MiR-503逆转肺癌耐药细胞株A549/DDP的耐药性及其机制研究

摘要

背景与目的临床上肺癌细胞往往出现对顺铂的耐药性，因此探讨肿瘤细胞的耐药机制，开发新的逆转耐药性的方法，对提高临床患者的受益有十分重要的意义。miRNA可通过其调控的目标基因，对多种与肿瘤细胞失控生长、抗凋亡、迁移和侵袭，甚至是肿瘤细胞对药物治疗的应答产生调控作用。本实验旨在探讨miR-503对肺癌顺铂耐药细胞株A549/DDP的耐药性逆转及其相关作用机制。方法应用MTS法检测miR-503对A549/DDP细胞顺铂耐受性的影响，流式细胞术检测肿瘤细胞凋亡率以及胞内罗丹明-123（Rhodamine-123, Rh-123）含量的变化， Western blot法和Real time PCR检测肿瘤细胞多药耐药蛋白MDR1、MRP1、Survivin和Bcl-2蛋白表达，以及Akt磷酸化的变化，应用双萤光报告基因技术检测细胞NF-κB和AP-1转录活性。结果与对照细胞组相比较，miR-503转染A549/DDP细胞株后，可明显增加细胞对顺铂的敏感性，使耐药逆转倍数增加为2.48倍，Rh-123含量升高2.49倍，细胞凋亡率提高10.3倍；在转录水平检测发现，与对照组相比较，miR-503转染的细胞中MDR1、MRP1、ERCC1、Survivin及Bcl-2等与肿瘤耐药相关基因的mRNA表达水平明显下调，而RhoE mRNA表达水平则明显升高（P<0.05）；进一步在蛋白水平亦证实MDR1、MRP1、ERCC1、Survivin、Bcl-2以及p-Akt的表达明显下降，RhoE的表达明显上升。结论miR-503可逆转A549/DDP对顺铂的耐药性，这一作用可能与抑制药物外排，负调控肿瘤耐药相关蛋白的表达，促进细胞凋亡有关。%Background and objective Cisplatin-resistance in lung cancer cells is general in clinic, hence it is sig-niifcant to investigate the mechanisms of cisplatin-resistant and develop new methods of reversing drug-resistance. Recent researches showed that miRNA could regulate cell growth, apoptosis, migration and invasion even in drug therapy in cancer by its target gene. hTe aim of this study is to investigate the effects and molecular mechanisms of miR-503 on reversing the cisplatin-resistance in lung cancer DDP-resistant cell line A549/DDP. Methods MTS assay was employed to determine the effect of miR-503 on A549/DDP’ sensitivity to cisplatin. Apoptosis rate and intracellular concentration of rhodamine-123 (Rh-123) were determined by lfow cytometry, the expression of multi-drugs resistant proteins MDR1and MRP1, ERCC1, RhoE, Survivin and Bcl-2 were determined by Western blot and real time PCR. hTe phosphorylation of Akt was analyzed by Western blot, the transcriptional activities of NF-κB and AP-1were detected by dual-luciferase reporter gene systems. Results MiR-503 was able to increase the cisplatin sensitivity of A549/DDP. Atfer treatment with miR-503, the reverse folds (RF) to cisplatin was 2.48 fold, the intracellular level of Rh-123 was 2.49 fold, the apoptosis rate was10.3 fold, the expressions of several drug-resistant related proteins, such as MDR1, MRP1, ERCC1, Survivin and Bcl-2 were downregulated signiifcantly, as shown by WB, in contrast, the level of RhoE was elevated, the mRNA epression of MDR1was18.5%, the mRNA epression of MRP1was 22.3%, the mRNA epression of ERCC1was18.6%, the mRNA epression of Survivin was 42.8%, the mRNA expression of Bcl-2 was 68.1%, the mRNA epression of RhoE was 206.5%, in addition, the phosphorylation of Akt decreased and transcrip-tional activities of NF-κB was 53.7%, AP-1was 47.4%compared with control group. Conclusion MiR-503 was able to reverse the cisplatin resistance of A549/DDP. MiR-503 processed this kind of effect by inhibiting the drug effux, downregulating the expression of drug-resistant related proteins and promoting cell apoptosis.