lncRNAs在NELL1促人脂肪干细胞成骨分化中的作用机制研究

Maxillofacial bone defects severely affect the patients' quality of life. Tissue engineering is expected to provide a new model of bone defect repair. However, how to efficiently and specifically induce osteogenic differentiation of seed cells is a key issue. Compared with bone morphogenetic protein 2 (BMP2) and some other common osteogenic growth factors, neural EGFL like 1 (NELL1) has a highly efficient osteoinductive effect, functions as a key mediator downstream of RUNX2, and cannot induce ectopic bone formation. Therefore, NELL1 has a more prominent value for bone regeneration. But the mechanism of NELL1 on osteogenic induction is still unclear. Recent reports demonstrated that some long noncoding RNAs (lncRNAs) were involved in the regulation of osteogenic differentiation of stem cells by interaction with microRNAs (miRNAs) and acting as competing endogenous RNAs (ceRNAs). Our preliminary experiments indicated that the expressions of several lncRNAs could be increased or decreased significantly during osteogenic differentiation of human adipose derived-stem cells (hASCs) induced by NELL1. However, up to now, to our knowledge, no study has been reported about the roles of lncRNAs in NELL1-induced osteogenic differentiation. Based on our previous transcriptome sequencing study, this project will use series experiments both in vitro and in vivo, and methods of mechanism study to confirm the key lncRNAs that regulate the NELL1-induced hASC osteogenic differentiation, identify the miRNAs that interact with lncRNAs, investigate the effects and mechanisms of the interacted lncRNAs-miRNAs regulation of hASC osteogenic differentiation, and explore the potential crosstalk of lncRNAs-miRNAs with key osteogenic signaling pathways. This project is expected to provide a novel potential way for bone regeneration.

颌面部骨缺损严重影响患者的生存质量，组织工程有望为骨缺损修复提供新的模式。但如何高效、特异地诱导种子细胞成骨分化是一核心问题。与BMP2等常用因子相比，NELL1不仅具有高效成骨诱导作用，且作用于RUNX2下游，无异位成骨效应，具有更突出的应用价值，但其机制尚不明了。最新文献报道lncRNAs参与调控干细胞成骨分化，并常与miRNAs以ceRNAs形式相互作用进行调控。课题组前期研究证实，在NELL1促人脂肪干细胞成骨分化中多个相关lncRNAs表达明显上调或下调，但尚未见该过程中lncRNAs的调控作用及机制研究。本课题拟通过高通量筛选、体外及体内系列实验验证、作用机制探索的技术路线确定在NELL1促人脂肪干细胞成骨分化中调控作用最优的lncRNAs，研究并鉴定与之交互作用的miRNAs，明确lncRNAs-miRNAs作用机制及其与成骨相关通路之间的交叉对话，为骨再生修复提供新的思路。

较大范围骨缺损的修复是临床亟待解决的难题，骨组织工程的发展为其提供了新的思路和方法。人脂肪干细胞（human adipose-derived stem cells，hASCs）具有易于采集、安全性高等优势广泛用于组织工程中。NELL-1是一种成骨特异性生长因子。lncRNA和miRNA可通过多种调控机制参与成骨分化过程，但其在NELL-1促hASCs成骨分化中的作用尚不清楚。该研究利用高通量测序检测得到了在NELL-1诱导hASCs成骨分化中的lncRNA和miRNA的表达谱，并通过生物信息学分析lncRNA和miRNA的功能。从体外及体内探讨lncRNA和miRNA对hASCs成骨分化的调控作用，为lncRNA和miRNA调控hASCs成骨分化的机制提供实验依据。项目进一步从ceRNA机制入手，研究lncRNA与miRNA的相互作用，可望利用该机制应用于骨再生修复。最终通过一系列实验筛选了关键的lncRNA和miRNA：lncRNA NIOAT和miR-370-3p，抑制lncRNA NIOAT和过表达miR-370-3p可促进hASCs成骨分化。研究表明了lncRNA NIOAT可能通过ceRNA机制解除对miR-29a-3p的抑制，使Wnt/β-catenin通路抑制因子APC下调，由此参与经典Wnt通路调节成骨分化。通过实验证实miR-370-3p与circ_0003204存在ceRNA关系，circ_0003204通过miR-370-3p / HDAC4轴调控hASCs成骨分化。体内结果证实了抑制lncRNA NIOAT 和circ_0003204可促进hASCs修复颅骨缺损效果。项目还揭示了hASCs体外成骨分化过程中的m6A修饰特征，构建了hASCs成骨分化中m6A相关PPI网络，预测了受m6A调控的关键基因，为骨再生修复提供新的思路。