Bell-Hensley Austin, Brito Victor Gustavo Balera, Cai Lei, Liu Jin, Feeney Kathryn, Zheng Hongjun, McAlinden Audrey
Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA.
Department of Orthopaedic Surgery, Washington University in St. Louis, St. Louis, MO, USA.
Osteoarthr Cartil Open. 2024 Nov 26;7(1):100550. doi: 10.1016/j.ocarto.2024.100550. eCollection 2025 Mar.
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 chondrogenesis.
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.
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 , , and . Of the genes significantly downregulated by miR-181a/b-1, aquaporin 9 () 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 via an indirect targeting mechanism.
Our findings demonstrate a pro-differentiation/anabolic function for miR-181a/b-1 during chondrogenesis that may be due, in part, to suppression of . Future studies are needed to elucidate the role of this membrane channel protein in regulating chondrocyte differentiation and homeostasis.
需要有效的骨关节炎治疗方法来增强软骨细胞的合成代谢/再生能力。研究软骨发育过程可能会为我们提供控制软骨细胞分化和合成代谢的方法，并最终告知我们如何有效治疗骨关节炎。微小RNA是作用广泛的表观遗传调节因子，已知会影响许多骨骼过程。我们小组之前的报告表明，miR-181a-1在软骨细胞分化过程中上调。本研究的目的是确定整个miR-181a/b-1簇如何调节软骨生成。
使用慢病毒技术在软骨祖细胞中过表达前体miR-181a/b-1。将转导的软骨祖细胞在低氧条件下培养为微团颗粒，并刺激其进行软骨分化五周。应用批量RNA测序和免疫染色来评估软骨分化和基质产生。
软骨颗粒切片的免疫染色显示，miR-181a/b-1增加了成熟的II型胶原蛋白表达，并降低了软骨祖细胞IIA型胶原蛋白异构体的表达。软骨生成第7天的批量RNA测序显示，促合成代谢基因如……等上调。在被miR-181a/b-1显著下调的基因中，水通道蛋白9（AQP9）是下调最明显的，其表达下降超过14倍。虽然AQP9是miR-181a/b的预测靶点，但我们的数据表明，这个微小RNA簇可能通过间接靶向机制抑制AQP9。
我们的研究结果表明，miR-181a/b-1在软骨生成过程中具有促分化/合成代谢功能，这可能部分归因于对AQP9的抑制。未来需要进一步研究来阐明这种膜通道蛋白在调节软骨细胞分化和体内平衡中的作用。
