RNA干扰抑制EphA2癌基因在结肠腺癌HCT-8细胞系中的表达

Retrovirus-mediated RNA inhibition of EphA2 gene expression in colon adenocarcinoma HCT-8 cells

目的利用RNA干扰(RNAi)技术抑制EphA2癌基因在结肠腺癌HCT8细胞系中的表达,以期为肿瘤的基因治疗提供新的思路和手段。方法构建逆转录病毒表达载体pSIRENEphA2,应用逆转录病毒载体介导的RNAi技术抑制EphA2癌基因在结肠腺癌细胞HCT8中的表达,同时利用Western免疫印迹和免疫组织化学SP法分别检测转染后HCT8细胞中EphA2蛋白的表达水平。结果从EphA2mRNA序列中筛选出有效的siRNA序列,并设计了互补双链寡核苷酸发夹结构,成功构建了EphA2siRNA的逆转录病毒表达载体,重组逆转录病毒载体pSIRENEphA2经酶切鉴定结果正确;测序分析证实插入的EphA2siRNA的方向及序列正确。Western免疫印迹和免疫组织化学方法分析,与对照空载体及未转染细胞相比,重组pSIRENEphA2转染的HCT8细胞中EphA2蛋白表达水平明显降低,差异具有统计学意义(P<0.001)。结论通过逆转录病毒载体介导的RNAi能够抑制HCT8细胞中EphA2的蛋白表达,为以EphA2为靶向的结直肠癌的基因治疗提供了新的思路和手段。

Objective To explore novel cancer gene therapy by retrovirus-mediated RNAi technique to suppress the endogenous EphA2 oncogene expression in colon adenocarcinoma HCT-8 cells. Methods Sequence information of EphA2 mRNA was selected and two complementary oligonucleotides with hairpin loop were designed. Retrovirus-mediated RNAi expression vector （pSIREN-EphA2） was then constructed and transfected into the HCT-8 cells. Inhibition of EphA2 protein expression was quantitatively determined by Western blot and immunohistochemistry assay （SP method ）. Results The construction of pSIREN-EphA2 vector was successful and confirmed by restriction enzyme analysis and DNA sequencing. The post- transfection level of EphA2 protein expressions was greatly reduced in HCT-8 cells transfected with pSIREN-EphA2, as compared with those of untransfected cells and the vector control （ P 〈 0. 001 ）. Condusions EphA2 protein expression in HCT-8 cell line can be suppressed using recombinant retrovirus-mediated RNAi technique. This approach may provide a novel gene therapy against colonic adenocarcinoma.