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. 2014 Jan;17(1):1-7.
doi: 10.3779/j.issn.1009-3419.2014.01.01.

[The reversing and molecular mechanisms of miR-503 on the drug-resistance to cisplatin in A549/DDP cells]

[Article in Chinese]
Yi Wu  1 , Lili Guo  1 , Jinghao Liu  1 , Renwang Liu  1 , Minghui Liu  1 , Jun Chen  1
Affiliations

[The reversing and molecular mechanisms of miR-503 on the drug-resistance to cisplatin in A549/DDP cells]

[Article in Chinese]
Yi Wu et al. Zhongguo Fei Ai Za Zhi. 2014 Jan.

Abstract
in English, Chinese

Background and objective: Cisplatin-resistance in lung cancer cells is general in clinic, hence it is significant 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. The 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 flow cytometry, the expression of multi-drugs resistant proteins MDR1 and MRP1, ERCC1, RhoE, Survivin and Bcl-2 were determined by Western blot and real time PCR. The phosphorylation of Akt was analyzed by Western blot, the transcriptional activities of NF-κB and AP-1 were detected by dual-luciferase reporter gene systems.

Results: MiR-503 was able to increase the cisplatin sensitivity of A549/DDP. After 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 was 10.3 fold, the expressions of several drug-resistant related proteins, such as MDR1, MRP1, ERCC1, Survivin and Bcl-2 were downregulated significantly, as shown by WB, in contrast, the level of RhoE was elevated, the mRNA epression of MDR1 was 18.5%, the mRNA epression of MRP1 was 22.3%, the mRNA epression of ERCC1 was 18.6%, 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 transcriptional activities of NF-κB was 53.7%, AP-1 was 47.4% compared with control group.

Conclusions: MiR-503 was able to reverse the cisplatin resistance of A549/DDP. MiR-503 processed this kind of effect by inhibiting the drug efflux, downregulating the expression of drug-resistant related proteins and promoting cell apoptosis.

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。

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Figures

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MiR-503在人肺癌细胞系中的表达。在A549/DDP细胞中转染pre-miR-503质粒48 h后，利用TaqMan MicroRNA分析试剂盒，通过Q-PCR检测miR-503的表达水平。未处理组：A549/DDP细胞；对照组：转染空白对照质粒的A549/DDP细胞；miR-503组：转染pre-miR-503质粒的A549/DDP细胞。实验数据以均数±标准差表示，n=10，*表示与对照组相比，P < 0.05。 The expression of miR-503 in human lung cancer cell lines. After transfection pre-miR-503 plasmids into A549/DDP cells for 48 h, the expression of miR-503 was detected by Q-PCR using TaqMan MicroRNA assay kit. Untreated group: the primary A549/DDP cells; Control group: A549/DDP cells transfected with control blank vector; miR-503 group: A549/DDP cell transfected with pre-miR-503 plasmids. Data was represented as Mean±SD, n=10, bars indicate SD, *compared to the control group (P < 0.05).
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A549/DDP细胞中miR-503对药物敏感性，Rh-123的胞内浓度及细胞凋亡的作用。A：MTS法结果显示与对照组相比，转染pre-miR-503后，细胞对顺铂的敏感性明显增加，数据以Mean±SD表示，n=10，*表示与对照组相比，P < 0.05；B：流式细胞术结果表明与对照组相比，转染pre-miR-503后，细胞内Rh-123的浓度增加，数据以Mean±SD表示，n=10，*表示与对照组相比，P < 0.05；C：流式细胞术结果表明与对照组相比，转染pre-miR-503后，细胞凋亡数目增加，数据以Mean±SD表示，n=10，*表示与对照组相比，P < 0.05。 The effect of miR-503 on drug sensitivity, intracellular level of Rh-123 and apoptosis in A549/DDP cells. A: The MTS assay indicated that the DDP-sensitivity in pre-miR-503 group was significantly increased while compared to the control group. Data was represented as Mean±SD, n=10, bars indicate SD, *compared to the control group (P < 0.05); B: The flow cytometry assay results showed that there was an increased intracellular level of Rh-123 in pre-miR-503 group compared with control group. Data was represented as Mean±SD, n=10, bars indicate SD, *compared to the control group (P < 0.05); C: The flow cytometry assay results showed that there was an increased apoptosis in pre-miR-503 group compared with control group. Data was represented as Mean±SD, n=10, bars indicate SD, *compared to the control group (P < 0.05).
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A549/DDP细胞中转染pre-miR-503后，耐药相关基因的蛋白表达水平。Western blot结果显示，与对照组相比，MDR1、MRP1、ERCC1、Survivin和Bcl-2的蛋白水平明显降低，而RhoE的蛋白水平明显增加，α-tubulin为内参。 The expression of different drug-resistance related genes in A549/DDP cells transfected with pre-miR-503. Compared to the control group, the expression of MDR1, MRP1, ERCC1, Survivin and Bcl-2 were dramatically down-regulated while the expression of RhoE was significantly up-regulated in pre-miR-503 group by western blot assay. α-tubulin was used as an internal control.
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MiR-503对Akt磷酸化水平的影响。Western blot结果显示，与对照组相比，转染pre-miR-503质粒后，Akt磷酸化水平降低，α-tubulin为内参。 The effect of miR-503 on phosphorylation of Akt in human lung cancer cell lines. Western blot assay showed that the phosphorylation of Akt was downregulated in pre-miR-503 group compared with control group. α-tubulin was used as an internal control.
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