miR-20a-5p在成骨细胞和脂肪细胞分化中的调控功能及机制研究

Although specific mcroiRNAs have been shown to be involved in osteoblast and adipocyte differentiation regulation, the potential role of other microRNAs in the processes and the involved mechanisms have yet to be explored. Our previous data showed that miR-20a-5p was reduced in primary cultured medullary mesenchymal stem cells after osteogenic treatment but was induced after adipogenic treatment. In vitro, overexpression of miR-20a-5p promoted mesenchymal cell line C3H10T1/2, preadipocyte 3T3-L1 and stromal ST2 cells to differentiate into adipocytes, however, inhibition of miR-20a-5p suppressed the above cells differentiated into mature adipocytes. We also found miR-20a-5p had stimulatory effects on osteogenic differentiation, depending on what treatment is done with the cells. Moreover, we identified that Kdm6b and Tgfbr2 were direct targets of miR-20a-5p. Kdm6b and Tgfbr2 were all negative regulators of adipogenesis, thus, inhibition of Kdm6b and Tgfbr2 expression might be one of the mechanisms through which miR-20a controls adipogenesis. Because miR-20a-5p had the same effects on osteogenic and adipogenic differentiation, its regulatory mechanisms and new targets should be further explored. In the present study, first, we will confirm the functions of miR-20a-5p in osteoblast and adipocyte differentiation by primary cultured medullary mesenchymal stem cells. Then, several new potential targets of miR-20a-5p have been predicted using TargetScanMouse and Pictar web tool, we will perform the dual luciferase assay to identify if these new putative targets are real. Gain-of function and loss-of-function studies will be performed to investigate the role of new predicted target genes on osteoblast and adipocyte formation , rescue study will be performed to confirm if miR-20a-5p played its roles through the new potential targets. To clarify the mechanisms that might regulate miR-20a-5p expression, the cis-regulatory elements in miR-20a-5p promoter will be analyzed; in addition, we will conclude the regulation and function of new predicted target genes, the roles and expression regulation of miR-20a-5p to get a complete regulatory feedback loop. Finally, in vivo, we will inject the primary cultured medullary mesenchymal stem cells in which miR-20a-5p was overexpressed or downregulated to murine tibial cavity. One or three months after the first injection, the mice will be sacrificed and we will investigate whether the change of miR-20a-5p level in vivo alters the bone turnover, bone mass, the numbers of osteoblast in tibia and adipocytes in the marrow. Our proposed studies will provide novel insights into the mechanisms of osteoblast and adipocyte differentiation and implicate potential novel approach for bone loss recovery.

miRNAs对成骨和脂肪细胞的分化调控已成为研究热点。本项目组前期研究发现骨髓间充质干细胞诱导成骨后miR-20a-5p表达下降，诱导成脂后表达升高。体外实验证实miR-20a-5p可能通过靶向调控Kdm6b以及Tgfbr2而促进前脂肪细胞系分化。本项目在此基础上，拟采用原代骨髓间充质干细胞完善miR-20a-5p影响成骨/成脂分化的研究；在其发挥调控功能的下游，筛选鉴定其新的靶基因，通过功能获得性和功能缺失性研究确证靶基因的功能，并通过拯救实验确定miR-20a-5p是否通过新的靶基因发挥调控作用；分析miR-20a-5p启动子区的顺式作用元件，揭示其在分化过程中表达变化的上游调控机制，深入阐明上游影响其发挥调控功能的分子机制；进一步探讨miR-20a-5p对小鼠活体骨再建、骨量及成骨/成脂分化的影响。本研究有助于进一步揭示miRNAs在骨组织细胞发育和骨再建等过程中的调控作用。

本项目组前期研究发现骨髓间充质干细胞诱导成骨后miR-20a-5p表达下降，诱导成脂后表达升高。体外实验证实miR-20a-5p可能通过靶向调控Kdm6b以及Tgfbr2而促进前脂肪细胞系分化。本项目成功分离培养小鼠原代骨髓间充质干细胞（BMSCs），构建了miR-20a-5p的慢病毒过表达重组质粒及敲减（sponge）重组质粒并包装相应慢病毒用于原代细胞的感染。我们发现miR-20a-5p在成脂分化前3天表达量逐渐升高，随后2天仍保持较高的表达水平。miR-20a-5p过表达慢病毒及mimics均可以提高小鼠BMSCs中miR-20a-5p的水平，促进BMSCs的成脂分化，且上调成脂标志性基因PPARγ、C/EBPα和a P2蛋白水平。相反，miR-20a-5p sponge慢病毒及inhibitor均可以降低小鼠BMSCs中内源mi R-20a-5p的水平，抑制BMSCs的成脂分化，同时下调成脂分化过程中关键基因的蛋白水平。根据靶基因预测软件Target Scan预测结果结合双荧光素酶和GFP报告基因分析实验证实Klf3是miR-20a-5p的直接靶基因。Klf3的沉默表达可以促进小鼠BMSCs向脂肪细胞分化，与miR-20a-5p过表达的作用相同；拯救实验结果提示miR-20a-5p可通过抑制Klf3发挥促进成脂分化的作用。我们还发现与单独的 Klf3 相比，Klf3、Kdm6 和 Tgfbr2 三者能更有效的减弱miR-20a-5p的促成脂作用。. 此外，miR-20a-5p可以促进BMSCs的成骨分化，表现为碱性磷酸酶染色增强。我们还鉴定Nfic基因为miR-20a-5p的直接靶基因。我们发现Nfic基因在小鼠的骨组织中特异性高表达，ALP染色和RT-qPCR检测可见，在BMSC中过表达Nfic可以显著促进细胞成骨分化，而抑制Nfic的表达则显著抑制骨髓基质细胞系ST2成骨分化；Nfic可能通过LRP5影响Wnt信号通路进而调控了成骨细胞的分化。通过生物信息学预测及双荧光素酶报告系统检测和染色体免疫共沉淀检测实验，我们证明Nfic可以与miR-20b-5p promotor结合并抑制其表达。. 本研究进一步揭示了miR-20b-5p在成骨/成脂细胞分化进程中的分子机制，有助于阐释miRNAs在骨组织细胞发育和骨再建等过程中的调控作用。