抑制卵巢癌A2780/Taxol细胞hsa-miR-27a表达对紫杉醇敏感性影响的研究

Inhibition of hsa-miR-27a expression sensitizes human ovarian cancer A2780/Taxol cells to drug paclitaxel

目的:研究抑制卵巢癌A2780/Taxol细胞hsa-miR-27a表达对紫杉醇敏感性的影响。方法:用浓度递增法建立卵巢癌耐紫杉醇细胞系A2780/Taxol;Stem-loop real-time PCR技术检测A2780/Taxol及亲本细胞A2780中hsa-miR-27a表达水平;利用脂质体Lipofectamine2000将化学合成的miR-27a抑制物及阴性对照(Negative Control,NC)转染A2780/Taxol细胞;分别用Real-time PCR和蛋白印迹法技术检测MDR1mRNA和P-glyco-protein(P-gp)蛋白表达;MTT检测A2780/Taxol对紫杉醇敏感性的变化;流式细胞术检测细胞内P-gp荧光底物罗丹明123(Rh-123)的荧光强度。结果:(1)成功建立了卵巢癌耐紫杉醇细胞系A2780/Taxol;(2)miR-27a在A2780/Taxol细胞中高表达,与A2780细胞相比,表达增高2.2倍。(3)P-gp蛋白在A2780/Taxol细胞中高表达,在A2780细胞中未检测出其表达;(4)转染miR-27a抑制物后,A2780/Taxol细胞的MDR1mRNA和P-gp表达下降,与转染NC组相比,P-gp表达降低36%;对紫杉醇的敏感性增加,IC50为0.53μmol/L,与转染NC组IC506.8μmol/L相比,有明显差异;(5)流式细胞仪结果显示,细胞内Rh-123荧光强度在转染抑制物的A2780/Taxol细胞为5.10±0.35,与A2780/Taxol细胞的2.95±0.39和转染NC的A2780/Taxol细胞的3.30±0.45相比,差异均有统计学意义。结论:hsa-miR-27a在卵巢癌耐紫杉醇A2780/Taxol细胞中高表达,可能通过间接调节P-gp的表达和功能,参与耐药。抑制miR-27a表达后,可以增加A2780/Taxol细胞对紫杉醇的敏感性,部分逆转耐药。

Objective：To study whether inhibition of hsa-miR-27a expression sensitizes human ovarian cancer A2780/Taxol cells to drug paclitaxel.Methods：A2780 cell line resistant to Taxol（A2780/Taxol） were established using stepwise selection method.Stem-loop real-time PCR was used to detect hsa-miR-27a expression.A2780/Taxol cells were transfected with the inhibitors of miR-27a or negative control by Lipofectamine 2000.The expressions of MDR1 mRNA and P-gp protein levels were measured by real-time PCR and Western blot respectively.MTT was used to analyze drug sensitivity.The fluorescence of remaining Rh-123 in cells was measured by FACS.Results：（1）A2780/Taxol cell line was established successfully.（2）MiR-27a was an average of 2.2-fold higher expression level in A2780/Taxol than that in A2780 cell lines.（3）P-gp protein was highly expressed in A2780/Taxol cells,but it was not detected in A2780 cells.（4）The expression levels of MDR1 mRNA and P-gp were down-regulated,the sensitivity to paclitaxel was enhanced,and the fluorescence intensity of Rh-123 was increased in A2780/Taxol cells transfected with inhibitors of miR-27a.Conclusion：Hsa-miR-27a is high expressed and may involve in drug resistance by indirect targeting MDR1/P-gp in A2780/Taxol cells.Inhibition of miR-27a can sensitize A2780/Taxol cells to paclitaxel,reverse drug resistance in part.