MiR-206与ANXA2的3'UTR靶向结合抑制乳腺癌侵袭

MiR-206 Inhibits Breast Cancer Invasion by Targeting the 3'UTR of ANXA2 mRNA

膜联蛋白A2(annexin A2,ANXA2)可促进人结直肠癌的侵袭和迁移。然而,ANXA2在乳腺癌中的作用以及调节机制尚缺乏系统的研究。本研究旨在探讨微小RNA-206(microRNA-206,miR-206)如何调节ANXA2基因的表达,进而影响乳腺癌的侵袭。通过基因预测软件Target Scan(Target Scan V5.2)找到与ANXA2的3'UTR区互补结合的miR-206。运用实时定量PCR(qRTPCR)检测不同乳腺癌细胞系中miR-206的表达水平,发现低侵袭性乳腺癌MCF-7细胞株miR-206表达量明显高于高侵袭性乳腺癌细胞株MDA-231、MDA-435和T47D。运用转染技术将miR-206质粒及miR-206抑制剂转入乳腺癌细胞系MDA-231后,qRT-PCR检测转染后各组细胞中miR-206的表达情况,结果显示转染成功。用Western印迹法检测各组细胞中ANXA2的表达情况,结果显示,miR-206负向调控ANXA2蛋白的表达。qRT-PCR显示,过表达乳腺癌细胞内miR-206后,ANXA2 mRNA基本没有变化。结果显示,miR-206是在翻译水平上影响ANXA2蛋白的表达。荧光素酶实验显示:miR-206能特异性地与ANXA2 mRNA的3'UTR结合,抑制其荧光素酶活性。Transwell侵袭实验检测各组细胞的侵袭能力。结果显示,过表达miR-206后,乳腺癌细胞体外侵袭能力明显减弱。综上所述,miR-206通过靶向结合癌基因ANXA2 mRNA的3'UTR区,抑制ANXA2蛋白翻译,从而抑制了乳腺癌细胞的侵袭。因此,miR-206有望成为抑制乳腺癌侵袭与治疗乳腺癌的新靶点和生物学标记物。

Human Annexin A2（ ANXA2） promotes the invasion and migration of colorectal cancer.However,the role and the regulatory mechanism of ANXA2 in breast cancer are still unclear. This study investigates how microRNA-206（ miR-206） regulates the expression of ANXA2 and thus affects invasion of breast cancer. MiR-206,which binds with the 3＇ UTR region of ANXA2,was found by a gene prediction software Target Scan（ Target Scan V5. 2）. Then quantitative real-time PCR（ qRT-PCR）showed that the expression levels of miR-206 was significantly higher in low invasive grade MCF-7 cells than other high invasive grade breast cancer cell lines： MDA-231,MDA-435 and T47 D. Subsequently,MDA-231 cells were transfected with miR-206 plasmids,miR-206 inhibitors and control vectors. After confirming the successful transfection of miR-206 by qRT-PCR,we examined ANXA2 protein levels by Western blots and ANXA2 mRNA levels by qRT-PCR. While ANXA2 protein abundance decreased,ANXA2 mRNA remained constant,suggesting that miR-206 affects the translation of ANXA2 mRNA.Mechanistically, double luciferase reporter assays were performed, and we found that miR-206 specifically binds to ANXA2 3＇ UTR and inhibits the relative mRNA＇s activity of luciferase.Consequently,the invasion ability of breast cancer cells was significantly decreased after overexpression of miR-206,as evidenced by Transwell invasion assay results. To sum up,miR-206 inhibits the translation of ANXA2 proteins by targeting the 3＇ UTR region of ANXA2 mRNA,thereby inhibiting the invasion of breast cancer cells. Therefore,we propose that miR-206 could be a biomarker of metastatic breast cancer and a new target for breast cancer therapy.