TRF2小干扰RNA载体的构建及表达

目的:构建TRF2小干扰RNA载体,观察其在胃癌细胞中的表达．方法:根据pSilencer3．1-H1载体要求设计两对小干扰RNA,退火后连接入载体相应位点．经双酶切及测序鉴定后分别转染多药耐药衍生胃癌细胞SGC7901／ADR和SGC7901/VCR． G418选择培养液培养2 mo选取转染组及对照组细胞克隆．MTT法检测转染对细胞生长增殖速度的影响．细胞用阿霉素和足叶乙甙处理 6h后Western blot法检测TRF2表达．结果:成功构建TRF2小干扰RNA载体,建立稳定转染细胞株,转染后TRF2表达明显降低,对细胞的生长增殖速度无明显影响(P>0．05)．结论:成功构建TRF2小干扰RNA载体及稳定转染细胞株．

Construction and expression of telomeric repeat binding factor 2 small interfering RNA eukaryotic vector

AIM: To construct telomeric repeat binding factor 2 (TRF2) small interfering RNA (siRNA) eukaryotic expression vector and evaluate its expression in gastric cancer cells. METHODS: Two pairs of hairpin siRNA template oligonucleotides for TRF2 based on pSilencer3.1-H1 vector were designed using the manufacturer's web-based tool. These oligonucleotides were annealed and ligated into vector respectively. After confirmation by double enzyme digestion analysis and DNA sequencing, positive recombinant plasmids were transfected into multidrug-resistant gastric cancer cells SGC7901/ADR and SGC7901/VCR respectively. Stable cell lines of G418-resistance were acquired after 2-month selection. The effect of transfection on cell growth was analyzed by MTT assay. The expression of TRF2 was detected by Western blot after the cells treated with adriamycin (ADR) or etoposide for 6h. RESULTS: We constructed two TRF2 siRNA eukaryotic expression vectors successfully and the transfected cells showed significantly suppressed TRF2 expression. The growth of cells were not significantly influenced by transfection (P>0.05). CONCLUSION: The TRF2 siRNA eukaryotic expression vectors are successfully constructed and stably expressed in gastric cancer cells.