MicroRNAs调控网络对OA软骨细胞分化的作用机制研究

Based on the miRNA and mRNA differetentially expression profiles respectively obtained in rat chondrocyte differentiation and osteoarthritis (OA) patients in our previous study, we further illustrates the mechanism of miRNA network regulation on chondrocyte differentiation. This project is going to obtain the differetentially expressed miRNAs in OA chondrocytes, compared with miRNA regulating chondrocyte differentiation, to screen candidate key miRNAs and varify them in various chondrocyte differentiation models. In the meantime, the signaling transduction pathways of these differetentially expressed genes are intervened to confirm the candidate key target genes. Bioinformatical methods are used to predict the relationship between candidate miRNAs and their targets, and these candidate miRNAs are further intervened to detect the epression alteration of the targets for confirmation of their relationship and draw the atlas of miRNA relgaiton network. In induced chondrocyte differentiation model and OA chondrocytes, miRNAs and target gene expression are intervened to observe cell biological behavior and specific gene expression. Eventuallay, above-mentioned target genes and miRNA levels are detected in OA/normal chondrocytes and plasma, combined with clinical and patholigcal indexes to analyze their relationship with OA condition. Our results will demonstrate the function of miRNA regulation network on chondrocyte differentiation, and provide novel molecular markers for OA diagnosis as well as targets for disease treatment.

我们在前期获得大鼠软骨分化过程的miRNAs和OA患者软骨差异基因表达谱基础上，拟利用Solexa测序获得OA软骨差异表达miRNAs，与调控软骨分化的miRNAs比较，筛查候选关键miRNAs，并在多种软骨细胞分化模型中验证；同时干预调控差异表达基因的信号转导通路，确定通路中分子为的候选关键靶基因；生物信息学方法预测候选miRNAs和候选靶基因的靶向关系，继而通过干预候选miRNAs，检测靶基因表达确定关系，绘制miRNAs调控网络图；在软骨细胞诱导分化模型及OA软骨细胞中，分别干预miRNAs及靶基因表达，观察细胞生物学行为和特异基因的表达；最后，检测OA及正常软骨和血清中上述靶基因和miRNAs含量，结合临床与病理指标分析与OA病情的关系。结果将找到关键节点miRNAs，阐释miRNAs调控网络对软骨细胞分化的作用，为发现OA新的分子标记物及治疗靶点奠定基础。

处于软骨形成关键节点的miRNAs与其靶基因，按照严格的分级响应机制，协同调控其介导的信号转导通路，构成复杂的调控网络，以实现对软骨细胞分化和凋亡的精细调节。发现并阐明这些关键miRNAs及其介导的调控网络，可能是发现OA新分子标记物及治疗靶点的重要途径。按照计划，我们通过多组学方法获得大鼠关节软骨发育过程的miRNAs和OA患者关节软骨差异基因表达谱，并与调控软骨分化的miRNAs比较，筛选出miR-181a-5p、-23b、-26a；生物信息学方法预测、报告基因及脱靶实验证实，其靶基因分别是调控硒蛋白表达的关键分子SECISBP2、催化软骨Aggrecan合成的关键酶HS6ST2、骨髓源性巨噬细胞炎性反应的重要受体TLR3；在TNF-α和IL-1β处理的软骨细胞系SW1353和C28I/2模型发现，并在软骨形成、软骨损伤、T2毒素、两代低硒等大鼠模型确认，这些关键分子参与的信号转导通路；干预这些信号通路，证实了miR-181a-5p和SECISBP2介导的软骨氧化应激调控网络、miR-23b和HS6ST2介导的软骨基质代谢调控网络、和miR-26a和TLR3介导的炎性反应转导调控网络；再检测OA及正常人的软骨和血清样本中上述靶基因和miRNAs含量，结合临床与病理指标，确定miR-181a-5p和miR-23b与OA病情程度密切相关；最后，使用miR-26a在体治疗大鼠关节炎，能明显缓解关节炎症状。另外，首次发现了若干novel ncRNA，并启动了相关分子在软骨形成和OA中的作用研究；并找到了对低硒环境因子敏感的调控软骨细胞染色体重塑的关键分子PRMT5、HDAC2、SFMBT2，分别实验确认了其维持软骨表型的机制。基本实现本项目预期的研究目标，开启全面了解miRNA调控网络对软骨细胞分化精细调控的机制，从相对宏观的角度阐释了软骨细胞分化、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奠定了坚实的理论基础。