ORIGINAL ARTICLE

Cell Research (2009): 487-496
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BMP-6 inhibits microRNA-21 expression in breast cancer through repressing δEF1 and AP-1

Jun Du*, Shuang Yang*, Di An1, Fen Hu, Wei Yuan, Chunli Zhai and Tianhui Zhu

Correspondence: Tianhui Zhu, Tel: +86-22-23505501; Fax: +86-22-23505501 E-mail: zhuth@nankai.edu.cn *These two authors contributed equally to this work.

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MicroRNAs (miRNAs), which are small noncoding RNA molecules, play important roles in the post-transcriptional regulation process. The microRNA-21 gene (miR-21) has been reported to be highly expressed in various solid tumors, including breast cancer. Bone morphogenetic protein-6 (BMP-6) has been identified as an inhibitor of breast cancer epithelial-mesenchymal transition (EMT) through rescuing E-cadherin expression. We initiated experiments to identify the relationships between miR-21 and BMP-6 in breast cancer progression. Real-time PCR analysis showed that miR-21 expression was very high in MDA-MB-231 cells that expressed little BMP-6. A reverse correlation between BMP-6 and miR-21 was also determined in breast cancer tissue samples. Moreover, BMP-6 inhibited miR-21 transcription in MDA-MB-231 cells. In order to investigate how BMP-6 inhibited the miR-21 promoter (miPPR-21), we constructed a series of miPPR-21 reporters. Luciferase assay results indicated that BMP-6 inhibited miPPR-21 activity through the E2-box and AP-1-binding sites. We also demonstrated that both δEF1 and TPA induced miR-21 expression. Using site-directed mutation and CHIP assay, we found that δEF1 induced miPPR-21 activity by binding to the E2-box on miPPR-21. Moreover, TPA triggered miPPR-21 activity through the AP-1 binding sites. BMP-6 treatment significantly reduced the binding of these factors to miPPR-21 by decreasing the expression of δEF1 and c-Fos/c-Jun. We also demonstrated that BMP-6-induced downregulation of miR-21 modified the activity of PDCD4 3'UTR and inhibited MDA-MB-231 cell invasion. δEF1 overexpression and TPA induction blocked this inhibitory effect of BMP-6. In conclusion, BMP-6-induced inhibition of miR-21 suggests that BMP-6 may function as an anti-metastasis factor by a mechanism involving transcriptional repression of miR-21 in breast cancer.

Cell Research (2009) 19:487–496. doi: 10.1038/cr.2009.34; published online 24 March 2009

Keywords: BMP-6, microRNA-21, AP-1, δEF1, breast cancer invasion