RNA去甲基化酶ALKBH5修饰的LncRNA-SSR经ceRNA网络调控力学负荷介导软骨基质降解的机制研究

过度力学负荷导致骨性关节炎（osteoarthritis，OA）软骨基质降解的分子机制未明。我们前期基因测序发现长链非编码(Lnc)RNA-SSR在严重膝内翻OA患者破损的内侧软骨中高表达；生物信息学和体内外实验表明LncRNA-SSR、miR-223-3p和炎症小体NLRP3 mRNA构成的ceRNA网络，在过度力学负荷介导的软骨基质降解中发挥关键调控作用。进一步MeRIP-qPCR分析发现靶向ALKBH5可调控软骨LncRNA-SSR m6A水平、NLRP3和基质降解酶类的表达。我们据此提出“ALKBH5通过调控LncRNA-SSR m6A水平影响其表达，并经ceRNA网络调控力学负荷介导的软骨基质降解过程”的科学假说。本课题拟利用细胞和动物力学负荷模型，阐明ALKBH5对LncRNA-SSR甲基化水平及其表达的具体调控机制，以期为OA生物力学病变机制提供新观点，为治疗OA提供新思路。

The development of osteoarthritis (OA) and cartilage matrix degradation is driven by mechanical stress. However, the underlying molecular mechanism remains unclear. Our previous study found that physical overloading was significantly associated with OA progression, and long coding RNA-SSR (LncRNA-SSR) was upregulated in damaged medial cartilage of severe genua varus OA patients. Bioinformatic analyses and experiments in vivo and vitro revealed that LncRNA-SSR functioned as a competitive endogenous RNA (ceRNA) for miR-223-3p to positively regulate NLRP3 inflammsome. The ceRNA network formed by LncRNA-SSR/miR-223-3p/NLRP3 could play a critical role in the regulation of mechanical stress induced cartilage matrix degeneration. Our further experiments found that ALKBH5, a demethylase, was upregulated in chondrocytes of severe genua varus OA patients and mechanical stress induced OA mice. Moreover, down regulating ALKBH5 induced low expression of LncRNA-SSR, NLRP3 and MMPs. Therefore, we suggest that ALKBH5 modifies LncRNA-SSR’s N6-methyladenosine (m6A) to enhance its expression, which induces cartilage matrix degradation through regulating LncRNA-SSR/miR-223-3p/NLRP3 ceRNA network. In the current study, we aim to determine the molecular mechanism of ALKBH5 regulating LncRNA-SSR expression and its m6A modification using cell and animal models. This project will enable us to uncover the underlying mechanism of mechanical stress-induced cartilage matrix degradation, and help to identify novel therapeutic targets to promote effective OA prevention.