PPARγ启动子突变引起股骨头坏死脂肪蓄积的基因组分子签名研究

The increasing ONFH incidence has made that the fat accumulation in damaged bone marrow cavity become the focus of ONFH pathogenesis, especially, as the main regulator of adipogenic differentiation, the new evidences of transcription factor PPARγ intruding in ONFH lipid metabolism disorder have been increasing. However, its molecular mechanism remains unclear. Recently, our group first found that the mutation of PPARγ promoter 2 significantly associated with ONFH risk and its lipid metabolism disorder, which first proposed the evidence that the promoter mutation of PPARγ involved in the development of ONFH. More significantly, the latest reports from《Nature》and《Cell》confirms that the mutation of PPARγ promoter region can change the genomic molecular binding spectrum of PPARγ in directly transcriptional regulation, which determines the individualized risk of obesity and insulin resistance. In order to explore the molecular link between the promoter mutation of PPARγ and the fat accumulation of ONFH, the project will construct the mutant and wild type expression vectors containing PPARγ promoter sequence, respectively, and transfect them into BMSCs to validate the activity of the mutant promoter and to investigate the effects of the mutant promoter on the PPARγgene expressions, the roles of the promoter mutation of PPARγ in the osteogenic and adipogenic transdifferentiation of BMSCs of ONFH. Most important, the project will screen the molecular spectrum of PPARγ transcriptional binding in whole genomic scale with using chromatin immunoprecipitation techniques and deep sequencing techniques. Through the bioinformatics analysis of the transcriptional binding molecular spectrum obtained from the genomic molecular signature technique, our results will first propose the genomic mechanism of fat accumulation of ONFH caused by the promoter mutation of PPARγ. Furthermore, on the basic of the bioinformatics analysis on the molecular spectrum, the project will select key signaling pathways and gene clusters to validate their roles of in the osteogenic and adipogenic differentiation during the occurrence and development of ONFH. The project will innovate the genomic molecular mechanisms of ONFH and suggest the potential molecular targets for the prevention and treatment of ONFH.

ONFH发病率持续攀升使病变骨髓腔脂肪蓄积成为ONFH机理的关注焦点，尤其作为成脂分化主控制器的PPARγ介入其脂肪蓄积的新证据日益凸显，但分子机制不清。项目团队新近发现PPARγP2启动子突变显著相关ONFH风险及其脂代谢紊乱，率先提出了PPARγ启动子突变介入ONFH发病的证据。更惹人注目的是《Nature》、《Cell》最新报告证实PPARγ启动子区突变可在基因组规模改变PPARγ直接转录调控的分子谱，从而决定个体肥胖、胰岛素抵抗的发病风险。为了阐述我们发现的PPARγ启动子突变与ONFH脂肪蓄积的分子联系，本项目拟分别构建突变型及野生型PPARγ启动子表达载体，观察启动子突变对其基因表达及ONFH成骨、成脂转分化的影响，应用CHIP-seg技术筛查PPARγ启动子突变后基因组规模转录调控结合分子谱，通过基因组分子签名首次解析PPARγ启动子突变引起ONFH脂肪蓄积的基因组机制。

脂代谢紊乱早已被公认为ONFH核心发病环节,但一直不清楚其脂肪蓄积的分子机理。近年对BMSCs分化微环境的研究,发现了关键转录因子 PPARγ在调节成脂、成骨转分化的主调控器作用。项目组前期发现PPARγ启动子rs2920502突变（CC型）显著增加ONFH风险，但PPARγ启动子突变是否以及通过何种分子机制引起ONFH的 BMSCs脂紊乱尚不清楚。本项目通过构建突变型PPARγ启动子表达载体，观察启动子突变对PPARγ基因表达及ONFH BMSCs成骨、成脂转分化的影响，通过基因组分子签名首次解析PPARγ启动子突变引起ONFH脂肪蓄积的基因组机制。.研究主要取得如下结果：.关于PPARγ 基因启动子突变对启动子活性的影响，应用PCR技术分别克隆了基因组rs2920502GG型与CC型的PPARγ基因启动子序列并插入荧光素酶报告基因慢病毒表达载体，发现启动子突变（CC型）显著增加启动子活性。.关于PPARγ 基因启动子突变对 ONFH 患者 BMSCs PPARγ基因表达及成骨、成脂分化影响的研究结果，首先完成了入组ONFH 病例BMSCsrs2920502（G/C）的基因分型，对不同基因分型BMSCs成脂、成骨诱导培养及PPARγ基因表达研究结果发现，ONFH组CC型BMSCs成脂分化能力亢进伴有PPARγ基因等成脂Markers过表达，成骨分化能力明显减低伴有成骨转录因子RUNX2等成骨Markers表达显著下调，率先阐述了PPARγ启动子突变促进成脂转录因子PPARγ基因表达，抑制ONFH BMSCs成骨分化、促进成脂分化的科学证据。.关于PPARγ 基因启动子突变引起转录调控的基因组分子签名结果，发现ONFH组 PPARγ 基因启动子突变结合的DNA序列数显著高于对照组，其转录组mRNA分子谱亦明显增加或减少，率先阐述了PPARγ 基因启动子突变引起ONFH的成脂成骨转分化障碍的基因组分子签名特点。 .关于PPARγ 基因启动子突变引起主要信号通路及其分子靶点的生信分析与验证的结果，发现主要涉及integnin、Wnt信号信号通路等，主要分子靶点为成骨分化标志物表达显著下调，成脂分化主控制器PPARγ及其转录共因子C/EBPα及 Adipsin表达显著上调，提出了PPARγ启动子突变引起ONFH基因组分子签名异常的关键信号通路与分子靶标。