Post-transcriptional regulation of MMP2 mRNA by its interaction with miR-20a and Nucleolin in breast cancer cell lines

Matrix-metalloproteinase-2 (MMP2) is a foremost MMP, governing invasion of breast cancer cells during metastasis. miR-20a was reported to induce mesenchymal to epithelial transition in MDA-MB-231 cells and its endogenous expression varies directly with invasiveness of breast cancer cells. The inverse and direct correlation of invasiveness with miR-20a and Nucleolin respectively led us to study the post-transcriptional regulation of MMP2 by miR-20a and mRNA stabilizing protein, Nucleolin. Thus, understanding the mechanism of its regulation will enable modification of the invasion potential. MMP2 was found to be higher in MDA-MB-231 than MCF-7 cells both at RNA and protein levels. RNA–protein co-immunoprecipitation assay with Argonaute 2 revealed that MMP2 undergoes miRNA-mediated post-transcriptional regulation. miR-20a decreased MMP2 expression as well as its enzymatic activity as found by zymogram assay. Reporter assay showed that miR-20a directly binds to its putative binding site in MMP2 3′-UTR as per in silico prediction. miR-20a additionally impeded MMP2 mRNA stability, and binding of stabilizing trans-factor Nucleolin to its 3′-UTR was confirmed by RNA–protein co-immunoprecipitation assay. Partial down-regulation of Nucleolin by Si-RNA resulted in the downregulation of MMP2 and Nucleolin over-expression rescued the inhibitory effect of miR-20a on MMP2 expression. Delineating the mechanism of post-transcriptional regulation of MMP2, two of its potent regulators, miR-20a and Nucleolin were identified. It was established for the first time that MMP2 is a direct target of miR-20a. The results also elucidated that Nucleolin binds to MMP2 3′ UTR and its abundance affects MMP2 expression.