miR-455双向调控MSCs成软骨分化和退变中表观遗传学去阻遏的机制研究

The epigenetic regulations, including histone acetylation, DNA Methylation, and miRNAs, have been proved to exert important effects on chondrogenesis of mesenchymal stem cells and degradation of cartilage. Our previous work identified that miR-455 has a rare dual-way effect to prompt chondrogenesis and cartilage degradation simultaneously, which can barely be explained with models of previous reports regarding miRNAs-chondrogenesis-degradation. We thus proposed a novel theoretical conjecture: "miR-455 exerts a derepressive effect to prompts chondrogenesis and cartilage degradation simultaneously via the downstream regulation of epigenetic mechanisms, including histone acetylation, DNA Methylation, etc.". Our preliminary results indicated that with specifical inhibition of the HDAC2, 3, or Dnmt3a expression, miR-455 might relieve the acetylation of histone and methylation of DNA, the following repression of transcription, and prompt chondrogenesis of mesenchymal stem cells. We planned to identify the whole target gene profile of miR-455 with new technique of miRTRAP, find the target genes related to epigenetic mechanisms from the whole profile, and verify the theoretical conjecture in cell-tissue-body levels. This project is the first to identify the whole target gene profile of a specific miRNA and the first to propose a novel model of miRNA regulation of stem cell differentiation.

组蛋白乙酰化、DNA甲基化修饰和miRNAs等表观遗传学机制在间质干细胞成软骨分化和软骨退变过程中发挥重要作用。课题组前期发现miR-455具备罕见的促成软骨/退变双向调控能力，无法用以往报道的miRNAs调控成软骨/退变的模型加以解释。我们认为miR-455可能通过调控下游表观遗传学机制，包括组蛋白乙酰化、DNA甲基化，发挥去阻遏作用，从而同时促进间质干细胞成软骨分化和软骨退变。预实验结果发现miR-455可能靶向抑制HDAC2、3或Dnmt3a表达，解除其组蛋白去乙酰化、DNA甲基化产生的广泛基因抑制，发挥阻遏间质干细胞成软骨分化的作用。本研究我们拟采用新技术miRTRAP鉴定miR-455的全部靶基因，从中找出表观遗传学调控相关的靶基因，从细胞-组织-整体层次证明我们的理论猜测。该项目将首次一次性获得特定miRNA的全部靶基因并提出miRNAs调控干细胞分化的新模型。

按照研究计划，项目组重点研究蛋白乙酰化及DNA甲基化修饰的表观遗传学在人骨髓间质干细胞成软骨分化和软骨退变过程中的作用机制。项目组研究发现miR-455-3p可抑制HDAC2/8的3′-UTR区域的活性，从而抑制HDAC2/8的表达进而促进COL2A1启动子区域的组蛋白H3乙酰化状态。使用HDAC抑制剂TSA不但可提高软骨特异性基因的表达水平及粘多糖的积聚，还可抑制IL-1β刺激后的MMP13的表达水平及促进SOX9的核转位，进一步敲低HDAC2/3/8还可以增加SOX9的表达及降低RUNX2等炎症特异性基因的表达水平。从而验证了本项目构想的可行性，miR-455-3p 通过直接靶向HDAC2/8来促进组蛋白H3乙酰化，从而促进早期的成软骨分化发挥重要的作用。. 同时项目组将有或无miR-455-3p过表达的人骨髓间充质干细胞成小球样本进行全基因组DNA甲基化芯片分析，发现miR-455-3p 在人骨髓间充质干细胞成软骨分化的中期高表达，在晚期低表达，而DNMT3A则呈相反的趋势。为进一步体内验证本项目的构想，本项目组还成功构建了miR-455-3p 基因敲除鼠，发现DNMT3A在miR-455-3p基因敲除鼠的血浆中的表达水平明显高于野生型小鼠，研究鉴定DNMT3A的3′-UTR是miR-455-3p直接作用靶点，全基因组DNA甲基化芯片分析提示过表达miR-455-3p可抑制的软骨特异性基因的甲基化水平，GO生信分析发现PI3K-Akt是去甲基化水平最明显的信号通路，从而验证了本项目的另一个构想：miR-455-3p通过PI3K-Akt信号通路靶向DNMT3A 3′-UTR来抑制DNA甲基化，从而促进人骨髓间充质干细胞在分化的早中期向软骨分化。本研究鉴定 了miR-455-3p的靶基因SOX9及RUNX2，从中找出表观遗传学调控相关的下游HDAC2/8及DNMT3A，获得特定miRNA 的靶基因并提出 miRNAs 调控干细胞分化的新模型。