miR-140-5p靶向作用LIFR调控成脂分化机制的研究

The research aims to demonstrate that the mechanisms of miR-140-5p targeting on LIFR in adipogenic differentitation. This study is divided into two parts (in vitro and vivo). It will be as an object of study that human bone marrow mesenchymal stem cells differentiate into adipoctyes in vitro. We will confirm the pre-experiments results that miR-140-5p target on the LIFR by real time quantitative PCR,lentiviral-mediated RNAi and western-blotting. Then we identify miR-140-5p target gene is LIFR by up\down regulation of miR-140-5p expression and further confirm the target sites by dual luciferase system.Subsequently,we analysis the JAK2-STAT3 signal pathway, the adipogenic phenotype and the gene expression of adipogenic characteristics by up\down regulation of miR-140-5p and LIFR expression. In vivo, miR-140-5p and LIFR of the different gene expression level are implanted in the nude mice subcutaneous where adipogenic differentitation will happen. After the newborn subcutaneous adipose tissue will be removed. We also make a analysis using the same assessment methods adopted in vitro. In this way, we can further clarify the regulatory mechanisms of adipogenic differentiation which miR-140-5p target on LIFR through JAK2-STAT2 signaling pathway in vitro and vivo experiments. It will provide a new theoretical basis for profound understanding and effective treatment of osteoporosis.

本项目以miRNA为视角，沿着miR-140-5p靶向作用LIFR的思路，朝着阐述miR-140-5p调控成脂分化机制的目标开展工作。研究分体内和体外二部分，体外试验以人类骨髓间充质干细胞成脂分化过程为对象，应用定量PCR、慢病毒介导的RNAi和免疫印迹等技术证实miR-140-5p靶向作用于LIFR这一预实验结果，采用上\下调miR-140-5p表达的方法验证LIFR是miR-140-5p的靶基因，并用荧光素酶报告系统鉴定靶位点；通过改变miR-140-5p、LIFR表达，检测JAK2-STAT3信号通路、成脂表型及成脂特征基因表达。体内试验将miR-140-5p和LIFR表达水平不同的hMSCs植入裸鼠皮下，获取新生组织采用与体外相同的评价指标，从体内和体外二方面阐述miR-140-5p靶向作用LIFR经JAK2-STAT3通路调控成脂分化的机制，为认识和治疗骨质疏松提供新的理论依据。

探索人骨髓间充质干细胞(hMSCs)成骨/成脂分化的分子机制对于防治骨质疏松症具有至关重要的意义。本项目以miRNA为视角，沿着miR-140-5p靶向作用LIFR的思路，朝着阐述miR-140-5p调控成脂分化机制的目标开展了工作。主要研究成果如下：1）完成了 miR-140-5p 及其预测的靶基因白血病抑制因子受体（LIFR） 在成脂分化过程中的变化趋势；运用慢病毒介导的过表达和siRNA干扰研究了 miR-140-5p 对成脂分化的影响。 2）发现了LIFR 上游 miRNA-377-3p 对hMSCs成脂分化具有重要的调控作用；并运用双荧光素酶报告系统、实时荧光定量 PCR 和免疫印迹等技术鉴定靶基因 LIFR 3′UTR 含有miRNA-377-3p 的靶位点。3）证实了LIFR 在 hMSCs 成脂分化中所扮演的重要角色；阐述了LIFR 也是链接细胞因子和 JAK-STATS、MAPK 信号通路之间关键核心点。4）在完成项目计划的基础上，我们探索了miRNA上游调控因子lncRNA可能在hMSCs成骨和成脂分化平衡调控中起到了重要作用，并通过对高通量数据的深入挖掘发现lncRNA TSC22D1-AS1可能以ceRNA模式抑制miR-765靶向作用BMP6调控成骨分化。该项目的研究结果系统阐明了miRNA-140-5p和miRNA-377-3p及其靶基因LIFR调控人骨髓间充质干细胞成脂分化的分子机制，为探索骨质疏松的发病机制提供了有价值的线索。