Department of Orthopaedics, The Affiliated Hospital of Nantong University, Nantong 226001, P.R. China.
Biochem J. 2021 Feb 26;478(4):721-734. doi: 10.1042/BCJ20200314.
Osteoporosis is a prevalent systemic skeletal disorder entailing bone fragility and increased fracture risk, often emerging in post-menopausal life. Emerging evidence implicates the dysregulation of microRNAs (miRNAs or miRs) in the progression of osteoporosis. This study investigated the effect of miR-199a-3p on osteoporosis and its underlying mechanism. We first examplished an ovariectomized (OVX)-induced rat osteoporosis model, and then isolated mesenchymal stem cells (MSCs) from bone marrow of the model rats. The overexpression and knock down of miR-199a-3p were conducted in OVX rats and MSCs to verify the role of miR-199a-3p on MSC differentiation. Calcium nodules were measured using alizarin red S (ARS) staining. RT-qPCR and Western blot assay were performed to measure the expression of miR-199a-3p, Kdm3a and osteogenic differentiation-related markers in rat tissues and cells. The correlation between miR-199a-3p and Kdm3a was confirmed using dual-luciferase reporter assay. The enrichment of Kdm3a at the Erk2 and Klf2 promoter was assessed using chromatin immunoprecipitation (ChIP) assay. Isolated MSCs were positive for CD29, CD44, CD90, and CD45, suggesting successful isolation of MSCs. There was increased expression of miR-199a-3p and inhibited osteogenic differentiation in OVX rats. Kdm3a was negatively targeted by miR-199a-3p. Our results also demonstrated that Kdm3a elevated the expression of Erk2 and Erk2 by promoting Erk2 and Klf2 demethylation, which further contributed to osteogenic differentiation. Overall, our results revealed a regulatory network of miR-199a-3p in osteogenic differentiation, highlighting miR-199a-3p as a potential target for therapeutic interventions in osteoporosis.
骨质疏松症是一种普遍的系统性骨骼疾病，涉及骨脆性增加和骨折风险增加，通常在绝经后出现。新出现的证据表明，微小 RNA（miRNA 或 miR）的失调参与了骨质疏松症的进展。本研究探讨了 miR-199a-3p 对骨质疏松症的影响及其潜在机制。我们首先建立了去卵巢（OVX）诱导的大鼠骨质疏松症模型，然后从模型大鼠的骨髓中分离间充质干细胞（MSCs）。在 OVX 大鼠和 MSCs 中进行 miR-199a-3p 的过表达和敲低，以验证 miR-199a-3p 对 MSC 分化的作用。使用茜素红 S（ARS）染色测量钙结节。进行 RT-qPCR 和 Western blot 分析，以测量大鼠组织和细胞中 miR-199a-3p、Kdm3a 和成骨分化相关标志物的表达。使用双荧光素酶报告基因检测证实 miR-199a-3p 与 Kdm3a 之间的相关性。使用染色质免疫沉淀（ChIP）分析评估 Kdm3a 在 Erk2 和 Klf2 启动子上的富集。分离的 MSCs 对 CD29、CD44、CD90 和 CD45 呈阳性，表明成功分离了 MSCs。OVX 大鼠中 miR-199a-3p 的表达增加，成骨分化受到抑制。Kdm3a 是 miR-199a-3p 的负靶标。我们的结果还表明，Kdm3a 通过促进 Erk2 和 Klf2 去甲基化来升高 Erk2 和 Erk2 的表达，从而进一步促进成骨分化。总的来说，我们的结果揭示了 miR-199a-3p 在成骨分化中的调节网络，突显了 miR-199a-3p 作为骨质疏松症治疗干预的潜在靶点。
