干扰REV3L基因表达逆转结肠癌的耐药性

Reversion of oxaliplatin resistance of colon carcinoma cells by REV3L gene intefering

目的探讨跨损伤DNA合成途径(TLS)关键基因REV3L的靶向下调对人耐药结肠癌细胞系耐药性的逆转作用。方法用RNA干扰技术(RNAi)沉默REV3L基因在人耐奥沙利铂结肠癌细胞系(THC8307/L-OHP)中的表达,以实时荧光定量PCR和免疫细胞化学检测REV3L基因mRNA及蛋白均低表达的细胞作为实验组。运用四甲基噻唑蓝(MTT)、流式细胞术和细胞形态学的方法检测肿瘤细胞耐药系数变化、耐药性相对逆转率及细胞凋亡的情况。结果转染mU6pro-siREV3质粒的实验组细胞REV3L基因的表达被成功抑制。基因低表达的细胞耐药系数明显低于空白对照组和阴性对照组,相对耐药逆转率显著高于阴性对照组(P<0.05)。细胞凋亡指标显示实验组细胞凋亡率显著高于两对照组(P<0.05)。结论下调REV3L基因的表达,可逆转人结肠癌细胞的耐药性并促进细胞凋亡,REV3L可以作为肿瘤基因治疗的潜在靶点。

Objective To find the effects of down-regulation of human translesion DNA synthesis （TLS） gene REV3L on sensitizing drug-resistant tumor cells. Methods REV3L expression in human Oxaliplatin resistant colon cancer cells （THC8307/L-OHP） was inhibited by interference RNA technology （RNAi）. Real-time qPCR and Im- munocytochemistry were used for judging the transfection efficiency by RNAi. The level of drug sensitivity was measured by MTY（Thiazolyl blue tetrazolium bromide）. The resistance index （RI） and relative reversion of drug resistance are calculated by statistical methods. The cell apoptosis of was determined by flow cytometry and cyto- morphology. Results THC8370/L-OHP cells transfected with mU6pro-siREV3 significantly reduced the cellular REV3L mRNA and protein levels （P 〈 0. 05）. The IC50 （ Inhibitory Concentration 50） by Oxaliplatin and the RI were markedly decreased （ P 〈 0. 05 ）. Depletion of cellular REV3L also enhanced apoptosis （ P 〈 0. 05 ）. Conclu- sions Depletion of REV 3 L from Oxaliplatin - resistant human colon cancer cells enhances the drug - sensitivity and induces apoptosis of tumor cells. Hence, REV3L is a potential target for tumor gene therapy.