MiR-146a调节TRAIL不敏感的乳腺癌细胞迁移及其分子机制

摘要

目的 研究miR-146a及其靶基因EGFR对乳腺癌细胞迁移的影响.方法 用终浓度为50、100、200和500 μg/L的重组可溶性TRAIL(rsTRAIL)持续刺激对TRAIL敏感的MDA-MB-231细胞4周,筛选得到TRAIL不敏感的乳腺癌细胞MDA-MB-231/TR.RT-PCR检测miR-146a的表达；Transwell实验以及划痕实验检测乳腺癌细胞的迁移能力；双荧光素酶报告基因分析以及Western blot鉴定MDA-MB-231/TR细胞中miR-146a与EGFR基因的靶向调控关系；Western blot检测DR4、DR5、IRAK1、CXCR4、p-IκBα、IκBα、caspase 8和caspase 3的蛋白表达水平；染色质免疫共沉淀技术(ChIP)分析MDA-MB-231和MDA-MB-231/TR细胞中NF-κB P65亚基与miR-146a启动子区的结合情况.结果 TRAIL细胞毒作用不敏感的人乳腺癌细胞系MDA-MB-231/TR中miR-146a的表达降低,并引起其靶基因EGFR的表达增加,最终导致TRAIL不敏感的乳腺癌细胞迁移能力增强.同时发现NF-κB参与miR-146a低表达的调控.结论 miR-146a对TRAIL不敏感的乳腺癌细胞迁移起重要的调节作用.%Objective To investigate the effection of miR-146a and its target gene EGFR on the migration of breast cancer cells.Methods Recombinant soluble TRAIL (rsTRAIL) was used at a final concentration of 50,100,200,and 500 μg/L to continually stimulate TRAIL-sensitive MDA-MB-231 cells for four weeks,and a TRAIL-insensitive breast cancer cell line named MDA-MB-231/TR was screened.MiR-146a expression level was evaluated using RT-PCR.Transwell cell invasion assay and wound-healing experiment were performed to examine the migration of breast cancer cells.The relationship between miR-146a level and EGFR expression was identified in MDA-MB-231/TR cells by dual luciferase reporter assay and Western blot.The expression of DR4,DRS,IRAK1,CXCR4,p-IκBα,IκBα and apoptosis-related proteins were detected by Western blot.Chromatin immunoprecipitation(ChIP) analysis revealed the binding activity of NF-κB P65 subunit to the binding sites of miR-146a promoter element in MDA-MB-231/TR cells.Results MiR-146a expression in a TRAIL-insensitive breast cancer cell line MDA-MB-231/TR was decreased significantly.Down-regulation of miR-146a increased its target EGFR expression,and eventually promoted breast cancer cell migration.Furthermore,we found that NF-κB regulated the low expres sion of miR-146a in the MDA-MB-231/TR cells.Conclusions miR-146a plays an important regulatory role in the migration of TRAIL-insensitive breast cancer cells.