软骨中一个新circRNA的功能及在骨关节炎中的作用研究

Circular RNAs (circRNAs) are a large class of non-coding RNAs that exist ubiquitously in the cytoplasm of eukaryotic cells; these endogenous RNAs are characterized by a stable structure and high tissue-specific expression. Compared with linear RNAs, circRNAs exhibit the remarkable characteristic of undergoing non-canonical splicing without a free 3′ or 5′ end. Recently, it was reported that circRNAs function as miRNA ‘sponges’ that naturally sequester and competitively suppress miRNA activity. It was demonstrated that circRNAs are involved in the development of several types of diseases, such as atherosclerosis and nervous system disorders. However, the role of circRNAs in cartilage and their overall contribution to OA pathogenesis are still unknown. .We found a novel circRNA which plays as a potential sponge of miR-140 in rats via performed proteomic sequencing and transcriptome sequencing, including mRNA, lncRNA, sRNA and non-polyA-RNA. The novel circRNA, named ciRS-140, is highly conserved in zebra fish, mouse, rat and human. In this study, the functions of ciRS-140 in cartilage will be clarified from the four dominating strategies. .Firstly, ISH method is applied to confirm the novel circRNA in normal knee joint cartilage in human. And RT-qPCR is used to detect its temporal and spatial specificity expression in several organs and tissues in inbred DA rats,which is a osteoarthritis (OA) susceptible inbred rats. .Secondly, CRISPR/Cas9 technology is available to knock out ciRS-140 in human chondrocytes C28/I2 and rats' chondrocytes RCJ3.1/C5.18 cell lines. Meanwhile the ciRS-140 is overexpression through Adenovirus vectors in the two cell lines. Then, the molecules phenotype of cartilage is checked with RT-qPCR and westren blotting. And the target gene of miR-140 is also detection by westren blotting. .Thirdly, three significative approachs, Dual-luciferase assays system, RNA-FISH with the probe of LAN-miR-140, and PAR-CLIP of targeting the AGO, are employed to demonstrate the interaction between ciRS-140 and miR-140..Finally, three types samples is collected for detecting the expression of ciRS-140 to investigate the relationship with OA, including knee joint cartilage tissues, synovia and serum samples of normal and OA patients. And the SNP of ciRS-140 is researched to correlation analysis of genetic susceptibility with OA. In addition, the ciRS-140 is overexpression and knocking out in primary chondrocytes of OA patients and established OA model by DA rats, to illuminate the role of ciRS-140 in OA..In summary, clarifying the ciRS-140 function in cartilage is the perfect prospect for biomarkers and potential therapeutic targets of OA. Deciphering the precise molecular mechanisms of circRNA function in OA will be critical for understanding OA pathogenesis and exploring new potential therapeutic targets.

单链共价闭合环状非编码RNA（circRNA）是细胞内的miRNA海绵。我们用多组学方法在大鼠软骨找到一个新circRNA，在多物种高度保守，可能通过调节游离miR-140丰度而精细调控软骨功能，暂称ciRS-140。为阐明其功能，拟对其：用索尾插接位点特异的探针，ISH法确认在人膝关节软骨达，观察在近交系DA大鼠表达的时空特异性；在人、大鼠软骨细胞系和DA大鼠软骨敲出和高表达，观察软骨分子表型，分析在软骨的生理功能；报告基因、LAN-miR-140探针的RNA-FISH和AGO的PAR-CLIP法，确实与miR-140的相互作用；检测在OA和正常人膝关节软骨、滑液及血清的表达，和与OA的关联分析，明确与OA的相关性；在人原代软骨细胞和DA大鼠OA模型敲出和高表达，观察软骨分子表型及miR-140靶蛋白表达，明确在OA中的作用。为OA的精准医疗寻找新的、有效的分子标记和治疗靶点奠定基础。

单链共价闭合环状非编码RNA（circRNA）是细胞内的miRNA海绵。我们用多组学方法在大鼠软骨找到一个新circRNA，在多物种高度保守，可能通过调节游离miR-140丰度而精细调控软骨功能，暂称ciRS-140。提出：系统地对其功能进行诠释，可能是全面阐明软骨生成和OA发生机制的关键。. 通过该项目的研究，我们确认ciRS-140即为hsa_circ_0088036，实验证实是miR-140-5p的海绵。hsa_circ_0088036 和miR-140-5p构成的调控网络，与miR-181a-5p与SBP2软骨氧化应激调控网络、miR-23b与HS2HT6软骨基质代谢调控网络、novel ncRNA与PRMT3染色质重塑网络、miR-26a参与的OA炎症反应调控网络相互作用，共同构成软骨发育调控的关键节点，对软骨损伤和修复有至关重要的作用。实验确认miR-181a-5p和miR-23b与OA病情程度密切相关，miR-26a在体治疗能明显缓解大鼠关节炎症状。. 对这个复杂调控网络的进一步研究，发现了OA软骨组织中染色质重塑与修饰的关键分子PRMT3、PRMT5、SFMBT2。并实验证实PRMT5对SOX9的R74K的甲基化，对软骨形成与表型维持过程中的关键调控机制。提出：SFMBT2是调控软骨细胞染色质重塑的关键分子的新理论，并得到NSFC81772410项目资助。.项目组通过OA及RA滑膜组织蛋白质组比较研究，发现了关节腔内OA及RA的差异表达蛋白并实验验证，为阐明RA发病机制及发现潜在性治疗靶点提供理论依据做了初步探索。. 本项目从相对宏观的角度阐释软骨细胞分化、骨骼发育及OA发病的分子机制，为临床发现OA新分子标记物、及治疗靶点，OA的精准医疗奠定了坚实的理论基础。.在本项目资助下，共发表文章18篇，其中包括SCI论文14篇，总IF：53.92（其中以本项目为第一标资助9篇，总IF：38.43）；中文核心4篇，以本项目为第一标资助3篇；其余均为第二标注。培养博士后2人，培养博士并获得学位5人（包括国际学生1名），培养硕士并获得学位5人。. 项目组在前述的基础理论研究基础上，致力于实现临床转化与应用努力。目前已经取得其他各级相关的应用研究资助2项，合计50万元。