miR-455-3p和C/EBPβ在间充质干细胞成软骨分化中阻抑退变的协同调控机制研究

The early degeneration of tissue engineering cartilage during forming and surviving is still a big problem for clinical application, and the key point of this early degeneration in tissue engineering cartilage is cell regulation. We have found that transcriptional (C/EBPβ etc) and post-transcriptional regulation were both involved in the chondrocyte degeneration, and we also found out that miR-455-3p was up-regulated over 2-fold during human MSCs chondrogenesis by miRNA microaray, which was also targeted C/EBPβ from bioinformatics. In this study, we investigate the mechanism of human MSCs chondrogenesis by ordered combinatorial regulation of miR-455-3p and C/EBPβ to improve chondrogenesis and retard chondrocyte degeneration at transcriptional and post-transcriptional level. Firstly, we will observe the expression kinetics of miR-455-3p, C/EBPβ and specific biomarker of cartilage to figure out their regulative rules and relationships with chondrogenesis and degeneration of tissue engineering cartilage. Next, we will clarify circulating co-regulation rules of these factors during hMSCs chondrogenesis at different stages and micro-environment. Lastly, we will prove the rule of retardment of degeneration and improvement of chondrogenesis by regulating this co-regulation mechanism of miR-455-3p and C/EBPβ,to provide the foundation for the control of degeneration of tissue engineering cartilage.

组织工程软骨在形成与生存中的较早退变仍困扰着临床应用，而较早退变的关键是细胞的调控。我们研究发现软骨细胞退变中转录后和转录（C/EBPβ等）协同调控起着重要作用，而最近我们对人MSCs进行 miRNA芯片筛选发现成软骨分化中miR-455-3p表达升高超过两倍，且生物信息学显示其与C/EBPβ有着对应靶点。本课题以miR-455-3p和C/EBPβ为切入点，研究转录后和转录调控子在人MSCs分化为成软骨细胞过程中阻抑退变、促进分化的协同调控机制：观察不同环境、不同阶段hMSCs成软骨分化时miR-455-3p、C/EBPβ和软骨特征性指标的表达动力学规律，及其与成软骨分化效果和退变效应相关性；研究miR-455-3p与C/EBPβ在hMSCs成软骨分化不同时期的协同调控机制；阐明两者的协同调控机制阻抑hMSCs向成软骨细胞分化中较早退变、促进分化的规律，为解决组织工程软骨退变提供理论基础。

项目以miR-455-3p切入点，研究转录后和转录调控子在人MSCs 分化为成软骨细胞过程中阻抑退变、促进分化的协同调控机制。目前研究开展按照计划完成，对研究内容均较好完成了工作并发表了相关SCI文章7篇。研究目前主要发现了MSCs成软骨分化中软骨细胞生长相关miRNA、C/EBPβ和Runx2循环协控，而miR-455-3p极可能抑制Runx2的表达，从而激活早期成软骨分化。我们也发现miR-455-3p的促成软骨作用在分化晚期明显减弱，这可能与软骨退变相关lncRNA的作用相关。我们进一步利用lncRNA芯片研究发现与miR-455-3p 有着密切相关性的有4个lncRNAs，共同组成ceRNA调控网络，并初步探讨证明了miR-455-3p的这种调控表现恰恰支持了lncRNA的竞争性内源RNA调控网络以及表观遗传学机制，这为后续研究的深入提供了基础。这些研究均证明了miR-455-3p协同阻抑MSCs向成软骨细胞分化中早期退变、促进分化的重要作用，将为解决骨关节炎关节软骨和软骨组织工程中间充质干细胞软骨分化后的退变这一难题提供了新的理论基础，具有比较重要的理论价值和科研应用价值。