microRNA对NFATc1/RANKL骨免疫信号通路的调控机制研究

项目名称： microRNA对NFATc1/RANKL骨免疫信号通路的调控机制研究

项目编号： No.81200793

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 医学二处

项目作者： 孟姝

作者单位： 四川大学

项目金额： 23万元

中文摘要： 活化T淋巴细胞分泌RANKL促进破骨细胞分化；miRNA参与调控破骨细胞分化。本课题旨在研究以RANKL和NFATc1为靶向的miRNA，以及活化T淋巴细胞中NFATc1对RANKL表达的调控作用。首先通过生物信息学分析预测可能与NFATc1/RANKL结合的miRNA，选取与miRNA微阵列分析结果一致的作为候选miRNA进行研究；采用萤光素酶报告基因技术，构建含有Luciferase报告基因和 NFATc1或RANKL 3'UTR序列的载体，验证候选miRNA是否与目的基因有效结合；构建pri-miRNA慢病毒载体转染细胞，检测miRNA的调控作用及下游效应。采用染色质免疫共沉淀技术和基因功能学实验，分析活化T淋巴细胞中NFATc1对RANKL表达的调控作用。本研究将揭示miRNA对NFATc1/RANKL信号通路的调控作用，阐明炎症性骨吸收的分子机制，为牙周炎的靶向治疗提供新的思路。

中文关键词： 微小RNA；破骨细胞；活化T细胞核因子-1；RANKL；

英文摘要： Activated T lymphocytes can secrete RANKL, which stimulates osteoclastogenesis. MicroRNAs regulate several cell process, including osteoclastogenesis. The present study is aimed to investigate microRNAs involved in regulation of RANKL and NFATc1 expression, and the role of NFATc1 in regulation of RANKL expression in activate T cells. First, bioinformatics analysis is applied to predict possible microRNAs which may bind to NFATc1 and RANKL transcripts. Then microRNA microarray assay can produce microRNA profiling of activated T cells. Both results are comparatively analyzed and coincident microRNAs are generated as candidate microRNAs for further investigation. The binding efficiency of candidate microRNAs with target gene transcripts, such as NFATc1 and RANKL, is tested by luciferase reporter gene assay.Pri-miRNA lentivirus constructs of candidate microRNA is built and used to transfect target cells, in order to determine the effects of microRNA. Chromatin immunoprecipitation, together with gain and loss of gene function analysis, is used to study the regulatory role of NFATc1 on RANKL expression.The research can reveal the microRNAs involved in regulation of NFATc1 expression in activated T cells, as well as RANKL in osteoclasts. The results can be applied to clarify the molecular mechanisms of inflammatory bon

英文关键词： microRNA；osteoclast；NFATc1；RANKL；