软骨相关lncRNAs与miR-320c构成ceRNA网络靶向调控锌离子转运体Slc39a8促进软骨再生、抑制退变的作用和机制研究

How to promote chondrogenesis and delay cartilage degeneration in chondrogenic differentiation of MSCs are crucial points for the cartilage tissue engineering. We reported important roles of miR-320 in cartilage development and degeneration in our previously studies. Zinc translporter Slc39a8 is a potential target of miR320c, and may play an important role in cartilage degeneration. The bioinformatics predictions indicate that 4 cartilage degeneration related lncRNAs could suppress the expression of miR-320c as ceRNAs, and the four lncRNAs also have some connections with Slc39a8 in chondrogenesis and cartilage degeneration. Thus, our hypothesis is: down regulation of degeneration related lncRNAs, upregulation of miR-320c could suppress the Slc39a8 expression, and lead to the promotion of MSCs chondrogenesis and the delay of cartilage degeneration. Multiple ways including over expression, RNA interference, as well as chromatin immunoprecipitation and dual-luciferase reporter system will be applied to demonstrate the ceRNA network. We will use the ceRNA network to promote the cartilage regenerations of rabbit knee joints cartilage defects filled with novel MSCs-seeded TCP-COL-HA scaffolds. This project could provide a new method for delaying the cartilage degeneration process as well as promoting the chondrogesis of MSCs in cartilage tissue engineering, and improve the cartilage tissue engineering with efficient benefits.

目前组织工程软骨的种子细胞成软骨分化效率低，且易发生退变。我们前期研究表明miR320c能促进成软骨分化、抑制退变；锌离子转运体Slc39a8是miR320c的潜在靶基因，与软骨退变有关；我们进一步实验及信息学分析发现芯片筛选的4个软骨退变lncRNAs可作为ceRNA竞争性结合miR320c，拮抗miR320c对Slc39a8的负性调控。因此，我们认为：抑制lncRNAs，过表达miR320c可抑制Slc39a8进而促进成软骨分化，抑制软骨退变。本研究将采用RNA干扰、过表达、ChIP及双荧光素酶报告基因等手段阐明软骨退变lncRNAs、miR320c和Slc39a8组成的ceRNA调控关系；在兔膝软骨缺损中植入复合MSC的TCP-COL-HA软骨支架修复缺损，研究这一ceRNA调控网络在软骨修复中的作用，为解决软骨组织工程中MSC成软骨分化效率低、分化后易退变的难题提供新思路。

按照研究计划，项目组重点研究在表观遗传学层面，以miR-320c代表的软骨分化相关微小RNA(miRNA)、Slc39a8及成软骨相关ncRNA作为ceRNA竞争结合miR-320c在MSC成软骨分化和软骨退变过程中的作用及机制。项目组研究发现miR-320c可抑制Slc39a8的3′-UTR区域的活性，从而抑制Slc39a8的表达进而促进MSC成软骨分化。同时发现与lncRNA同为ncRNA的软骨退变相关circRNA可作为ceRNA竞争性结合miR-320c从而调控MSC成软骨分化及软骨退变过程，抑制circCRIM1可降低其与miR-320c的竞争性结合作用从而提高MSC软骨特异性基因的表达水平及粘多糖的积累，促进MSC成软骨分化及体内组织工程软骨的形成。. 同时，在表观遗传学层面，项目还研究了与miR-320c共同筛选出的另一成软骨分化相关miR-193b-3p通过抑制HDAC3的表达进而促进COL2A1等启动子区域促进组蛋白H3的乙酰化状态进而调控MSC成软骨分化及软骨退变的作用机制。. 半月板主要是由纤维软骨细胞构成，本项目还研究了软骨分化相关miRNA在半月板的损伤、退变过程中的表达变化规律以及miRNA在骨关节炎患者的滑膜细胞中的表达变化规律。结果提示软骨分化相关miRNA的表达变化可能影响半月板的损伤、退变过程。软骨分化相关miRNA在骨关节炎患者滑膜细胞中表达的升高可能对软骨的退变起一定的保护作用。以上研究结果为未来治疗软骨退变相关疾病提供了一些新的潜在干预靶点。