miRNA-451逆转非小细胞肺癌A549/DDP细胞对顺铂的耐药性及其机制研究

目的:探讨microRNA-451(miR-451)逆转非小细胞肺癌A549/DDP细胞对顺铂的耐药性及其可能的作用机制.方法:应用实时荧光定量PCR法(real-time fluorescent quantitative PCR,qRT-PCR)检测耐DDP细胞株A549/DDP及其亲本细胞株A549中miR-451的表达差异,同时检测A549/DDP细胞转染miR-451 mimic后miR-451表达的变化;分别应用MTT法、克隆形成实验、流式细胞术检测转染后A549/DDP细胞对DDP药物敏感度,细胞增殖能力,联合DDP处理后细胞凋亡变化;Western印迹法检测转染后A549/DDP细胞Akt、p-Akt( Ser473)、bcl-2和bax的表达变化.结果:miR-451在耐DDP细胞株A549/DDP中的表达量显著低于敏感细胞株A549 (P ＜0.05),A549/DDP细胞转染miR-451 mimic24h后,细胞中miR-451的表达水平较对照组明显提高(P＜0.05).在A549/DDP细胞中过表达miR-451可产生以下效应:相较于对照组,DDP对A549/DDP/miR-451细胞的半数抑制浓度(half inhibition concentration,IC50)减低(P＜0.05),A549/DDP/miR-451细胞的增殖能力减弱,A549/DDP/miR-451经过DDP处理后细胞凋亡增多(P＜0.05).Western印迹法结果显示,与对照组比较,转染miR-451 mimic的A549/DDP细胞p-Akt ( Ser473)、bcl-2表达水平降低;bax表达水平升高.结论:miR-451可能通过诱导细胞凋亡,抑制细胞增殖,上调bax蛋白及下调p-Akt( Ser473)、bcl-2蛋白表达而逆转A549/DDP细胞对DDP的耐药性.

miRNA-451 resensitizes non-small cell lung cancer A549/DDP cells to cisplatin resistance and the mechanisms involved

Objective:To explore the effect of microRNA-451(miR-451) in reversing cisplatin(DDP) resistance of non-small cell lung cancer(NSCLC) A549/DDP cell line and elucidate its related mechanisms.Methods:The real-time fluorescent quantitative PCR(qRT-PCR) was used to detect miR-451 expression in A549 cells,DDP-resistant A549/DDP cells,and A549/DDP cells transfected with miR-451 mimic or miR-NC.The difference in miR-451 expression was compared between the groups.MTT assay,colony formation assay and flow-cytometric analysis were sequentially used to detect transfection of A549 / DDP cells to DDP drug sensitivity,cell proliferation ability,apoptosis on treatment with DDP.The protein expression of Akt,p-Akt(Ser473),bcl-2 and bax in transfection A549 / DDP cells were detected by western blotting.Results: The miR-451 expression level in A549 / DDP cell line was significantly lower than that in A549 cell line(P < 0.05).The miR-451 expression was significantly increased at 24h after transfection than that of the control group(P < 0.05).Ectopic miR-451 expression in A549 / DDP cells could produce the following effects: compared to the control group,the half inhibition concentration(IC 50) of DDP in A549 / DDP / miR-451 cells lower(P < 0.05),proliferation of A549 / DDP / miR-451 cells diminished(P < 0.05),apoptosis on treatment with DDP increased(P < 0.05).Furthermore,the protein expressions of p-Akt(Ser473),bcl-2 were downregulated and expression of bax was upregulated in miR-451 mimic transfected group than those in miR-NC group.Conclusion: miR-451 reverses the resistance of A549 / DDP cells to DDP through induction of apoptosis,diminished cell proliferation,down-regulating the expressions of p-Akt(Ser473),bcl-2 proteins and up-regulating the expression of bax protein.