The Raf kinase inhibitor protein (RKIP) is a tumor suppressor that protects against metastasis and genomic instability. RKIP is downregulated in many types of tumors, although the mechanism for this remains unknown. MicroRNAs silence target genes via translational inhibition or target mRNA degradation, and are thus important regulators of gene expression. In the current study, we found that miR-224 expression is significantly upregulated in breast cancer cell lines, and especially in highly invasive MDA-MB-231 cells, compared to human normal breast epithelial cells. In addition, miR-224 inhibits RKIP gene expression by directly targeting its 3'-untranslated region (3'-UTR). Moreover, metastasis, as assayed by Transwell migration, 3D growth in Matrigel, and wound healing, was enhanced by ectopic expression of miR-224 and inhibited by miR-224 downregulation. Promotion of metastasis in response to miR-224 downregulation was associated with derepression of the stroma-associated RKIP target genes, CXCR4, MMP1, and OPN, which are involved in breast tumor metastasis to the bone. Taken together, our data indicate that miR-224 play an important role in metastasis of human breast cancer cells to the bone by directly suppressing the RKIP tumor suppressor.
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