癌变·畸变·突变 ›› 2021, Vol. 33 ›› Issue (6): 435-441,460.doi: 10.3969/j.issn.1004-616x.2021.06.006

• 论著 • 上一篇    下一篇

乳腺癌中miR-221、miR-222表达水平对GATA3和FOXA1蛋白的影响及其作用机制

方煜彤1,2, 张群琛1,2, 洪超群2,3, 陈春发1,2, 陈炯玉2,3, 张任栋1,2, 吴俊东1,2   

  1. 1. 汕头大学医学院附属肿瘤医院乳腺中心, 广东 汕头 515041;
    2. 汕头大学医学院附属肿瘤医院中心实验室, 广东 汕头 515041;
    3. 广东省乳腺癌诊治研究重点实验室, 广东 汕头 515041
  • 收稿日期:2021-07-16 修回日期:2021-09-08 出版日期:2021-11-30 发布日期:2021-12-04
  • 通讯作者: 吴俊东,E-mail:wujun-dong@163.com E-mail:wujun-dong@163.com
  • 作者简介:方煜彤,E-mail:fyt1996@126.com。
  • 基金资助:
    汕头市科技计划项目(170828211930352,[2018]120);吴阶平医学基金会临床科研专项资助基金(320.6750.2021-10-34);2019年广东省科技专项资金项目(汕府科2019-132)

Mechanisms of miR-221 and miR-222 on expression of GATA3 and FOXA1 and their effects in breast cancer

FANG Yutong1,2, ZHANG Qunchen1,2, HONG Chaoqun2,3, CHEN Chunfa1,2, CHEN Jiongyu2,3, ZHANG Rendong1,2, WU Jundong1,2   

  1. 1. The Breast Center, Cancer Hospital of Shantou University Medical College, Shantou 515041;
    2. Department of Central Laboratory, Cancer Hospital of Shantou University Medical College, Shantou 515041;
    3. Guangdong Provincial Key Laboratory for Breast Cancer Diagnosis, Shantou 515041, Guangdong, China
  • Received:2021-07-16 Revised:2021-09-08 Online:2021-11-30 Published:2021-12-04

摘要: 目的: 探讨乳腺癌中miR-221和miR-222(miR-221/222)表达水平对GATA结合蛋白3(GATA3)和叉头框蛋白A1(FOXA1)表达的影响及其作用机制。方法: 收集汕头大学医学院附属肿瘤医院2020年1月—2021年5月经手术切除的86例乳腺浸润性导管癌及其癌旁组织标本,分别采用茎环引物实时荧光定量PCR(qPCR)和免疫组织化学方法检测癌组织和相应癌旁组织中miR-221/222的表达水平及GATA3、FOXA1蛋白的表达水平,分析其与临床病理指标的关系。进一步在5种乳腺癌细胞系(MCF-7、T47D、SKBR3、MDA-MB-231和BT-549)中转染miR-221/222 mimics后,采用qPCR检测各细胞中miR-221/222的表达水平;采用Western blot检测转染后MCF-7细胞中GATA3、FOXA1、雌激素受体-α(ER-α)和钙黏蛋白E(E-cadherin)的表达;用细胞划痕和迁移侵袭实验检测转染后MCF-7细胞修复和迁移侵袭能力。结果: 在乳腺癌组织中,miR-221/222的表达水平明显高于癌旁正常乳腺组织(P=0.00)。miR-221/222表达水平在FOXA1、GATA3、ER-α及孕激素受体(PR)阴性表达组均显著高于阳性表达组(P<0.01);在Her-2阳性和高表达Ki-67的乳腺癌中较Her-2阴性和低表达Ki-67组显著升高(P<0.05);在三阴性和Her-2阳性亚型乳腺癌中显著高于Luminal A和Luminal B1型乳腺癌(P=0.00);而miR-221/222水平与患者年龄、月经状况、肿瘤大小、组织学分级、淋巴结转移及TNM分期无显著相关关系(P>0.05)。在5种乳腺癌细胞系中,miR-221/222在MCF-7细胞中均低表达。与对照组相比,在MCF-7细胞中瞬时转染miR-221/222 mimics后miR-221/222表达显著升高(P<0.05),且内源性的GATA3和FOXA1蛋白表达被抑制(P<0.01),上皮细胞相关分子ER-α和E-cadherin表达显著下降(P<0.01);划痕修复能力和迁移侵袭能力均显著增强(P<0.01)。结论: miR-221/222可能通过下调GATA3和FOXA1的表达促进乳腺癌的迁移和侵袭。

关键词: 乳腺癌, miR-221, miR-222, GATA结合蛋白3, 叉头框蛋白A1, 茎环引物实时荧光定量PCR

Abstract: OBJECTIVE: To investigate effects and mechanisms of miR-221 and miR-222 levels on expression of GATA3 and FOXA1 in invasive ductal carcinoma of breast cancers. METHODS: Invasive ductal carcinoma of breast cancer and their adjacent tissue samples were collected from January 2020 to May 2021 in the Cancer Hospital of Shantou University. Expression levels of miR-221/222 were detected by Stem-loop real-time PCR and expression of GATA3 and FOXA1 were detected by immunohistochemistry in both tissues. Correlations between expression levels of miR-221/222,GATA3 and FOXA1,as well as the relation between their expression levels and clinicopathological characteristics were analyzed. Furthermore,expression levels of miR-221/222 were detected by qRT-PCR in five breast cancer cell lines and MCF-7 cells after transfection with miR-221/222 mimics. Meanwhile,protein expression of GATA3,FOXA1,ER-α and E-cadherin were analyzed by Western blot. The abilities cell scratch repair,and migration and invasion of the cells were detected by both wound healing and transwell assays. RESULTS: Among the 86 cases,expression levels of miR-221 and miR-222 in breast invasive ductal carcinoma were significantly higher than that in adjacent tissues (P=0.00). Expression levels of miR-221/222 were significantly higher in the negative expression group of GATA3,FOXA1,ER-α and PR compared with the positive expression group (P<0.01). miR-221/222 expression levels were significantly increased in breast cancer of positive expression of Her-2 and high expression of Ki-67 compared with the negative expression of Her-2 and low expression of Ki-67 (P<0.05). miR-221/222 expression levels were also significantly higher in triple negative breast cancer and Her-2 positive subtypes than that in Luminal A and Luminal B1 subtypes (P=0.00). No association was found between miR-221/222 expression levels with age,menopausal status,tumor size,histological grade,lymph node metastasis,and TNM stage. In all 5 breast cancer cell lines,expression of miR-221/222 was one of the lowest in MCF-7 cells. Compared with the control group,the expression of miR-221/222 was significantly increased after transfection with miR-221/222 mimics in MCF-7 cells (P<0.05),and endogenous GATA3 and FOXA1 protein expressions were inhibited in cells (P<0.01). Expression of ER-α and E-cadherin decreased significantly (P<0.01). The ability of scratch repair and migration and invasion were significantly enhanced (P<0.01). CONCLUSION: miR-221/222 might promote migration and invasion in breast cancers by down-regulating expressions of GATA3,FOXA1,and ER-α.

Key words: breast cancer, miR-221, miR-222, GATA3, FOXA1, stem-loop qPCR

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