MicroRNA-34Suppressed Breast Cancer Invasion And Metastasis By Directly Targeting Fra-1

miR-34family (a, b and c) have attracted much attention because of their identification as p53target genes and their involvement in p53-mediated processes, such as cell cycle arrest and apoptosis. Functionally, miR-34was found to affect tumor cell apoptosis, senescence, proliferation and invasion. Recently, emerging evidence showed that miR-34a is involved in progression of various types of cancers including colon cancer, glioblastoma, pancreatic cancer, hematological malignancies and lung cancer, these findings indicate that miR-34may function importantly in human carcinogenesis, however, the possible roles and mechanisms of miR-34in human breast cancer are still not well established. In this study we determined the functions of miR-34in breast cancer and the mechanisms involved.First we examined expression of miR-34a and miR-34c in a series of human mammary tumor cell lines and primary breast cancer and their corresponding nontumorous tissues, we found that miR-34a/c expression is significantly decreased in metastatic breast cancer cells and human primary breast tumors with lymph node metastases, suggesting that miR-34a and miR-34c deregulation in breast cancer might be correlated with metastasis.Next, we performed the Transwell assay to investigatge the cell motility and invasiveness after re-expression of miR-34a/c, overexpression of miR-34a and miR-34c substantially suppressed the motility and invasiveness of MDA-MB-231and Hs578T cells. To further study whether miR-34a and miR-34c could inhibit metastatic behaviors in vivo, we constructed miR-34a/c expression cassette pLVX-EGFP-34ac lentiviral expression vector which expresses EGFP and miR-34a/c simultaneously. Only1of9mice implanted with the lenti-34ac-derived MDA-MB-231cells had lung metastasis, strikingly, however,7out of10mice implanted with the cells derived from lenti-EGFP exhibited remarkably metastasis to the lungs. The number of pulmonary metastatic nodules decreased significantly in the lenti-34ac group compared with control group, suggesting that miR-34ac is a metastatic inhibitor in vivo. Then we employed three strategies and identified Fra-1as a potential downstream target of miR-34a/c. We constructed pISO-Fra-1-3’UTR-WT which has a full length Fra-1mRNA3’UTR and pISO-Fra-1-3’UTR-MUT which has several nucleotide substitutions in the core binding sites of miR-34a/c. We identified Fra-1mRNA as a direct target of miR-34a/c through dual-luciferase reporter system. Consistent with these results, we observed a clear decrease in endogenous Fra-1protein and mRNA in MDA-MB-231and Hs578T cells transfected with miR-34a/c mimics.Furthermore, we explored whether miR-34a/c functions in cell migration and invasion via Fra-1regulation. Knock-down of Fra-1significantly decreased the motility and invasiveness of MDA-MB-231and Hs578T cells, while ectopic expression of Fra-1significantly increases MDA-MB-231cell migration and invasion, demonstrating the positive role for Fra-1in the migration and invasion in breast cancer.’Rescue’ experiments wherein Fra-1was overexpressed could partially abrogate miR-34a/c mediated inhibition of migration and invasiom in MDA-MB-231and Hs578T cells, and re-expression of miR-34a/c pool profoundly reduced cell migration and invasion in MDA-MB-231shGFP control cells but failed to do so in MDA-MB-231shFra-1cells, reinforcing the idea that Fra-1is a direct and functional target of miR-34a/c.Finally, we analysed of Fra-1in expanded human mammary epithelial cell lines and breast cancer samples, and found Fra-1expression was increased in metastastic cell lines and breast tumors with lymph node metastases, a significant inverse correlation between expression of Fra-1and miR-34a was also found in these cells and samples, indicate that miR-34a down-regulation may be associated with the increase of Fra-1protein levels in metastatic breast tumorsCollectively, these results suggest that miR-34functions as a metastasis suppressor in vitro and in vivo by targeting Fra-1oncogene and suggest a therapeutic application of miR-34in breast cancer.