CAJ | 학술논문

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。
배경여목적림상상폐암세포왕왕출현대순박적내약성，인차탐토종류세포적내약궤제，개발신적역전내약성적방법，대제고림상환자적수익유십분중요적의의。miRNA가통과기조공적목표기인，대다충여종류세포실공생장、항조망、천이화침습，심지시종류세포대약물치료적응답산생조공작용。본실험지재탐토miR-503대폐암순박내약세포주A549/DDP적내약성역전급기상관작용궤제。방법응용MTS법검측miR-503대A549/DDP세포순박내수성적영향，류식세포술검측종류세포조망솔이급포내라단명-123（Rhodamine-123, Rh-123）함량적변화， Western blot법화Real time PCR검측종류세포다약내약단백MDR1、MRP1、Survivin화Bcl-2단백표체，이급Akt린산화적변화，응용쌍형광보고기인기술검측세포NF-κB화AP-1전록활성。결과여대조세포조상비교，miR-503전염A549/DDP세포주후，가명현증가세포대순박적민감성，사내약역전배수증가위2.48배，Rh-123함량승고2.49배，세포조망솔제고10.3배；재전록수평검측발현，여대조조상비교，miR-503전염적세포중MDR1、MRP1、ERCC1、Survivin급Bcl-2등여종류내약상관기인적mRNA표체수평명현하조，이RhoE mRNA표체수평칙명현승고（P<0.05）；진일보재단백수평역증실MDR1、MRP1、ERCC1、Survivin、Bcl-2이급p-Akt적표체명현하강，RhoE적표체명현상승。결론miR-503가역전A549/DDP대순박적내약성，저일작용가능여억제약물외배，부조공종류내약상관단백적표체，촉진세포조망유관。
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.