MicroRNA-193b-3p regulates chondrogenesis and chondrocyte metabolism by targeting HDAC3.
MicroRNA-193b-3p regulates chondrogenesis and chondrocyte metabolism by targeting HDAC3.
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MicroRNA-193b-3p 通过靶向 HDAC3 调节软骨形成和软骨细胞代谢
作者：
Meng F;Li Z;Zhang Z;Yang Z;Kang Y;Zhao X;Long D;Hu S;Gu M;He S;Wu P;Chang Z;He A;Liao W
组蛋白脱乙酰酶 3 (HDAC3) 在抑制人软骨细胞中软骨特异性基因表达中发挥着关键作用。本研究的目的是确定 microRNA-193b-3p (miR-193b-3p) 在软骨形成和软骨细胞代谢过程中是否调节 HDAC3 的表达。方法：在人间充质干细胞 (hMSC) 软骨形成模型中，在白介素-1β (IL-1β) 处理的原代人类中评估 miR-193b-3p 的表达。 软骨细胞（PHC）以及未降解和降解的软骨。 hMSCs 和 PHCs 用 miR-193b-3p 或其反义抑制剂转染。通过进行荧光素酶报告基因测定，证实了 miR-193b-3p 与其 HDAC3 mRNA 3'-非翻译区 (3'-UTR) 中的推定结合位点之间的直接相互作用。用 miR-193b-3p 转染软骨细胞，然后用抗乙酰化组蛋白 H3 抗体进行染色质免疫沉淀测定。为了在体内研究 miR-193b-3p 转染的 PHC，将它们接种在磷酸三钙-胶原蛋白-透明质酸 (TCP-COL-HA) 支架中，然后植入裸鼠体内。此外，还测量了正常对照和骨关节炎（OA）患者样本中的血浆外泌体 miR-193b-3p。 结果：与未降解软骨相比，软骨形成和肥大 hMSC 中 miR-193b-3p 表达升高，而退化软骨中表达显着降低。此外，miR-193b-3p 抑制含有 HDAC3 3'-UTR 的报告构建体的活性，抑制 HDAC3 表达，并促进 COL2A1、AGGRECAN、COMP 和 SOX9 启动子中的组蛋白 H3 乙酰化。 HDAC 抑制剂曲古抑菌素 A (TSA) 治疗可增加软骨特异性基因表达并增强 hMSC 软骨形成。 TSA 还增加了 IL-1β 处理的 PHC 中 AGGRECAN 的表达并降低了 MMP13 的表达。此外，在裸鼠皮下植入PHC接种的TCP-COL-HA支架8周后，我们发现与对照条件下相比，miR-193b过表达强烈增强体内软骨形成。我们还发现 OA 患者的血浆外泌体 miR-193b 水平低于对照受试者。结论：这些发现表明 miR-193b-3p 直接靶向 HDAC3，促进 H3 乙酰化，并调节 PHC 中的 hMSC 软骨形成和代谢。
Histone deacetylase 3 (HDAC3) plays a pivotal role in the repression of cartilage-specific gene expression in human chondrocytes. The aim of this study was to determine whether microRNA-193b-3p (miR-193b-3p) regulates the expression of HDAC3 during chondrogenesis and chondrocyte metabolism.Methods: miR-193b-3p expression was assessed in a human mesenchymal stem cell (hMSC) model of chondrogenesis, in interleukin-1β (IL-1β)-treated primary human chondrocytes (PHCs), and in non-degraded and degraded cartilage. hMSCs and PHCs were transfected with miR-193b-3p or its antisense inhibitor. A direct interaction between miR-193b-3p and its putative binding site in the 3′-untranslated region (3′-UTR) of HDAC3 mRNA was confirmed by performing luciferase reporter assays. Chondrocytes were transfected with miR-193b-3p before performing a chromatin immunoprecipitation assay with an anti-acetylated histone H3 antibody. To investigate miR-193b-3p-transfected PHCs in vivo, they were seeded in tricalcium phosphate-collagen-hyaluronate (TCP-COL-HA) scaffolds, which were then implanted in nude mice. In addition, plasma exosomal miR-193b-3p in samples from normal controls and patients with osteoarthritis (OA) were measured.Results: miR-193b-3p expression was elevated in chondrogenic and hypertrophic hMSCs, while expression was significantly reduced in degraded cartilage compared to non-degraded cartilage. In addition, miR-193b-3p suppressed the activity of reporter constructs containing the 3′-UTR of HDAC3, inhibited HDAC3 expression, and promoted histone H3 acetylation in the COL2A1, AGGRECAN, COMP, and SOX9 promoters. Treatment with the HDAC inhibitor trichostatin A (TSA) increased cartilage-specific gene expression and enhanced hMSCs chondrogenesis. TSA also increased AGGRECAN expression and decreased MMP13 expression in IL-1β-treated PHCs. Further, 8 weeks after implanting PHC-seeded TCP-COL-HA scaffolds subcutaneously in nude mice, we found that miR-193b overexpression strongly enhanced in vivo cartilage formation compared to that found under control conditions. We also found that patients with OA had lower plasma exosomal miR-193b levels than control subjects.Conclusions: These findings indicate that miR-193b-3p directly targets HDAC3, promotes H3 acetylation, and regulates hMSC chondrogenesis and metabolism in PHCs.
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