Osteoarthritis and Cartilage Open (Mar 2025)
MicroRNA-181a/b-1 enhances chondroprogenitor anabolism and downregulates aquaporin-9
Abstract
Objective: Effective osteoarthritis treatments that enhance the anabolic/regenerative capacity of chondrocytes are needed. Studying cartilage development processes may inform us of approaches to control chondrocyte differentiation and anabolism and, ultimately, how to effectively treat OA. MicroRNAs are broad-acting epigenetic regulators known to affect many skeletal processes. Previous reports from our group indicated that miR-181a-1 is upregulated during chondrocyte differentiation. The goal of this study was to determine how the entire miR-181a/b-1 cluster regulates in vitro chondrogenesis. Design: Precursor miR-181a/b-1 was over-expressed in cartilage progenitor cells using lentiviral technology Transduced cartilage progenitor cells were cultured as micromass pellets in hypoxic conditions and stimulated to undergo chondrogenic differentiation for five weeks. Bulk RNA-sequencing and immunostaining was applied to evaluate chondrogenic differentiation and matrix production. Results: Immunostaining of cartilage pellet sections showed that miR-181a/b-1 increased mature type II collagen and decreased expression of the chondroprogenitor type IIA collagen isoform. Bulk RNA-Seq at day 7 of chondrogenesis revealed upregulation of pro-anabolic genes such as COL2A1, COL9A2/3, COL11A2 and SNORC. Of the genes significantly downregulated by miR-181a/b-1, aquaporin 9 (AQP9) was the top hit which decreased in expression by over 14-fold. While a predicted target of miR-181a/b, our data showed that this miRNA cluster likely suppresses AQP9 via an indirect targeting mechanism. Conclusions: Our findings demonstrate a pro-differentiation/anabolic function for miR-181a/b-1 during in vitro chondrogenesis that may be due, in part, to suppression of AQP9. Future studies are needed to elucidate the role of this membrane channel protein in regulating chondrocyte differentiation and homeostasis.