miR-34a调控人脂肪基质细胞成骨向分化的作用和机制研究

Human adipose-derived stromal cell (hASCs) is one of the ideal seed cells for bone tissue engineering. The researches of my study group indicate the inhibition of retinoblastoma binding protein 2 (RBP2), a histone demethylase that specifically catalyzes demethylation of dimethyl or trimethyl histone H3 lysine 4, can significantly improve the in vitro and in vivo osteogenic capability of hASCs. Therefore, RBP2 is a potential drug target during the molecular targeted regulation of osteogenic differentiation of hASCs, and intervention of the expression of RBP2 can potentially modulate the osteogenic differentiation of hASCs. However, upstream factors which control the functions of RBP2 are still unknown and the ambiguity in these aspects prohibits the developing of targeted drugs which aim at effective induction of the osteogenic differentiation of hASCs. Recent researches show miRNAs, other important epigenetic factors, have emerged as key mediators in the osteoblastic differentiation of mesenchymal stem cells, but the effects of miRNAs on the modulating functions of RBP2 during the osteogenic differentiation of hASCs are not reported. Therefore, the association between miRNA and RBP2, the effects of miRNAs on the modulating functions of RBP2 during the osteogenic differentiation of hASCs are worth launching an early and frontier investigation because the knowledge will do great benefits to develop targeted pharmaceuticals. This project includes 3 main subjects listed as follows: 1) To select and determine the keynote RBP2-targeted miRNA (miR-34a) which specifically modulate the expression of RBP2 by using the bioinformatics and to verify these results at cell biology level of hASCs; 2) to investigate the molecular mechanisms which govern the modulation modes of miR-34a on the expression and functions of RBP2 and other keynote signal molecules, and further effectively induce the osteoblastic differentiation of hASCs; 3) To evaluate the value of miR-34a working as a more effective drug target than RBP2 in promoting the osteogenic differentiation of hASCs through overexpression and knockdown experiments of miR-34a, RBP2, and other keynote factors in vitro and in vivo. This project is conducive to determine the modulation function of miR-34a during the osteoblastic differentiation of hASCs, to verify the value of miRNA as a more effective target drug than RBP2 for effective osteogenic induction of hASCs, and will definitively benefit the clinical translation of bone tissue engineering based on hASCs.

人脂肪基质细胞是骨组织工程理想的种子细胞之一。本课题组首先发现抑制组蛋白去甲基化酶RBP2的活性可显著促进该细胞的成骨分化，但在此过程中上游因素调控RBP2的机制尚不明确，因而制约了靶向药物开发的进程。已知miRNA在干细胞成骨分化中发挥重要作用，但其如何影响RBP2进而影响该细胞成骨的调控尚未见报道，故明确上述两种因素间的关联对未来前沿性地开发促进该细胞成骨的靶向药物有重大意义。项目内容包括：运用生物信息学方法和细胞实验筛选并确定靶向调控RBP2的主要miRNA分子miR-34a；比较分别过表达或敲低miR-34a和RBP2等基因影响细胞体内体外成骨分化的效应；探讨miR-34a调控人脂肪基质细胞成骨分化的机制（包括RBP2及其他通路）。本项目利于明确miR-34a对细胞成骨的调控作用，利于明确miRNA作为药物靶点实现高效调控人脂肪基质细胞成骨的价值，利于实现骨组织工程技术的临床转化。

本研究运用生物信息学方法筛选RPB2转录本的3′UTR上是否存在miR-34a潜在的结合位点，并且利用双荧光素酶报告基因实验，实时定量PCR（qRT-PCR），Western blot实验在人脂肪间充质干细胞（hASCs）中验证RBP2为miR-34a直接作用的新靶基因。慢病毒过表达或敲低miR-34a后，体外实验（包括碱性磷酸酶染色和定量、茜素红染色和定量、von Kossa染色和成骨相关基因表达的qRT-PCR检测）以及裸鼠背部皮下异位成骨的体内实验显示，miR-34a具有促进hASCs成骨向分化的能力。为了深入研究miR-34a调控该细胞成骨向分化的机制，通过过表达或敲低miR-34a以及分别敲低RBP2和NOTCH1基因，利用qRT-PCR和Western blot检测各自下游可能靶基因的表达变化，探讨hASCs成骨分化中miR-34a及其靶基因的表达水平和相互作用关系，从而验证假想的RBP2/NOTCH1/CYCLIN D1调控网络在miR-34a调控hASCs成骨向分化的作用和机制。总之，miR-34a通过RBP2/NOTCH1/CYCLIN D1共调控网络提高hASCs的成骨分化能力，提示miR-34a可作为骨再生修复中潜在的治疗靶点或作为小分子调控骨再生。在上述研究基础之上，本项目还发现miR-375促进人脂肪间充质干细胞成骨向分化的作用和机制；并研究了长链非编码RNA MIR31HG，MEG3和MIAT在人脂肪间充质干细胞成骨向分化中的作用和机制；上述系列研究为以干细胞为基础的骨再生修复提供了潜在的治疗靶点。