Department of Joint Surgery, First Affiliated Hospital of SunYat-sen University, Guangzhou, Guangdong 510080, China.
Theranostics. 2018 Apr 15;8(10):2862-2883. doi: 10.7150/thno.23547. eCollection 2018.
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. 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 , 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. 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 , , , and 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 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. These findings indicate that miR-193b-3p directly targets HDAC3, promotes H3 acetylation, and regulates hMSC chondrogenesis and metabolism in PHCs.
组蛋白去乙酰化酶 3（HDAC3）在人类软骨细胞中抑制软骨特异性基因表达中起关键作用。本研究旨在确定 microRNA-193b-3p（miR-193b-3p）是否在软骨生成和软骨细胞代谢过程中调节 HDAC3 的表达。在人骨髓间充质干细胞（hMSC）软骨生成模型中、白细胞介素 1β（IL-1β）处理的原代人软骨细胞（PHC）中以及未降解和降解软骨中评估了 miR-193b-3p 的表达。hMSC 和 PHC 转染 miR-193b-3p 或其反义抑制剂。通过进行荧光素酶报告基因测定，证实 miR-193b-3p 与其假定的结合位点在 HDAC3 mRNA 的 3'-非翻译区（3'-UTR）之间存在直接相互作用。在使用抗乙酰化组蛋白 H3 抗体进行染色质免疫沉淀测定之前，用 miR-193b-3p 转染软骨细胞。为了研究转染 miR-193b-3p 的 PHC，将它们接种在三钙磷酸盐-胶原-透明质酸（TCP-COL-HA）支架中，然后将其植入裸鼠中。此外，还测量了来自正常对照者和骨关节炎（OA）患者的血浆外泌体 miR-193b-3p。与非降解软骨相比，在软骨生成和肥大的 hMSC 中 miR-193b-3p 的表达升高，而在降解软骨中表达显著降低。此外，miR-193b-3p 抑制包含 HDAC3 3'-UTR 的报告构建体的活性，抑制 HDAC3 的表达，并在 、 、 和 启动子中促进组蛋白 H3 乙酰化。用组蛋白去乙酰化酶抑制剂曲古抑菌素 A（TSA）处理可增加软骨特异性基因表达并增强 hMSC 软骨生成。TSA 还增加了 IL-1β 处理的 PHC 中的 AGGRECAN 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 的过表达强烈增强了 软骨形成。我们还发现，OA 患者的血浆外泌体 miR-193b 水平低于对照者。这些发现表明，miR-193b-3p 直接靶向 HDAC3，促进 H3 乙酰化，并调节 hMSC 软骨生成和 PHC 中的代谢。
