近年骨质疏松症（骨密度）的全基因组关联研究鉴定了262个基因（212个SNPs）与骨密度相关联，未来的挑战就是理解这些基因和SNPs如何影响骨质疏松症的易感性。本研究先通过生物信息学方法对这些基因（SNP）的功能进行计算分析，包括序列保守性、转录因子/miRNAs结合、剪接、信号通路与分子网络分析，然后对重要的发现用实验的方法进行验证；通过敲降/过量表达GLI3对成骨细胞分化和骨形成的影响阐明我们发现的GLI3基因及其介导的Hh信号途径调控骨密度的机制；用RNA-seq技术研究成骨分化过程中miRNA表达谱，鉴定不同时期差异表达的miRNA及其在成骨细胞分化和骨形成中的作用。通过把计算与实验、功能与机理研究紧密结合起来，系统阐明全基因组关联研究鉴定的骨质疏松症易感基因（SNP）的功能和调控骨密度的机制与分子网络，这对于揭示骨质疏松症的分子病理学发病机理有重要意义。

Genome-wide association studies of osteoporosis have identified 262 genes and 212 SNPs that are significantly associated with bone mineral density.Future challenge of osteoporosis genetics is to clarify how these genes and SNPs influenc susceptibility of osteoporosis. Aims of this proposal are: 1) first to characterize the function of osteoporosis-associated genes and SNPs by bioinformatics,including sequence conservation, binding of transcription factors and miRNA, splicing, signal pathways, molecular network, and then to volidate the important findings by experimental approaches. 2)to reveal the mechanism of the GLI3 gene identified by us and Hh signals in the regulation of bone mineral density by the effect of the GLI3 gene knock down/overexpression on osteoblast differentiation and bone formation. 3) to identify the differentially expressed miRNA in osteoblast differentiation by RNA-seq technology. Systematic elucidation of the function and mechanism of the genes and SNPs identified by genome-wide association studies in regulating bone mineral density by the combination of computation and experiment, function and mechanism, have significant implication for the understanding of molecular pathophysiology of osteoporosis.

本项目的研究目标是对全基因组关联研究鉴定的骨质疏松症易感基因和SNPs进行计算分析和功能研究，阐明它们调控骨密度的分子机制。系统分析了骨质疏松症关联SNPs的序列保守性、对转录因子/miRNAs结合、蛋白质磷酸化以及剪接的影响，运用RegulomeDB和3DSNP对它们进行了功能注释，对骨质疏松症关联基因进行了信号通路富集和蛋白质分子互作网络分析；通过离体和活体实验在hFOB1.19、U2OS、Saos-2和293T四种细胞中证明rs1026364能影响miR345与USF3的结合、rs75901553能影响miR-98-5p与SOST的结合、rs7220711的A/T等位基因能差异结合转录因子MAFF/MAFK, rs1107748能差异结合转录因子CTCF。USF3是一个功能未知的骨质疏松症易感新基因(转录因子)，采用TALEN技术制备了Usf3基因敲除小鼠，获得的Usf3-/-小鼠骨量明显减少；在U2OS细胞中敲降和过表达USF3，证明USF3基因具有促成骨、抑破骨的功能。进一步的机制研究发现USF3拮抗TWIST1通过GWAS lead SNP rs2908007调控Wnt16 的表达, 拮抗CREB1通过GWAS lead SNP rs4531631调控Rankl 的表达。还发现miR345抑制骨形成，Runx3和Smad1是其靶基因。