miRNA-139-5p通过靶向调控CXCR4/CXCL12信号通路逆转非小细胞肺癌A549细胞顺铂耐药

miRNA-139-5p reverses cisplatin resistance in non-small cell lung cancer cells A549 through targeted regulation of CXCR4/CXCL12 signaling pathway

目的:探讨miRNA-139-5p对非小细胞肺癌A549细胞顺铂(cisplatin,DDP)耐药的影响,及其可能的分子机制。方法:采用DDP浓度递增法诱导A549细胞建立DDP耐药细胞株A549/DDP,将miR-139-5p mimics、无义序列(mimics-NC)、CXCR4过表达质粒(pcDNA3.1-CXCR4)、pcDNA3.1空载质粒(Vector)转染至A549/DDP细胞中,将细胞分为mimics-NC组(转染无义序列)、mimics组(转染miR-139-5p mimics)、mimics+Vector组(共转染miR-139-5p mimics和空载质粒)和mimics+CXCR4组(共转染miR-139-5p mimics和CXCR4过表达质粒),另设置空白对照组(blank)。不同浓度DDP处理,MTT法检测细胞增殖活性,并计算IC 50值和耐药指数(resistance index,RI);qRT-PCR法检测细胞中miR-139-5p和CXCR4 mRNA表达水平;流式细胞术检测细胞凋亡率;Western blot法检测细胞中耐药相关蛋白P-糖蛋白(P-gp)、多药耐药相关蛋白1(MRP1)及CXCR4/CXCL12信号通路相关蛋白表达水平;双荧光素酶报告基因实验验证miR-139-5p与CXCR4的靶向关系。结果:不同浓度DDP处理24 h后,耐药株A549/DDP及其亲本A549细胞IC 50值分别为(208.87±27.89)μmol/L和(31.66±6.30)μmol/L,RI=6.59。与亲本A549细胞比较,耐药株A549/DDP中miR-139-5p的表达水平明显降低(P<0.05),而CXCR4 mRNA和蛋白表达水平明显升高(P<0.05)。与mimics-NC组比较,mimics组A549/DDP细胞增殖活性明显降低(P<0.05),细胞凋亡率明显升高(P<0.05),细胞中CXCR4 mRNA和蛋白以及P-gp、MRP1、PI3K(p110α)和p-AKT/AKT等蛋白表达水平显著降低(P<0.05)。与mimics+Vector组比较,mimics+CXCR4组A549/DDP细胞增殖活性显著升高(P<0.05),细胞凋亡率显著降低(P<0.05),而细胞中CXCR4、P-gp、MRP1、PI3K(p110α)和p-AKT等蛋白表达水平显著升高(P<0.05)。双荧光素酶报告基因实验证实,miR-139-5p靶向负调控CXCR4表达。结论:miRNA-139-5p通过靶向下调CXCR4表达,提高非小细胞肺癌DDP耐药细胞株A549/DDP对DDP的药物敏感性。

Objective:To explore the effect of miRNA-139-5p on cisplatin(DDP)resistance in non-small cell lung cancer cells A549 and its possible molecular mechanism.Methods:DDP concentration increasing method was used to induce A549 cells to establish DDP-resistant cell line A549/DDP.The miR-139-5p mimics,nonsense sequence(mimics-NC),CXCR4 overexpression plasmid(pcDNA3.1-CXCR4),and pcDNA3.1 empty plasmid(Vector)were transfected into A549/DDP cells.Then the cells were divided into mimics-NC group(transfected with nonsense sequences),mimics group(transfected with miR-139-5p mimics),mimics+Vector group(cotransfected with miR-139-5p mimics and Vector plasmid),and mimics+CXCR4 group(cotransfected with miR-139-5p mimics and CXCR4 overexpression plasmid).A blank control group(blank)was set.After treatment with different concentrations of DDP,MTT method was used to detect the cell proliferation activity,and IC 50 value and drug resistance index(RI)were calculated.qRT-PCR method was used to detect the expression levels of miR-139-5p and CXCR4 mRNA in cells.Flow cytometry was used to detect cell apoptosis rate.Western blot method was used to detect the expression of drug resistance-related protein P-glycoprotein(P-gp),multidrug resistance-related protein 1(MRP1)and CXCR4/CXCL12 signaling pathway related proteins in cells.Dual-luciferase reporter gene experiment verified the targeting relationship between miR-139-5p and CXCR4.Results:After 24 hours of treatment with different concentrations of DDP,the IC 50 values of the drug-resistant strain A549/DDP and its parent A549 cells were(208.87±27.89)μmol/L and(31.66±6.30)μmol/L,with RI=6.59.Compared with the parental A549 cells,the expression level of miR-139-5p in resistant strain A549/DDP cells was significantly reduced(P<0.05),while the expression levels of CXCR4 mRNA and protein were significantly increased(P<0.05).Compared with the the mimics-NC group,the proliferation activity of A549/DDP cells in the mimics group was significantly reduced(P<0.05),and the apoptosis rate was significantly increased(P<0.05),and the expression level of CXCR4 mRNA and protein in the cells,as well as P-gp,MRP1,PI3K(p110α)and p-AKT/AKT were markedly reduced(P<0.05).Compared with the mimics+Vector group,the proliferation activity of A549/DDP cells in the mimics+CXCR4 group was significantly increased(P<0.05),and the apoptosis rate was markedly reduced(P<0.05),while the expression levels of CXCR4,P-gp,MRP1,PI3K(p110α)and p-AKT proteins were significantly increased(P<0.05).The dual-luciferase reporter gene experiment confirmed that miR-139-5p targeted and negatively regulated CXCR4 expression.Conclusion:miRNA-139-5p improves the drug sensitivity of non-small cell lung cancer DDP resistant cell lines A549/DDP to DDP by targeting down-regulation of CXCR4 expression.