miR-125a-5p靶向RRM2调控A549/DDP细胞的顺铂耐药性  

MicroRNA-125a-5p targets ribonucleotide reductase regulatory subunit M2 to inhibit cisplatin resistance of A549/DDP cells

作　　者：姜永杰 庞敏 龚海英 黄语嫣 蒋莉[1,2] JIANG Yongjie;PANG Min;GONG Haiying;HUANG Yuyan;JIANG Li(Affiliated Hospital of North Sichuan Medical College,Sichuan Nanchong 637000,China;North Sichuan Medical College,Sichuan Nanchong 637000,China)

机构地区：[1]川北医学院附属医院,四川南充637000 [2]川北医学院,四川南充637000

出　　处：《现代肿瘤医学》2023年第2期227-234,共8页Journal of Modern Oncology

基　　金：四川省科技计划项目(编号:2020YFS0572);四川省医学会项目(编号:2021HR24)。

摘　　要：目的:探究微小RNA-125a-5p(microRNA-125a-5p, miR-125a-5p)对非小细胞肺癌A549/DDP细胞顺铂敏感性的影响及其机制。方法:使用ENCORI在线数据库分析miR-125a-5p在非小细胞肺癌癌组织与癌旁组织中的表达水平;采用实时荧光定量聚合酶链反应(real-time quantitative PCR,RT-qPCR)的方法检测人肺上皮正常细胞(BEAS-2B)与非小细胞肺癌细胞系(A549、A549/DDP、SK-MES-1)中miR-125a-5p的表达水平;使用RT-qPCR与蛋白免疫印迹法(Western blot)检测A549、A549/DDP细胞中核糖核苷酸还原酶M2(ribonucleotide reductase regulatory subunit M2,RRM2)的表达情况;生物信息学方法分析miR-125a-5p与RRM2的靶向调控序列;双荧光素酶基因报告实验与Western blot测定miR-125a-5p与RRM2在A549/DDP细胞中的调控关系;细胞计数试剂盒(CCK-8)检测细胞的存活率与半数抑制浓度(half inhibitory concentration, IC_(50));流式细胞术检测细胞凋亡率。结果:在非小细胞肺癌组织与细胞系中miR-125a-5p的表达水平显著降低(P<0.05);与A549细胞相比,A549/DDP细胞中RRM2的表达水平显著升高(P<0.05);过表达miR-125a-5p部分恢复了顺铂对A549/DDP细胞的抑制作用,使细胞存活率显著降低(P<0.05),凋亡率显著增加(P<0.05);双荧光素酶报告实验证实miR-125a-5p能够靶向结合RRM2(P<0.05);过表达RRM2逆转了miR-125a-5p对A549/DDP的作用,降低了A549/DDP对顺铂的敏感性(P<0.05)。结论:miR-125a-5p可通过靶向下调RRM2基因的表达,从而部分恢复A549/DDP细胞对顺铂的敏感性。Objective:To explore the influence of microRNA-125a-5p(miR-125a-5p)on the cisplatin sensitivity of A549/DDP cells in non-small cell lung cancer and its mechanism.Methods:The expression level of miR-125a-5p in non-small cell lung cancer tissues and adjacent tissues was analyzed through ENCORI online database.Besides,the expression levels of miR-125a-5p in human lung epithelial normal cells(BEAS-2B)and non-small cell lung cancer cell lines(A549,A549/DDP,and SK-MES-1)were detected through real-time quantitative PCR(RT-qPCR).The expression of ribonucleotide reductase regulatory subunit M2(RRM2)in A549 and A549/DDP cells was detected through RT-qPCR and Western blot.Moreover,the targeted regulatory sequence of miR-125a-5p and RRM2 was analyzed by bioinformatics methods.The regulation of miR-125a-5p and RRM2 in A549/DDP cells was determined by double luciferase gene reporter assay and Western blot.The cell survival rate and half inhibitory concentration(IC)were detected by cell counting kit(CCK-8),and the cell apoptosis rate was determined by flow cytometry.Results:The expression level of miR-125a-5p in non-small cell lung cancer tissues and cell lines was significantly decreased(P<0.05).Compared with A549 cells,the expression level of RRM2 in A549/DDP cells was significantly increased(P<0.05).The over-expression of miR-125a-5p partially restored the inhibitory effect of cisplatin on A549/DDP cells.As a result,the cell survival rate was significantly decreased(P<0.05),while the apoptosis rate was significantly increased(P<0.05).Double luciferase gene reporter assay confirmed that miR-125a-5p could achieve targeted combination with RRM2(P<0.05).The over-expression of RRM2 reversed the effect of miR-125a-5p on A549/DDP,and reduced the sensitivity of A549/DDP to cisplatin(P<0.5).Conclusion:miR-125a-5p can partially restore the sensitivity of A549/DDP cells to cisplatin through the targeted down-regulation of RRM2 gene expression.

关 键 词：微小RNA-125a-5p 核糖核苷酸还原酶M2 顺铂 非小细胞肺癌 耐药性 

分 类 号：R734.2[医药卫生—肿瘤]