miR-409-3p靶向调控MTF2基因在鼻咽癌细胞紫杉醇耐药机制中的作用  

The role of miR-409-3p in taxol-resistance of nasopharyngeal carcinoma cells by targeting MTF 2 and its mechanism

作　　者：肖剑 陈亦龙 谭国林[1] 宋业勋[1] XIAO Jian;CHEN Yilong;TAN Guolin;SONG Yexun(Department of Otolaryngology-Head and Neck Surgery,Third Xiangya Hospital of Central South University,Changsha 410013,Hunan Province,China;Department of Otolaryngology-Head and Neck Surgery,Ningxiang People’s Hospital,Ningxiang 410600,Hunan Province,China)

机构地区：[1]中南大学湘雅三医院耳鼻咽喉头颈外科,湖南长沙410013 [2]宁乡市人民医院耳鼻咽喉头颈外科,湖南宁乡410600

出　　处：《肿瘤》2021年第5期341-352,共12页Tumor

基　　金：国家自然科学基金(编号:81702706)。

摘　　要：目的:探讨微RNA(microRNA,miR)-409-3p及其靶基因MTF2在鼻咽癌细胞对紫杉醇耐药中的作用以及可能的作用机制。方法:采用CCK-8法和集落形成实验检测鼻咽癌亲本细胞CNE-1和5-8F以及耐药细胞CNE-1/Taxol和5-8F/Taxol对紫杉醇的敏感性,实时荧光定量PCR法检测miR-409-3p在亲本细胞和耐药细胞中的表达水平。采用脂质体法将miR-409-3p-抑制子(inhibitor)分别转染CNE-1/Taxol和5-8F/Taxol细胞,采用实时荧光定量PCR法验证转染效率,CCK-8法和集落形成实验检测鼻咽癌细胞对紫杉醇耐药与miR-409-3p之间的相关性。利用Targetscan、miRDB、miRTarBase和miRWalk数据库预测miR-409-3p可能的下游靶基因,并采用荧光素酶报告基因检测miR-409-3p是否直接靶向结合MTF2基因。采用实时荧光定量PCR法和蛋白质印迹法检测转染miR-409-3p-inhibitor(以转入NC-inhibitor为对照)后对MTF2 mRNA和蛋白表达水平的影响。采用实时荧光定量PCR法和蛋白质印迹法检测MTF2 mRNA和蛋白在鼻咽癌亲本细胞和紫杉醇耐药细胞中的表达水平。采用脂质体转染法将pcDNA3.1-MTF2(以转入pcDNA3.1-NC为对照)转入CNE-1/Taxol和5-8F/Taxol细胞,CCK-8法和集落形成实验检测过表达MTF2后耐药细胞对紫杉醇药物敏感性的变化,FCM检测MTF2过表达后5-8F/Taxol细胞凋亡率的变化。结果:鼻咽癌亲本细胞对紫杉醇的敏感性明显高于耐药细胞(P<0.05)。鼻咽癌耐药细胞中miR-409-3p的表达水平均高于鼻咽癌亲本细胞(P<0.001)。利用Targetscan、miRDB、miRTarBase和miRWalk数据库进行预测以及荧光素酶报告基因检测证实,miR-409-3p靶向调控MTF2基因。下调鼻咽癌耐药细胞中miR-409-3p的表达水平后,细胞对紫杉醇的耐药性下降(P<0.05),而MTF2的表达水平增加(P<0.01);鼻咽癌亲本细胞中的MTF2表达水平高于鼻咽癌耐药细胞(P<0.01);上调鼻咽癌耐药细胞中MTF2的表达水平后,细胞对紫杉醇的耐药性下降(P<0.05);上调鼻咽癌耐药细Objective:To investigate the role of microRNA (miR)-409-3p and its target gene MTF2 in taxol-resistance of nasopharyngeal carcinoma cells and its mechanism.Methods:The sensitivity of nasopharyngeal carcinoma parental cells (5-8F and CNE-1) and taxol-resistant cells (5-8F/Taxol and CNE-1/Taxol) to taxol was detected by CCK-8 assay and colony formation assay.The expression level of miR-409-3p in parental and taxol-resistant cells was detected by real-time fluorescent quantitative PCR.The CNE-1/Taxol and 5-8F/Taxol cells were transfected with miR-409-3p-inhibitor by liposome,then the transfection efficiency was verified by real-time fluorescent quantitative PCR,and the CCK-8 assay and colony formation assay were used to detect the correlation between miR-409-3p and taxol-resistance in nasopharyngeal carcinoma cells.Targetscan,miRDB,miRTarBase and miRWalk databases were used to explore the downstream target gene of miR-409-3p,and the luciferase reporter gene assay was used to verify the targeting relationship between miR-409-3p and MTF2 gene.The expression levels of MTF2 mRNA and protein after transfection with miR-409-3p-inhibitor (transfected with NC-inhibitor as the control) were detected by real-time fluorescent quantitative PCR and Western blotting.CNE-1/Taxol and 5-8F/Taxol cells were transfected with pcDNA3.1-MTF2 (transfected with pcDNA3.1-NC as the control) by liposome.The sensitivity of taxol-resistant cells to taxol after MTF2 overexpression was detected by CCK-8 assay and colony formation assay,and the apoptosis rate of 5-8F/Taxol cells after MTF2 overexpression was detected by FCM.Results:The sensitivity of nasopharyngeal carcinoma parental cells to taxol was significantly higher than that of taxol-resistant cells (P < 0.05).The expression level of miR-409-3p in taxol-resistant cells was higher than that of parental cells (P < 0.001).It was verified that the miR-409-3p could target MTF2 gene by using Targetscan,miRDB,miRTarBase and miRWalk databases for prediction and luciferase reporter gene detection.T

关 键 词：鼻咽肿瘤 微RNAS MTF2基因 抗药性 肿瘤 细胞凋亡 

分 类 号：R739.63[医药卫生—肿瘤]