miR-196b对成骨细胞和脂肪细胞分化的调节作用及其机制研究

Several types of microRNAs have recently been defined as important regulators in adipocyte differentiation, the role of other microRNAs in the processes and the mechanisms involved have yet to be explored. Our preliminary data showed that miR-196b was reduced in primary cultured and established murine stromal progenitor cell line after osteogenic treatment but was induced after adipogenic treatment. Supplementing miR-196b activity inhibited murine ST2 stromal cells to differentiate into osteoblasts, evidenced by alleviated alkaline phosphatase staining and reduced expression of osteoblast-specific genes including Runx2, osterix, osteocalcin and bone sialoprotein. Moreover, Supplementing miR-196b activity induced ST2 stromal cells to differentiate into mature adipocytes, along with the induction of adipocyte-specific transcription factors peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein-α (C/EBPα), and the marker gene adipocyte protein 2 (aP2). Conversely, inhibition of the endogenous miR-196b repressed ST2 cells to fully differentiate. Several potential targets of miR-196b were predicted using Targetscan and Pictar web tool. In the present study we will further investigate the regulatory role of miR-196b on osteoblast and adipocyte differentiation using various cell lines and primary cultured stromal cells. Then the dual luciferase assay will be done to identify direct targets of miR-196b. Gain-of-function and loss-of-function studies will be performed to investigate the role of the putative target genes on osteoblast and adipocyte formation. Finally, female mice will be subjected to ovariectomy or sham operation, followed by intravenous injection of either miR-196b antagomir to knockdown the level of miR-196b or its negative control that never targets any miRNA. 6 weeks after the 1st injection, the mice will be sacrificed and we will investigate whether the change of miR-196b level in vivo alters the bone turnover and bone mass of the sham mice and recover the bone loss in the OVX mice. In brief, bone density and bone mass of tibias and femurs will be studied by using micro-CT, X-ray, and histomorphological approaches. The numbers of active osteoblast in the tibia and adipocytes in the marrow as well as the serum levels of osteocalcin and carboxy-terminal collagen crosslinks will be measured. The bone formation rate and mineral apposition rate will be determined as well. Our proposed studies will provide novel insights into the mechanisms of osteoblast and adipocyte development and implicate potential novel approach for bone loss recovery.

前期研究发现骨髓间充质干细胞诱导成骨后miR-196b表达下降，诱导成脂后miR-196b表达上升。在骨髓基质干细胞系ST2中上调miR-196b后向成骨细胞分化减弱，而向脂肪细胞分化增强；相反，在ST2下调miR-196b后向脂肪细胞分化减弱，提示miR-196b对成骨细胞和脂肪细胞分化起调节作用。本研究在此基础上，将通过细胞分子生物学研究进一步确证间充质干细胞向成骨细胞和脂肪细胞分化过程中miR-196b所起的调节作用；采用双荧光素酶报告基因技术筛选鉴定miR-196b的直接靶基因；采用功能获得性研究和功能缺失性研究明确靶基因对成骨细胞和脂肪细胞分化的调控作用及其机制；观察改变小鼠体内miR-196b水平对骨组织成骨细胞和骨髓脂肪细胞数量、整体骨再建和骨量的影响以及对去卵巢小鼠骨质疏松是否具有改善作用等。本研究将为揭示miRNA在骨组织细胞发育和骨再建等过程中的作用及机制提供理论依据。

在前期研究中，我们采用 miRNA 芯片和 RT-qPCR 证明原代培养的骨髓基质细胞和基质细胞系 ST2 诱导成骨后 miR-196b-5p表达下降，诱导成脂后miR-196b-5p 表达上升，提示miR-196b-5p 可能对前体细胞的定向分化起调节作用。本项目首先研究了miR-196b-5p前体细胞定向分化的调控作用。将人工合成的 miR-196b-5p mimics或miR-196b-5p inhibitor或其阴性对照转染前体细胞系，或者构建miR-196b过表达和抑制性慢病毒感染原代BMSCs，随后进行成骨/成脂诱导。结果表明miR-196b-5p 抑制成骨分化而促进成脂分化。在机制研究中，我们根据生物信息学软件预测结果并结合luciferase实验、Western blotting技术筛选鉴定出Tgfbr1、Tsc1和Sema3a可以被miR-196b-5p直接靶向调控。通过对SEMA3A和TSC1的功能研究，我们发现SEMA3A促进前体细胞成骨分化，而TSC1则促使前体细胞成脂分化。miR-196b-5p mimics转染细胞后TSC1蛋白减少，mTOR通路被激活。将Tsc1过表达载体与miR-196b mimics共转染ST2细胞可减弱miR-196b-5p mimics对成脂分化的促进作用；mTOR通路抑制剂Rapamycin也可减弱miR-196b-5p mimics对成脂分化的促进作用。以上结果表明miR-196b-5p通过靶向抑制TSC1翻译而增强mTOR通路活性，从而促进前体细胞成脂分化，而通过靶向抑制SEMA3A翻译抑制前体细胞成骨分化。.在体作用研究方面，我们制备了在成骨细胞特异性表达miR-196b的转基因小鼠（Col1-196b-5p），分析了转基因鼠表型。6月龄转基因小鼠X-ray 显示长骨干骺端近段骨量显著减少，μCT 分析也证实干骺端发生严重骨丢失，小梁骨显著减少，与野生型对照组相比，miR-196b转基因鼠小梁骨容积和骨小梁数目显著 减少，而小梁分离度和结构模型指数显著增加。表明miR-196b-5p在小鼠体内抑制成骨细胞分化，减少骨量。.综合我们的研究结果，miR-196b-5p抑制骨髓基质干细胞成骨分化而促进其成脂分化，其表达紊乱可引起骨稳态异常和骨丢失，miR-196b-5p可能成为骨质疏松等骨代谢疾病防治的新靶点。