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目的通过RNA干扰(RNAi)技术沉默DNA聚合酶polβ的表达,观察其对食管癌细胞顺铂(cDDP)耐药性的影响。方法构建靶向polβ基因的siRNA重组慢病毒pRNAT-U6.2/Lenti-polβ1、pRNAT-U6.2/Lenti-polβ2及阴性对照载体pRNAT-U6.2/Lenti-polβC,将其感染人食管鳞癌顺铂耐药细胞系EC9706/cDDP,通过RT-PCR、蛋白质印迹分析和免疫荧光实验观察细胞polβ的表达情况,采用MTT法观察不同浓度顺铂对细胞生长的抑制作用并计算50%细胞生长抑制所需的药物浓度(IC50)和耐药指数(RI)。结果靶向polβ的siRNA重组慢病毒pRNAT-U6.2/Lenti-polβ1和pRNAT-U6.2/Lenti-polβ2感染EC9706/cDDP后均可下调polβ的mRNA和蛋白表达水平,其中前者效果更为显著。顺铂以剂量依赖方式抑制EC9706/cDDP细胞增殖,感染pRNAT-U6.2/Lenti-polβ1、pRNAT-U6.2/Lenti-polβC的EC9706/cDDP细胞及未感染EC9706/cDDP细胞对顺铂的IC50分别为55.71、62.41、63.11μg/mL,耐药指数分别为13.9、15.5、15.7,其中前者与后二者相比差异有统计学意义(P<0.05)。结论 polβ的表达与食管鳞癌细胞系EC9706/cDDP顺铂耐药性有关,沉默其表达可部分逆转EC9706/cDDP细胞对顺铂的耐药性。
Objective To silence the expression of DNA polymerase polβ by RNA interference (RNAi) technique and observe its effect on cisplatin (cDDP) resistance in esophageal cancer cells. Methods Recombinant lentiviral vector pRNAT-U6.2 / Lenti-polβ1, pRNAT-U6.2 / Lenti-polβ2 and negative control vector pRNAT-U6.2 / Lenti-polβC targeting polβ gene were constructed and transfected into human esophageal squamous cell carcinoma The cisplatin-resistant cell line EC9706 / cDDP was used to observe the expression of polβ by RT-PCR, Western blotting and immunofluorescence assay. The inhibitory effect of different concentrations of cisplatin on cell growth was observed by MTT method and the cell growth was calculated by 50% Inhibition of the required drug concentration (IC50) and resistance index (RI). Results The mRNA and protein expression levels of polβwere both down-regulated by siRNA targeting recombinant retroviral vector pRNAT-U6.2 / Lenti-polβ1 and pRNAT-U6.2 / Lenti-polβ2 after EC9706 / cDDP infection, of which the former effect was more significant . Cisplatin inhibited the proliferation of EC9706 / cDDP cells in a dose-dependent manner. The IC50 of cisplatin in EC9706 / cDDP cells infected with pRNAT-U6.2 / Lenti-polβ1, pRNAT-U6.2 / Lenti-polβC and non-infected EC9706 / Respectively, 55.71,62.41,63.11μg / mL, drug resistance index was 13.9,15.5,15.7, of which the former was significantly different from the latter two (P <0.05). Conclusion The expression of polβ is related to the resistance of esophageal squamous cell carcinoma cell line EC9706 / cDDP to cisplatin. Silencing its expression can partially reverse the drug resistance of EC9706 / cDDP cells to cisplatin.