MiR-519d-3p REDUCES OSTEOCLASTOGENESIS BY LOWERING MMP2 AND SHIFTING OSTEOBLAST SIGNALING TOWARD OSTEOPROTEGERIN.

Abstract

Background: Paracrine crosstalk between tumor cells and bone-resident cells drives osteoclastogenesis in the metastatic bone niche. miR-519d-3p has been linked to suppression of matrix metalloproteinases.

Aim: To test whether osteoblastic miR-519d-3p restrains tumor-conditioned osteoclastogenesis by lowering MMP2 expression and shifting signaling toward osteoprotegerin (OPG).

Materials and methods: Mouse osteoblasts (MC3T3-E1) were transfected with miR‑519d-3p mimic, inhibitor, or corresponding negative controls. Osteoblast-conditioned medium (OB-CM) was mixed 1:1 with MDA-MB-231 tumor-conditioned medium (231-CM) and applied to bone-marrow-derived macrophages for tartrate-resistant acid phosphatase (TRAP) staining. Osteoblastic MMP2 and OPG mRNAs were quantified by RT-qPCR, and a dual-luciferase system compared the wild-type vs seed-mutant MMP2 3'UTR reporters.

Results: Relative to their controls, miR-519d-3p mimic OB-CM produced fewer TRAP-positive multinucleated osteoclasts, whereas inhibitor OB-CM produced more. In osteoblasts, the mimic decreased MMP2 mRNA, whereas the inhibitor increased it, and the changes in OPG mRNA were modest. Dual-luciferase assays did not show a reproducible wildtype-over-mutant selective repression by miR-519d-3p, indicating context-dependent or indirect regulation of MMP2.

Conclusion: Elevating osteoblastic miR-519d-3p in vitro associated with lower MMP2 and an anti-osteoclastogenic paracrine output, suggesting a tractable microRNA-based approach to modulate the metastatic bone niche and warranting further validation in vivo.