MiR-146a调节TRAIL不敏感的乳腺癌细胞迁移及其分子机制

基础医学与临床 ›› 2013, Vol. 33 ›› Issue (7): 881-887.

MiR-146a调节TRAIL不敏感的乳腺癌细胞迁移及其分子机制

刘丹¹,姜明红²,刘敏²,高静³,刘彦信³,郑德先³

1. 北京协和医学院基础医学研究所
2. 中国医学科学院基础医学研究所医学分子生物学国家重点实验室
3. 中国医学科学院基础医学研究所

收稿日期:2013-05-09 修回日期:2013-05-14 出版日期:2013-07-05 发布日期:2013-06-26
通讯作者: 郑德先 E-mail:zhengdx@pumc.edu.cn

The molecular mechanism of regulation of TRAIL-insensitive breast cancer cell migration by miR-146a

Received:2013-05-09 Revised:2013-05-14 Online:2013-07-05 Published:2013-06-26

摘要/Abstract

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、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。

关键词: 关键词：TRAIL, 乳腺癌细胞, miR-146a, 迁移

Abstract: 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, respectively, 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, DR5, IRAK1, CXCR4, p-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. Result 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 expression of miR-146a in the MDA-MB-231/TR cells. Conclusion miR-146a plays an important regulatory role in the migration of TRAIL-insensitive breast cancer cells.

Key words: Key words: TRAIL, breast cancer cells, miR-146a, migration

刘丹姜明红刘敏高静刘彦信郑德先. MiR-146a调节TRAIL不敏感的乳腺癌细胞迁移及其分子机制[J]. 基础医学与临床, 2013, 33(7): 881-887.

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