significantly inhibited tumor growth in nude mice （P〈0.01） and depressed enolase-1 protein expression in viv. Conclusion： Small interfering RNA （siRNA） plasmids that express specific sequences of the enolase-1 gene can be successfully transfected into SGC-7901 cells in vivo and in vitro. Gastric carcinoma can be effectively inhibited by the plasmid pMSCVU6/ENO-1si2.