小干扰RNA靶向EGFR基因逆转卵巢癌细胞顺铂耐药的研究

目的：探讨载体表达的小干扰RNA（siRNA）影响卵巢癌耐药细胞株EGFR基因的表达并逆转其顺铂耐药的可行性。方法：体外构建EGFR小发卡状RNA（shRNA）的表达质粒,脂质体法介导将其转染入SKOV3/DDP细胞。实验分为正常对照组、空质粒转染组、非特异性转染组和特异性转染组。采用逆转录聚合酶链反应（RT-PCR）检测EGFR mRNA的表达;使用免疫细胞化学法（ICC）检测EGFR蛋白的表达;使用四甲基偶氮唑蓝法（MTT）测定各组细胞对顺铂的半数抑制浓度（IC50）。结果：EGFR shRNA转染组细胞EGFR mRNA的表达与其他两组相比明显减弱（P〈0.01）,EGFR蛋白表达明显下调（P〈0.01）;顺铂敏感性比正常对照组提高了约2.5倍。结论：针对EGFR合成的siRNA能够有效地抑制EGFR mRNA和蛋白的表达,并能恢复其对顺铂的敏感性。应用RNAi技术,能够逆转卵巢癌细胞对化疗药物的耐药性。

The Study on Reversal Effect of EGFR Gene Silencing by RNA Interference on Resistance of SKOV3/DDP Cell to Cisplatin

Objective： To explore the effect of vector-based small Interfering RNA （siRNA） on the expression of EGFR in drug-resistant ovarian cancer cell line, and its role in reversing the cisplatin resistance. Methods： EGFR shRNA recombinant plasmid was synthe- sized in vitro and then transfected into SKOV3/DDP cell by liposome. The study included the normal control cell line, the empty plasmid transfectant group, nonspecific and specific transfectant group. RT-PCR and immunocytochemistry were performed respectively to detect the level ofEGFR mRNA and its protein. The half inhibitory concentration （IC50） ofcisplatin in each group was measured by methyl thiazolyl tetrazolium （MTT） assay. Results： The expression of EGFR mRNA in shRNA transfectant group was less than that in the other three groups, which was obviously down-regulated （P〈0.01）, and the same to the protein expression （P〈0.01）, while the chemosensitivity to cisplatin was increased by 2.5 times compared with the normal control group. Conclusions： EGFR shRNA can effectively inhibit the expression ofEGFR mRNA and its protein, and recover the chemosensitivity to cisplatin. Therefore, it can reverse the drug resistance of ovarian cancer cells to chemotherapeutic agents.