成骨分化负调控分子RNF185在骨形成中的功能及作用机制研究

项目名称： 成骨分化负调控分子RNF185在骨形成中的功能及作用机制研究

项目编号： No.81472095

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 周颖

作者单位： 中国人民解放军总医院

项目金额： 72万元

中文摘要： 泛素连接酶RNF185是采用RNAi技术进行高通量筛选获得的一个潜在的成骨特异性的抑制因子。我们的前期研究显示，RNF185过表达可以下调成骨标志物ALP、Osx和Runx2的表达，抑制碱性磷酸酶活性，从而抑制小鼠颅骨来源的MC3T3-E1细胞的成骨分化。荧光素酶报告基因分析显示野生型RNF185下调β-catenin的转录活性，而酶活突变体S39/S42则表现为显性负效应，推测RNF185依赖泛素连接酶活性，降解Wnt/β-catenin信号通路的关键分子，实现抑制成骨分化的功能，拟采用报告基因实验、免疫共沉淀、GST沉降、泛素化等体内外实验加以验证。并在此基础上，采用RNA干扰技术评估RN185的siRNA的体内成骨能力。该项研究将有助于阐述调控成骨分化和骨形成的分子机制，有可能为进一步开发合成代谢疗法治疗骨质疏松症提供理论基础。

中文关键词： 骨形成；成骨分化；泛素化；信号转导

英文摘要： biquitin ligase RNF185 is identified during a high throughout siRNA library (targeting 5,000 human genes) screen to identify the endogenous repressors of osteogenic specification,which when silenced could initiate differentiation of hMSCs into osteoblasts.Here we show that RNF 185 modulates osteogenic differentiation of mouse calvaria-derived MC3T3-E1 cells. Overexpression of RNF185 inhibited expression of osteoblast-specific genes,alkaline phosphotase (ALP) activity and matrix mineralization. Furthermore, dual-luciferase reporter assay confirmed that β-catenin as bona fide target of wild-type RNF185, while mutant S39/S42 revealed the dominant-negative effects. Therefore, We postulated that RNF185 inhibits bone formation by inhibiting the Wnt/β-catenin signaling pathway dependent of the ubiquitin ligase activity. In the following study, we design in vitro and in vivo experiments including co-immunoprecipatation、GST pull-down、ubiquitanation assay 、using a delivery system specificlly targeting bone formation surfaces and so on to verify our hypothesis. Besides clarifying the molecular mechanisms of RNF185 regulating bone formation,our findings might suggest that pharmacological inhibition of RNF185 could represent a therapeutic strategy for enhancing bone formation in vivo, which would provide for the development of anabolic therapies for treatmen of osteoporosis.

英文关键词： bone formation;osteogenic differentiation;ubiquitination;signalling