氧化应激通过let-7家族导致骨衰老中骨髓间充质干细胞分化缺陷的机制研究

Oxidative stress (OS)-induced differentiation defect of bone marrow mesenchymal stem cells (BMSC) is a key factor of bone aging. Recent studies of our group and others confirmed that a number of microRNAs play important role in BMSC differentiation, while abnormal expression of these microRNAs lead to defect of bone formation during osteoporosis. But whether OS inhibits osteogenic differentiation of BMSCs through miRNA remains unknown. Our previous study showed that several members of let-7 family were significantly downregulated in aged BMSCs. Reactive oxygen species could inhibit the expression of let-7 family. Because let-7 family can regulate osteogenic differentiation of BMSCs and are closely related with multiple age-related degenerative diseases, these results suggest that OS might induce differentiation defect of aged BMSCs through let-7 family. Therefore, take advantage of normal aged mouse and accelerated senescence mouse models and methods of microRNA mimic/inhibitor transfection, bioinformatics analysis, luciferase reporter system, signaling transduction examination, this project is subjected to determine the members of let-7 family leading to osteogenic differentiation defect of aged BMSCs, confirm key target genes of let-7, unclose the function and molecular pathway that OS regulating let-7 family expression. Our study aims to get further understanding of the mechanism that OS inducing bone aging, explore the function of let-7 family in BMSC senescence, and develop novel strategy for age-related osteoporosis treatment.

氧化应激（OS）导致的骨髓间充质干细胞（BMSC）成骨分化缺陷是骨衰老的关键原因。国际及本团队研究证实microRNA在BMSC分化中发挥关键作用，其表达异常与骨质疏松中骨形成下降密切相关，但OS是否通过microRNA抑制BMSC成骨分化尚不清楚。我们前期研究发现多个let-7家族成员在衰老BMSC中显著下降，而氧自由基可抑制let-7表达。因为let-7家族调控BMSC成骨分化，并与多种衰老疾病相关，提示OS可能通过let-7导致衰老BMSC分化缺陷。因此本课题拟基于衰老和早衰小鼠模型，利用miRNA模拟/抑制物转染、生物信息学分析、荧光素酶报告体系、信号通路检测等方法，确定导致衰老BMSC成骨分化异常的let-7家族成员，验证let-7作用的靶基因，阐明OS调控let-7的作用及信号途径，从而深入认识OS导致骨衰老的机制、明确let-7在BMSC衰老中的作用、寻找新的骨衰老治疗靶点

氧化应激（OS）导致的骨髓间充质干细胞（BMSC）成骨分化缺陷是骨衰老的关键原因。国际及本团队研究证实microRNA在BMSC分化中发挥关键作用，其表达异常与骨质疏松中骨形成下降密切相关，但OS是否通过microRNA抑制BMSC成骨分化尚不清楚。我们前期研究发现多个let-7家族成员在衰老BMSC中显著下降，而氧自由基可抑制let-7表达。因为let-7家族调控BMSC成骨分化，并与多种衰老疾病相关，提示OS可能通过let-7导致衰老BMSC分化缺陷。因此本课题基于衰老和早衰小鼠模型，利用miRNA模拟/抑制物转染、生物信息学分析、荧光素酶报告体系、信号通路检测等方法，发现let-7a、let-7c、let-7d等多个成员在骨质疏松来源BMSCs中表达均异常升高，因为let-7a是let-7家族中最为保守的miRNA，并且表达量在其家族中也是最高的，所以接下来重点对let-7a的功能开展研究。通过基因聚类（GO）分析和KEGG信号通路分析，发现let-7a可参与细胞多种功能的调控。利用let-7a特异的模拟物mimic和抑制剂inhibitor，证实了let-7a可以抑制BMSCs的成骨分化和免疫调节能力。利用抑制剂纠正骨质疏松来源BMSCs中let-7a的异常表达，有效恢复了BMSCs的成骨分化能力。并在体外细胞学实验中发现抑制骨质疏松BMSCs中异常升高的let-7a可以有效恢复其对T细胞的迁移和凋亡诱导能力。证明通过抑制let-7a的异常表达可有效恢复衰老BMSCs的功能，并进一步发现let-7a可以靶向结合于Fas和FasL mRNA的3‘UTR区域，从而抑制Fas和FasL的蛋白表达，通过过表达let-7a可以抑制BMSCs中MCP-1的分泌，降低其免疫调控能力，从而证明let-7a是通过Fas/FasL分子途径影响BMSCs的功能。通过上述研究，我们确定导致衰老BMSC成骨分化异常的let-7家族成员，验证let-7作用的靶基因，阐明OS调控let-7的作用及信号途径，从而为进一步深入认识OS导致骨衰老的机制、明确let-7在BMSC衰老中的作用、寻找新的骨衰老治疗靶点提供了重要的实验支持。