MiR-365: a mechanosensitive microRNA stimulates chondrocyte differentiation through targeting histone deacetylase 4.

Mechanical stress plays an essential role in tissue development and remodeling. In this study, we determined the role of microRNA in chondrocyte mechanotransduction. Using microarray, we identified miR-365 as a mechanoresponsive microRNA in parallel to mechanical induction of Indian hedgehog (Ihh) in primary chicken chondrocytes cultured in 3-dimensional collagen scaffoldings under cyclic loading (1 Hz, 5% elongation). Interestingly, expression of miR-365 is elevated in the prehypertrophic zone of the growth plate, coinciding with the Ihh expression region in vivo. MiR-365 significantly stimulates chondrocyte proliferation and differentiation. MiR-365 increases expression of Ihh and the hypertrophic marker type X collagen, whereas anti-miR-365 inhibits the expression of these genes. We identified histone deacetylase 4 (HDAC4), an inhibitor of chondrocyte hypertrophy, as a target of miR-365. MiR-365 inhibits both endogenous HDAC4 protein levels as well as the activity of a reporter gene bearing the 3'-untranslated region of HDAC4 mRNA. Conversely, inhibition of endogenous miR-365 relieves the repression of HDAC4. Mutation of the miR-365 binding site in HDAC4 mRNA abolishes miR-365-mediated repression of the reporter gene activity. Overexpression of HDAC4 reverses miR-365 stimulation of chondrocyte differentiation markers including Ihh, Col X, and Runx2. Moreover, inhibition of miR-365 abolishes mechanical stimulation of chondrocyte differentiation. Taken together, miR-365 is the first identified mechanically responsive microRNA that regulates chondrocyte differentiation via directly targeting HDAC4.