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

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。

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

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.