miR-23a在氟诱导成骨活性增强中的作用及调控机制研究

Skeletal fluorosis is the main lesion of endemic fluorosis, and can damage the life quality and working capacity of the residents living in the endemic areas. Many studies have proved that enhancement of bone formation is one of the important pathological damage and pathogenesis of it. In this project, we hypothesize that miR-23a may play a important role in fluoride-induced osteogenic activity enhancement based on the following experimental supports: (1) Fluoride could increase ALP and BGP level in UMR-106 cells; (2) Results of miRNAs array showed that miR-23a increased in NaF treated cells; (3) MiR-23a being closely associated with bone metabolism also was found in literatures. Hence, the further research plan was designed to clarify the hypothesis. Firstly, cells and SD rats will be treated with different dose of fluoride for different period, miR-23a in cells and bone of rats will be measured with qRT-PCR method. Relationship between miR-23a and fluoride dose / exposure period will be analyzed. Secondly, miR-23a mimic and inhibitor will be transfected into UMR-106 cells to increase / decrease miR-23a; also, miR-23a agomir and antagomir will be injected into SD rats to meet the same aim. By analysis the change of biomarker representing osteogenic activity, the role of miRNA-23a in fluoride-induced osteogenic activity will be clarified. Thirdly, 1-2 target gene of miR-23a will be selected to study deeply with bioinformatics and proteomics analysis; further, dual-Luciferase reporter gene system, small interference RNA, over expression will be used to confirm the regulation of miR-23a to its target gene, and how their interaction regulates the osteogenic activity enhancement induced by fluoride. This project will contribute practical value and theoretical significance to the process in exploring the molecular mechanism of skeletal fluorosis, especially in the aspects of finding molecular therapeutic target.

氟骨症是地方性氟中毒的主要特征性病变，多数研究证实骨形成活跃是其主要病理损害之一 。本项目在首先证实染氟细胞ALP和BGP升高、miRNAs芯片发现miR-23a表达上调的基础上，拟进一步明确 miR-23a在氟诱导成骨活性增强中的作用及调控机制。内容包括：（1）采用qRT-PCR技术在细胞和动物水平验证染氟条件下，miR-23a的表达及其与染氟的剂量效应和时间效应关系；（2）采用miRNA模拟物与抑制物实现细胞和动物miR-23a上调/下调，检测成骨活性标志物变化，明确miR-23a在氟诱导成骨活性增强中的作用；（3）生物信息学分析和差异蛋白质组学结合，确定1-2个miR-23a靶基因，采用荧光素酶报告基因法、RNA干扰、过表达等技术，明确miR-23a对靶基因的调控及其相互作用对氟诱导成骨活性增强的调控作用。对进一步揭示氟骨症发生的分子机制，探索分子治疗靶点具有重要意义。

氟骨症是地方性氟中毒的主要特征性病变，多数研究证实骨形成活跃是其主要病理损害之一。本项目在首先证实染氟细胞ALP和BGP升高、miRNAs芯片发现miR-23a表达上调的基础上，拟进一步明确miR-23a在氟诱导成骨活性增强中的作用及调控机制。内容包括：（1）采用qRT-PCR技术在细胞和动物水平验证染氟条件下，miR-23a的表达及其与染氟的剂量效应和时间效应关系；（2）采用miRNA模拟物与抑制物实现细胞和动物miR-23a上调/下调，检测成骨活性标志物变化，明确miR-23a在氟诱导成骨活性增强中的作用；（3）生物信息学分析和差异蛋白质组学结合，确定1-2个miR-23a靶基因，采用荧光素酶报告基因法、RNA干扰、过表达等技术，明确miR-23a对靶基因的调控及其相互作用对氟诱导成骨活性增强的调控作用。对进一步揭示氟骨症发生的分子机制，探索分子治疗靶点具有重要意义。研究结果显示：（1）NaF作用下，UMR-106细胞存在差异表达的miRNA，其中miR-23a在 NaF作用下的UMR-106细胞中表达升高，靶基因生物信息学结果显示miR-23a参与成骨分化、骨代谢调控，说明miR-23a可能参与NaF作用下的成骨细胞调控。（2）氟化钠染毒大鼠模型建立成功，且miR-23a表达变化趋势与染氟剂量和大鼠骨相改变有一定联系。（3）miR-23a能促进染氟UMR-106细胞中成骨活性基因的表达，且DUSP5是miR-23a的靶基因。（4）miR-23a可能通过DUSP5介导的ERKs通路调节染氟UMR-106细胞的成骨分化。本项目在验证miR-23a的基因芯片结果的基础上，通过构建rno-miR-23a功能获得性及功能缺失性的UMR-106细胞染氟细胞模型及氟化钠染毒大鼠模型，进一步明确了 miR-23a 表达变化与氟诱导成骨活性之间的关系。