RNA干扰EGFR基因对卵巢癌细胞化疗敏感性的影响

EFFECT OF SILENCING EGFR GENE EXPRESSION BY RNA INTERFERENCE ON THE CHEMOSENSITIVITY OF OVARIAN CANCER CELL LINE skov3

目的构建表皮生长因子受体(EGFR)基因的小干扰RNA(siRNA)质粒,转染人卵巢癌细胞株sk-ov3后检测RNA干扰(RNAi)对EGFR基因表达及细胞对化疗药物敏感性的影响。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细胞。实验分为正常对照组、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EGFR shRNA转染skov3细胞后细胞对顺铂敏感性的改变。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(F=87.73,q=16.81、15.33,P<0.01),EGFR蛋白表达明显下调(F=69.29,q=14.71、13.57,P<0.01);顺铂敏感性比正常对照组提高了约3倍。结论靶向EGFR基因的序列特异性shRNA可有效抑制skov3细胞中EGFR基因的表达,增强skov3细胞对顺铂的敏感性。

Objective To construct expression vector of EGFR specific siRNA and transfect it into human ovarian cancer cell line skov3 and then investigate the effect of RNA interference （RNAi） on the target gene expression and cheraosensitivity of the cell line. Methods EGFR shRNA was synthesized in vitro and then transfected into skov3 cell to set up the EGFR shRNA transfectant cell fine. The normal control cell fine and non-specific transfectant ceil fine were also included in the study. RT-PCR and imraunocytocheraistry were performed to detect the inhibitive effect of RNAi on raRNA level and protein level, respectively. The cheraosensitivity to cisplatin was detected by MTT method. Results The expression of EGFR raRNA of sequence specific shRNA targeting EGFR was obviously down-regulated （F=87.73;q=16.81,15. 33;P〈0.01） and the same happened to the protein expression （F=69.29;q=14.71,13.57;P〈0. 01）, while the cheraosensitivity to cisplatin was increased by three tiraes. Conclusion The EGFR specific shRNA expression vector can effectively suppress the expression level of EGFR gene of skov3 cells, and increases its cheraosensitivity to cisplatin.