RUNX2靶向作用IPO8调控成骨细胞分化机制研究

This research aims to demonstrate that the mechanisms of RUNX2 targeting on IPO8 through miRNA-guided gene-silencing pathways regulate osteoblast differentiation in vitro induced from human mesenchymal stem cells (hMSCs). We will doubly confirm our preliminary finding that the IPO8 is one of target genes of RUNX2, contributing the differentiation of osteoblast from hMSC. Subsequently, we will set up three differentiation test groups: normal induction group, the group with RUNX2 interference, and the group with IPO8 interference. With each group, 4 time-points (d7, d14. d21 and d28) plus a common hMSC control will be collected, their phenotypes will be analyzed, mRNA_seq and miRNA profiling will be performed, and biologically duplicate tests will be performed. We will explore their paired and dynamic expression relationship. Combing with miRNA target genes forecast and network analysis, we will obtain the candidate miRNAs and their candidate target genes involved in the regulation of RUNX2 and IPO8, of which the candidate miRNAs overlapped with the RNA-seq analysis output will be the most-possible target genes. These candidate miRNAs and the most-possible target genes will be confirmed by RT-PCR, Western blot (for encoding mRNA), and the SuperLight Luciferase Reporter Gene assay. This research will demonstrate the molecular mechanisms of RUNX2 regulating the osteoblast differentiation by targeting on IPO8 via miRNA-guided gene-silencing pathways, including the profile of the target genes. It will provide a theoretical basis for profound understanding and effective treatment of degenerative bone diseases such as osteoporosis.

本项目以miRNAs为视角，沿着RUNX2靶向作用IPO8借助miRNAs调控成骨细胞分化的思路，朝着系统阐述RUNX2调控成骨分化机制的预期目标开展工作。以人类骨髓间充质干细胞成骨定向诱导分化过程为研究对象，首先证实RUNX2靶向作用IPO8（预实验结果）及其调控成骨分化的作用；随后运用miRNA和RNA测序技术动态检测正常对照、IPO8和RUNX2干扰三组细胞的分化过程，同时分析细胞表型特征的变化，对检测数据进行配对和动态关系研究，再结合生物信息预测法进行分析处理，获得参与RUNX2靶向作用IPO8调控成骨分化的候选miRNAs及其靶基因，用实时定量PCR和荧光素酶报告基因等方法鉴定候选者，确认miRNAs及其对应靶基因，从而在miRNAs水平上阐述RUNX2靶向作用IPO8调控成骨分化的分子机制。此机制具有重要的理论意义及临床应用价值，为深刻认识和有效治疗骨质疏松奠定理论基础。

人类骨髓间充质干细胞（human Mesenchymal Stem Cells, hMSCs）具有定向分化为人类成骨细胞和脂肪细胞的能力，如果成人骨骼中的这两种细胞分化比例失衡（成骨细胞减少、脂肪细胞增多），将导致骨量下降，是骨质疏松发生发展的关键因素。RUNX2是调控人类成骨细胞分化的关键转录因子，明确RUNX2靶向作用IPO8参与调控人类成骨细胞分化的作用机制有重要意义，并为更深层次的研究人类成骨与成脂细胞分化的平衡调控奠定基础，进而为骨质疏松的诊断防治提供新的思路及方法。.通过IPO8基因启动子的克隆及转录活性分析、RUNX2对IPO8 基因启动子的调节作用、IPO8 基因启动子区 rs35100176 位点变异对基因表达的影响和在Saos-2中RUNX2调控IPO8转录的研究，获得了以下研究结果：. 1.成功确定IPO8基因转录起始位点，酶切结果证实IPO8基因5'端非编码区6条续减片段正确插入到荧光素酶报告基因载体pGL3-Basic，成功构建具有转录活性的IPO8启动子片段，为进一步研究IPO8表达调控机制奠定了基础。. 2. 成功突变和剔除IPO8 启动子上RUNX2结合位点，破坏RUNX2结合位点导致IPO8 启动子活性在Saos-2细胞中显著下降（ P<0.05）。转录因子RUNX2在Saos-2细胞与IPO8 启动子有效结合，下调RUNX2的表达导致IPO8 转录水平下降（P<0.05）；证实转录因子RUNX2正性调控 IPO8 基因的转录。. 3. 证实IPO8 基因启动子区 rs35100176 位点的 CCT 碱基插入变异能显著抑制启动子活性，从而影 响 IPO8 基因的转录。. 4.证实 RUNX2调控IPO8基因转录并参与成骨细胞分化。