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

Abstract

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.

Evidence that miR‐146a attenuates aging‐ and trauma‐induced osteoarthritis by inhibiting Notch1, IL‐6, and IL‐1 mediated catabolism

“…We demonstrate in this study that miR‐146a may be a key factor that regulates OA pathogenesis by responding to aging, inflammation, and mechanical loading, thereby potentially linking these diverse OA‐driving factors. We were the first to identify miR‐146a as a mechanoresponsive microRNA along with miR‐365 through cytomechanical screening of microRNA microarray (Guan, Yang, Wei & Chen, 2011). Since then, multiple studies have supported the role of miR‐146a as a mechano‐miR (Huang, Crawford, Higuita‐Castro, Nana‐Sinkam & Ghadiali, 2012; Li et al., 2012).…”

“…Articular cartilage tissue sections were then stained with Safranin O/fast green or hematoxylin and eosin (H&E) or analyzed by immunohistochemistry. Primary human chondrocytes were isolated from human articular cartilage and were seeded into 6‐well plates for IL‐1β treatment, transfection, or seeded into 3D collagen sponges and subjected to 5%, 10%, and 20% elongation, 1 Hz cyclic loading for 24 hr as described previously (Guan et al., 2011; Yang et al., 2016). …”

“…18S ribosomal RNA was used as an internal control gene to normalize the mRNA levels. Relative transcription levels were calculated as previously described (Guan et al., 2011; Yang et al., 2016). In situ hybridization of miR‐146a was performed according to the manufacturer's instructions.…”

MicroRNAs and post-transcriptional regulation of skeletal development

“…Other miRNAs regulate cell differentiation by targeting chromatin epigenetic modifiers (Tuddenham et al 2006, Guan et al 2011. For instance, miR-365 stimulates chondrocyte differentiation through Hdac4 repression, thereby increasing the levels of Ihh and Col X (markers of pre-hypertrophic chondrocytes and hypertrophic chondrocytes respectively; Guan et al 2011).…”

MicroRNAs in farm animals

“…In chicken limb mesenchymal cells, miR-221 inhibits slug degradation by targeting mdm2 to regulate chondrogenic differentiation (Kim et al, 2010), and miR-142-3p regulates chondrogenesis by directly modulating the disintegrin and metalloprotease 9 (ADAM9) gene (Kim et al, 2011b). In primary chicken chondrocytes, miR-365 is a mechanically responsive miRNA that stimulates chondrocyte differentiation by targeting histone deacetylase 4 (HDAC4), an inhibitor of chondrocyte hypertrophy (Guan et al, 2011). miR-34a negatively regulates chondrogenesis by targeting EphA5 and suppressing the migration of chondroblasts (Kim et al, 2011a) or the reorganization of the actin cytoskeleton, which is essential for chondrocyte differentiation (Kim et al, 2012).…”