MYSM1调节miR-129的表达在间充质干细胞免疫调控功能中的作用及机制研究

Mesenchymal stem cells (MSCs) have been shown to be kind of important stem cells and immunoregulatory cells. In spite of their broad application, their unstable immunoregulatory function and unclear molecular mechanism limit their utilization in tissue engineering and regenative medicine. Our published data (Immunity 2011, Sci Rep 2016, et al.) have showed that histone H2A deubiquitinase MYSM1 controlled the self-renewal of stem cells and the generation and function of immune cells. So far, the influence of MYSM1 on the immunoregulatory function of MSCs remains unknown. Our preliminary data indicated that MSCs showed decreased immunoinhibitory function and enhanced pro-inflammatory cytokines secretion after knockdown of MYSM1 in MSCs.And miR-129 maybe one of the targets of MYSM1. Hence, this proposed study will mechanistically investigate the immunoregulatory function of MSCs through using conditional knockout mice, silencing or over expression MYSM1 with CRISPR/Cas9 system and studying the related animal model. ChIP-seq and RNA-seq techniques will be used to elucidate the target gene of MYSM1.This study will help to determine the biological function of MYSM1, thus contribute to a fundamental understanding of the biological function for the entire group of newly identified histone deubiquitinases. This study will also shed light on the fundamental understanding of the immunomodulatory capacity of MSCs at the epigenetic and chromatin level. Finally, this study will provide new theoretical guidance for the application of MSCs in tissue engineering and regenative medicine.

间充质干细胞（MSCs）具有强大的免疫调节功能，但其免疫调节作用不稳定、调控分子机制不清楚、大大限制了其在医学领域中的应用。我们已发表的工作（Immunity 2011,Sci Rep 2016等）表明MYSM1表观调控干细胞的自我更新及免疫细胞的发育和功能。MYSM1 对MSCs免疫调节功能的影响未见报道。我们的初步研究结果提示干扰MYSM1表达后，MSCs的免疫抑制作用减弱；miR-129可能是MYSM1在MSCs中的调控靶基因。为此本课题拟借助条件敲除鼠、CRISPR/Cas9系统干扰或过表达MYSM1，研究MYSM1对MSCs免疫调控功能的影响，通过ChIP实验和RNA-seq等深入探讨MYSM1调控miR-129的调节机制，最终明确MYSM1对MSCs免疫调节功能的作用规律。本课题有助于从表观遗传和染色质水平理解MSCs的免疫调控功能；为MSCs更有效应用于临床提供新的理论指导。

通过本项目研究，发现在TNFα和IFNγ的联合刺激下，脂肪来源间充质干细胞（ASCs）中MYSM1的mRNA和蛋白随着刺激表达升高。干扰MYSM1后，ASCs抑制T细胞增殖能力减弱，体内抑制炎症性肠炎能力降低，抑制肿瘤生长能力升高。进一步的研究表明MYSM1能结合到miR150前体的启动子区调控miR150的转录进而调控miR150在间充质干细胞中的表达水平。此外MYSM1还能调控巨噬细胞的极化，MYSM1敲除后巨噬细胞的增殖、凋亡均增加，而且巨噬细胞容易向M1型极化，以上结果说明，MYSM1在间充质干细胞和巨噬细胞中均发挥重要的免疫调控作用。在TNFα和IFNγ的联合刺激下，miR129-5p的表达显著升高，干扰miR-129-5p后，ASCs表达炎性因子的水平显著下降，而iNOS的表达及NO分泌显著升高，体外抑制T细胞增殖能力显著增强，体内显著缓解炎症性肠炎，但移植后能促进肿瘤生长，进一步研究发现，miR-129-5p通过调控Stat1的磷酸化调控炎性因子表达。miR-129-5p不仅调控ASCs的免疫调控功能，还通过抑制DKK3的表达促进MSC的成骨分化。MYSM1和miR-129-5p分别调控ASCs的免疫条件功能，有意思的是干扰MYSM1表达后，miR-129-5p的表达升高；过表达miR-129-5p后，MYSM1的表达显著下降，荧光素酶实验结果表明，MYSM1是miR-129-5p的直接调控靶基因，以上结果提示MYSM1和miR-129-5p相互抑制，后续将进一步研究二者的相互拮抗作用在ASCs中的详细机制。通过本项目研究，课题组共发表SCI论文8篇，影响因子大于8的1篇，大于5的1篇，共计影响因子约40，核心期刊论文3篇，综述2篇，授权专利4项，实审专利4项。