miR-200c通过下调TUBB3基因表达逆转老年肺癌患者顺铂耐药性的机制  

miR-200c reverses cisplatin resistance in elderly lung cancer patients by downregulating TUBB3 gene

作　　者：赵云龙 李少军 陈平[1] 刘阳[2] ZHAO Yun-Long;LI Shao-Jun;CHEN Ping;LIU Yang(Department of Thoracic Surgery, Fourth Medical Center, Chinese PLA General Hospital, Beijing 100048, China;Department of Thoracic Surgery, First Medical Center, Chinese PLA General Hospital, Beijing 100853, China)

机构地区：[1]解放军总医院第四医学中心胸外科,北京100048 [2]解放军总医院第一医学中心胸外科,北京100853

出　　处：《中华老年多器官疾病杂志》2020年第6期433-438,共6页Chinese Journal of Multiple Organ Diseases in the Elderly

摘　　要：目的探讨miR-200c逆转肺癌细胞顺铂耐药性的作用机制。方法miR-200c模拟物转染肺癌A549细胞后,采用实时定量PCR法检测miR-200c的表达;采用细胞活力测定(MTS)法检测miR-200c对肺癌A549细胞顺铂耐受性的影响。采用流式细胞术(FCM)检测肿瘤细胞凋亡率的变化。采用Western blotting检测肿瘤细胞耐药蛋白survivin和Bcl-2蛋白的表达。通过生物信息学分析发现miR-200c下游靶分子TUBB3表达存在差异后,构建TUBB33′UTR的野生型和突变型荧光素酶报告载体,然后利用双荧光素酶活性分析miR-200c对TUBB3基因表达的结合位点和调控作用。通过Western blotting检测miR-200c对TUBB3蛋白表达的调控作用以及Bcl-2蛋白和肿瘤细胞耐药蛋白survivin的表达。采用SPSS 19.0统计软件分析实验数据。结果与对照组相比,miR-200c转染肺癌细胞株后,肺癌细胞对顺铂的敏感性呈明显增加趋势[IC50分别为(37.3±3.1)和(15.3±3.3)μmol/L,P<0.01]。过表达miR-200c的A549/DDP组的细胞凋亡率明显高于NC组(P<0.01)。而肿瘤耐药相关基因Bcl-2和survivin蛋白表达降低。转染miR-200c mimics的A549/DDP细胞中TUBB3蛋白和mRNA的表达均下调(P<0.01)。双荧光素酶活性分析显示miR-200c对TUBB3基因表达具有调控作用(P<0.01)。这些结果均证实TUBB3为miR-200c的下游靶基因。pcDNA-TUBB3质粒转染后,过表达miR-200c的A549/DDP细胞株survivin和Bcl-2蛋白表达较miR-200c mimics组上调。结论miR-200c可通过下调TUBB3基因的表达逆转肺癌顺铂耐药细胞的耐药性。Objective To investigate the underlying mechanism of miR-200c reversing the drug resistance of lung cancer cells to cisplatin(DDP).Methods After transfection of miR-200c mimics into lung cancer A549 cells,the expression of miR-200c was detected by real-time PCR.MTS assay was employed to measure the cell viability to observe the tolerance of A549 cells to DDP.Flow cytometry was used to measure the apoptosis rate of the cells,and Western blotting was applied to test the expression of survivin and Bcl-2 protein.After bioinformatics analysis showed that TUBB3,the downstream target molecule of miR-200c,was expressed differentially,luciferase reporter vectors containing wild type and mutant TUBB33′UTR were constructed,and the regulation effect and binding site of miR-200c to TUBB3 was studied by double luciferase reporter assay.The regulatory effect of miR-200c on the expression of TUBB3 protein,and on the expression of survivin and Bcl-2 protein were detected by Western blotting.SPSS statistics 19.0 was used to perform the statistical analysis.Results Compared with the control group,miR-200c transfection significantly increased the sensitivity of lung cancer cells to cisplatin[IC:(37.3±3.1)vs(15.3±3.3)μmol/L,P<0.01].And the overexpression of miR-200c also resulted in the increased apoptosis rate of expression of A549/DDP cells(P<0.01),and decreased expression of survivin and Bcl-2 protein.The expression of TUBB3 at protein and mRNA levels was down-regulated in A549/DDP cells transfected with miR-200c mimics(P<0.01).Double luciferase reporter assay showed that miR-200c could regulate the expression of TUBB3(P<0.01).All these results confirmed that TUBB3 was the downstream target gene of miR-200c.After pcDNA-TUBB3 plasmid was transfected,the expression of survivin and Bcl-2 protein in A549/DDP cells with miR-200c overexpression was up-regulated than that in the cells transfected with miR-200c mimics.Conclusion miR-200c can reverse the drug resistance of DDP-resistant lung cancer cells by down-regulating TUBB3 expre

关 键 词：MIR-200C TUBB3基因表达 调控机制 

分 类 号：R734.2[医药卫生—肿瘤]