环状RNA FGFR1/miR-6756-5p/FASN轴向调控脂肪细胞早期分化过程机制研究

From the perspective of circRNA, this project aimed to systematically elaborate the mechanism of axial action regulating early-stage adipocyte differentiation based on the idea of circFGFR1 mediating FASN gene through targeting miR-6756-5p (axial action). Firstly, with bone marrow mesenchymal stem cells of healthy adults as subjects, mutual targeting in induced adipocyte differentiation was determined by luciferase, double FISH, real-time quantitative PCR and immunoblot assay; adipogenic ability was evaluated though analyzing the phenotypic characteristics of adipogenic differentiation using gene overexpression (interference); the pathway of axial action regulating adipogenic differentiation and the mechanism of circFGFR1/miR-6756-5p/FASN axial action regulating adipogenic differentiation were elaborated from the functional level. Then, with bone marrow mesenchymal stem cells of patients with osteoporosis as subjects, circFGFR1/miR-6756-5p expression was interfered or overexpressed in induced adipocyte and osteoblast differentiation to verify the correlation of axial regulating mechanism with osteoporosis through evaluating adipogenic and osteogenic ability by analyzing the phenotypic characteristics of adipocyte and osteoblast differentiation, providing novel ideas for the diagnosis and treatment of osteoporosis.

本项目以circRNA为视角，沿着ceRNA调控模式的思路，朝着系统阐述circFGFR1/miR-6756-5p/FASN轴向调控脂肪细胞早期分化过程的目标开展工作。研究从健康成人和骨质疏松症患者hMSCs两个层次展开，首先以健康成人hMSCs为对象，在成脂分化过程中，运用双荧光素酶报告基因和双FISH技术等鉴定相互间的靶向关系；采取目标基因上下调及回补实验，通过分析成脂分化能力，从功能上阐明轴向作用调控脂肪细胞早期分化的途径，阐述circFGFR1通过miR-6756-5p靶向作用FASN基因调控脂肪细胞分化的机制。再以骨质疏松症患者hMSCs为研究对象，在成脂/成骨诱导分化过程中干扰circFGFR1或上调miR-6756-5p表达，验证轴向调控机制与骨质疏松存在关联，通过对成脂/成骨分化能力的分析，确定其抑制骨质疏松症患者成脂分化、改善成骨能力，为骨质疏松症诊断和治疗提供新的思路。

本项目以circRNA为视角，系统阐述circRNA FGFR1（circFGFR1）调控成脂分化过程。研究从健康成人和骨质疏松症患者hMSCs两个层次展开，首先以健康成人hMSCs为实验对象，在成脂诱导分化过程中，运用RNA pull down、LC-MS、RIP和目标基因干扰等方法，通过分析成脂分化表型特征评价成脂分化能力，从功能上证实了circFGFR1通过结合IGF2BP2蛋白增强FASN mRNA稳定性来调控成脂分化过程。再以骨质疏松症患者hMSCs为研究对象，在成脂诱导分化过程中干扰表达circFGFR1，确定其抑制骨质疏松症患者hMSCs成脂分化能力。本项目同时对前期hMSCs成脂分化过程中转录组数据进一步分析验证，①筛选获得20个与成脂分化相关的潜在关键基因，其中有4个基因下调。并证实凝血因子II凝血酶受体(F2R)能促进成脂分化。②在成脂分化过程中获得19个与脂滴形成相关的差异miRNAs及其靶向的35个mRNA。其中5个miRNA，包括hsa-miR-146a-3p、hsa-miR-4495、hsa-miR- 4663、hsa-miR-6069和hsa-miR-675-3p是最新的脂滴形成潜在生物标志物，分别靶向ACSL1、APOB、METTL7A、PLIN1和PLIN4。③首次分析了hMSCs成脂分化过程中细胞代谢的转录谱。最富集的PI3K-Akt信号通路在成脂分化过程中通过靶向潜在的关键基因，如FABP4、PKC1、SCD和SLC2A1和启动葡萄糖有氧糖酵解、精氨酸和脯氨酸代谢、谷胱甘肽代谢和花生四烯酸代谢，刺激并直接调控细胞代谢。同时证实了PCK1是参与成脂分化过程中细胞代谢的关键基因。以上结果丰富了成脂分化的分子机制研究，为肥胖、2型糖尿病和骨质疏松等代谢性疾病的发病机制研究和筛选诊断和治疗新靶点提供理论基础。