BMP-6 Inhibits MicroRNA-21 Expression in Breast Cancer Through Repressing DeltaEF1 and AP-1

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 deltaEF1 and TPA induced miR-21 expression. Using site-directed mutation and CHIP assay, we found that deltaEF1 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 deltaEF1 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. deltaEF1 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.