miR-125a-5p靶向RRM2调控A549/DDP细胞的顺铂耐药性

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，IC50）；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。

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

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 (IC50) 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＜0.05).Conclusion:miR-125a-5p can partially restore the sensitivity of A549/DDP cells to cisplatin through the targeted down-regulation of RRM2 gene expression.