Matrix Vesicle microRNAs Regulate Growth Plate via Local Factor Production

Objectives: Local cell communication regulates proliferation and differentiation of chondrocytes within growth plate cartilage during endochondral bone formation, including extracellular matrix (ECM) mineralization. Matrix vesicles (MVs) are key players in the mineralization process. We found that MVs are selectively enriched with microRNAs (miRNA). The aim of this study was to determine the regulatory potential of specific miRNA that are enriched in MVs.
Methods: GC cells were isolated by enzymatic digestion from costochondral growth zone cartilage harvested from 5-week-old male Sprague Dawley rats (under approval of VCU’s IACUC). GC cells were cultured and fourth passage cells were transfected for 24 hours with specific miRNA mimics. Cells were harvested 48 hours post transfection completion. Proliferation (PicoGreen; EdU) and alkaline phosphatase specific activity (TNAP) were assayed. Protein levels for Ihh, osteoprotegerin (OPG), RANKL, VEGF and TGF-β1 were measured by ELISA. All experiment groups had an n=6 cultures per variable; results were validated in repeat experiments. A one-way analysis of variance with Tukey’s multiple comparison test was used. Significant differences have p < 0.05 and were determined using R v 3.4.3.
Results: Cell proliferation (total DNA; EdU incorporation) increased in GC cultures transfected with miR-122 while other miRNA had no effect. Cell layer TNAP was reduced by miR-122 and miR-451. OPG was increased by miR-122 and miR-451; RANKL was undetected for all groups; and VEGF production was increased by miR-22. miR-22 increased Ihh and latent TGF-β1, but not active TGF-β1.
Conclusions: Selective packaging of microRNA within MVs indicates that MVs are functioning in the role of cell signaling, likely in an autocrine and paracrine fashion. Our data indicate that miRNAs enriched in MVs are active components in the regulation of growth plate chondrocytes. Specific microRNA may be valid candidates for modulating the growth plate and its ECM production and turnover.