miRNA靶向调控CyclinD1/CDK4在氟致成骨细胞增殖活跃中的作用及机制研究

The endemic fluorosis, which is widely prevalent in Guizhou province, has been consecutively listed in our country’s “Twelfth Five-year Plan” and “Healthy China 2020” Strategic Plan as an important prevented disease. The pathogeny of endemic has been found out, however, the pathogenesis is not clear, which threatens people’s health and hinders the development of economy and society. The endemic fluorosis has become a public concern which needs to be solved promptly. This research is based on the findings in our previous studies that fluoride can upregulate expression of CyclinD1 and CDK4 mRNA and protein. This research makes full use of miRNA microarray technology and bioinformatics analysis to screen and verify miRNA targeting CyclinD1 / CDK4 in population with endemic fluorosis induced by coal-burning. This research also establishs an in vitro model of fluorosis with human primary osteoblasts by overexpression and suppression related miRNA, and conducts research of mechanism of related miRNA regulation by targeting CyclinD1 / CDK4. Its purpose is to reavle the effects of related miRNA in actively proliferating osteoblasts induced by fluoride. This research tries to explore skeletal fluorosis mechanism systematically with miRNA perspective in epigenetics based on DNA methylation and histone acetylation, and then to provide new scientific evidence for the prevention and therapy of fluorosis.

广泛流行在贵州省的地方性氟中毒（简称地氟病）是持续列入我国“十二五”和“健康中国2020行动计划”的重点防治疾病，该病虽然病因清楚，但致病机制未明，严重危害居民健康，影响经济社会发展，是我省亟待解决的重大公共卫生问题。本项目在既往研究发现氟暴露上调CyclinD1和CDK4转录与表达基础上，利用miRNA芯片技术，结合生物信息学分析，在燃煤污染型地方性氟中毒人群中筛选并验证靶向调控CyclinD1/CDK4的miRNA。同时构建人原代成骨细胞氟中毒模型，过表达和抑制相关miRNA，展开相关miRNA靶向调控CyclinD1/CDK4的机制研究，进而揭示其对氟所致成骨细胞增殖活跃的影响。在既往DNA甲基化和组蛋白乙酰化研究基础上，继续从表观遗传学的miRNA调控角度展开系统研究，探索氟骨症发生机制，为地方性氟中毒防治提供新的科学依据。

地方性氟中毒是全球性的公共卫生问题，主要临床表现为氟斑牙和氟骨症，尤其氟骨症是影响患者劳动力和生存质量最突出的问题。氟中毒病因清楚，但致病机制未明。成骨细胞增殖活跃和骨转换加速是氟骨症的特征性病变。本项目在既往研究发现氟暴露上调CyclinD1和CDK4表达基础上，利用miRNA测序技术，结合生物信息学分析，在燃煤污染型氟中毒人群中筛选验证靶向调控CyclinD1/CDK4的miRNA。同时构建人原代成骨细胞氟中毒模型，展开相关miRNA调控CyclinD1/CDK4的机制研究，并明确其在氟所致成骨细胞增殖活化中的作用。研究发现：1)miRNA测序及生物信息学分析筛选出靶向CyclinD1的miR-4755-5p、miR-486-3p、let-7c-5p以及靶向CDK4的miR-122-5p；2)miR-4755-5p直接结合于CyclinD1 3'-UTR调控其蛋白表达；3)miR-486-3p直接靶向CyclinD1在转录后水平调控其表达，并通过TGF-β1/Smad2/3信号通路在转录水平调控CyclinD1表达；4)let-7c-5p直接靶向CyclinD1在转录后水平调控其表达，并通过Wnt9a/β-catenin信号通路在转录水平调控CyclinD1表达；5)miR-122-5p直接结合于CDK4 3'-UTR调控其蛋白表达。并进一步挖掘了miRNA测序获得的数据，增加了与骨损伤相关的其他miRNA的筛选及生物信息学分析，为课题组后续研究提供了方向。本研究从miRNA角度阐释了氟骨症的发生，深化了对氟致病机制的认识。同时基于miRNA的临床应用价值，本研究筛选出来的miRNA有望成为氟中毒早期诊断和治疗的新靶点。