软骨lncRNAs、miR-320c及Runx2组成ceRNA网络维持MSC成软骨分化稳态的机制研究

How to delay the progress of cartilage degeneration in chondrogenesis is a crucial point for the cartilage tissue engineering. We reported the important roles of lncRNA and miR-320 in cartilage development and degeneration in our previously studies［Osteroarthritis and Cartilage, 2015, 2016]. We found the inverse correlation between four lncRNAs and miR-320c expression in the normal and OA cartilage. The bioinformatics predictions indicate that the four cartilage degeneration related lncRNAs could suppress the expression of miR-320c as ceRNAs, and the four lncRNAs have some connection with Runx2 in chondrogenesis. These four cartilage degeneration related lncRNAs, miR-320c and Runx2 could co-regulate the chondrogenesis and cartilage degeneration process. We also found the opposite expression pattern between Runx2 and miR-320c in the late-stage of chondrogenesis of ATDC5. As predicted by Target Scan, We know that Runx2 is a potential target gene of miR-320c. Therefore, on the basis of previous study, the aim of this project is to study the relation ship in cartilage degeneration related lncRNAs、miR-320c and Runx2 in the chondrogenesis and cartilage degeneration process, and study the role of lncRNA/miR-320c/Runx2 ceRNA network in promoting chondrogenesis and delaying cartilage degeneration. This project could provide a new method for stopping the cartilage degeneration process in cartilage tissue engineering and improving the cartilage tissue engineering with efficient benefits.

如何在促成软骨分化同时抑制早期退变发生是目前组织工程软骨研究的难题和关键。我们报道了软骨退变相关lncRNA以及miR-320在软骨的发育与退变中的重要作用。目前预实验结果显示, 4个退变相关lncRNA与miR-320c在OA软骨和正常软骨中的表达趋势相反，而信息学发现这4个lncRNAs均与Runx2相关，可能通过与miR-320c和Runx2构成的ceRNA调控环参与软骨发育与退变的调控。预实验结果也发现miR-320c和Runx2在成软骨分化的退变期呈现此消彼长的“镜像”关系，生物信息学分析也提示Runx2是miR-320c的靶基因。因此，本项目基于以上基础及理论，旨在采用体内外实验探讨软骨退变相关lncRNAs、miR320c组成的ceRNA调控网络与Runx2协同促进MSC成软骨分化，抑制早期退变发生的作用机制，为解决软骨组织工程中MSC成软骨分化后的退变难题提供新的理论基础。

本项目围绕组织工程软骨研究目前的关键点如何促成软骨分化同时抑制早期退变发生进行一系列的探索研究。在本项目研究中我们一方面研究发现间充质干细胞成软骨分化及软骨退变相关的lncRNA HOTTIP通过海绵吸附 miR-455-3p促进 CCL3的表达从而诱导软骨退变，阐明了lncRNA的内源性竞争RNA在成软骨分化及软骨退变过程中的调控网络。另一方面充分研究了间充质干细胞成软骨分化及软骨退变密切相关的miR-320c的下游靶点，发现miR-320c分别通过靶向CDK6、β-catenin来抑制NF-κB及Wnt信号通路活性从而调控间充质干细胞成软骨分化及与软骨退变。进一步通过外泌体芯片筛选及相关试验验证发现过表达miR-320c的人骨髓间充质干细胞（hBMSCs）来源的外泌体可促进成软骨分化，提示过表达miR-320c的hBMSCs的外泌体可能是早期逆转软骨退变及修复软骨的组织工程学靶向治疗方法之一。本项目的研究成果为解决软骨组织工程中MSC成软骨分化后退变的难题提供全新的理论基础及参考。