miRNA在IL-17促骨巨细胞瘤侵袭中的作用和机制研究

Giant cell tumor of bone (GCTB) is a benign, locally aggressive, osteolytic tumor that causes significant bone destruction at the epiphysis of long bones. The exact molecular mechanisms of GCTB progression remain largely unknown. Recently, we reported that the IL-17A was increased in GCTB patients and played an important role for GCTB progression through promotion of bone resorption by stimulating RANKL expression, and proliferation of stromal cells. In this proposal, we will further investigate the role of miRNA in the IL-17 mediated- GCTB progression. Firstly, we will screen differential expression of miRNAs in GCTB samples through miRNA array, and based on correlation analysis, pick up some microRNAs closely associated with IL-17 level. Furthermore, we will confirm whether these miRNAs can regulate IL-17 expression, or are regulated by IL-17, and determine the role of these microRNAs in IL-17- mediated bone resorption and tumor cell proliferation through in vitro and vivo assays. Then, through bioinformatic analysis, including TargetScan and miRanda, we predict the miRNA- targeted transcript factor, which regulate IL-17 expression, and confirm this via luciferase reporter assay, gain-of-function, and loss-of-function assays. Finally, we will further analyse the association between the identified miRNA and clinical parementers, including patient prognosis and tumor pathology. Thus, through combination basic and clinic researches, our project will open a new avenue for molecular mechanism of GCTB, and maybe provide an useful candidate marker for progression and therapeutic target for GCTB.

我们前期在《Clin Cancer Res》杂志首次报道了IL-17在骨巨细胞瘤中的重要作用。基于miRNA是调控基因表达的重要方式，在骨巨细胞瘤中还未见报道，因此本课题拟研究miRNA在IL-17促骨巨细胞瘤侵袭中的作用及机制。利用芯片筛选肿瘤组织miRNA差异表达谱，相关性分析确定与IL-17最相关的miRNAs；体外miRNA抑制及过表达实验明确IL-17的上下游miRNA分子，并研究候选miRNA在肿瘤细胞溶骨破坏以及增殖中的作用。TargetScan和miRanda等生物信息学手段预测miRNA直接结合的并能够调节IL-17表达的靶分子，荧光素酶报告实验结合功能获得和缺失实验进一步验证。结合临床标本，分析所鉴定的miRNA分子和患者预后及肿瘤临床病理指标间的关系。我们的研究结果不仅可能为骨巨细胞瘤的侵袭进展揭示新的分子机制，也可能为临床诊治、预后判断提供新的生物学指标和靶点。

本项目组在前期研究中首次报道了IL-17在骨巨细胞瘤生长和侵袭中的重要作用，但其miRNA在其调控中所起的作用及机制尚未见报道。因此本项目组设计了本课题，旨在利用分子生物学方法对在 IL-17促骨巨细胞瘤侵袭过程中起调控作用的miRNAs进行筛选，并对其具体机制进行研究。我们发现miR-125a表达水平在骨巨细胞瘤组织中显著上升，并与肿瘤复发呈正相关。体外实验和动物实验均显示，miR-125a可以刺激间质细胞成瘤，并促进肿瘤增长。且骨巨细胞瘤组织中，miR-125a的表达水平与IL-17a和β-catenin的表达水平成正相关。进一步研究发现，miR-125a是通过靶向抑制TET2、Foxp3、APC、GSK3β等IL-17a和β-catenin负性调节基因对肿瘤的侵袭性能进行调控。且IL-17a表达的升高，反过来又可以通过激活NF-kB信号通路，对miR-125a的表达进行正向调节。动物实验还表明，同时沉默IL-17a和miR-125a，可以显著抑制肿瘤生长。临床患者回顾性分析同样证实，miR-125a的表达水平，是患者复发的风险因素。由此我们认为miR-125a通过抑制TET2、Foxp3、APC、GSK3β等负性调节靶基因，对IL-17a和β-catenin产生正向调控，而IL-17a又可以提高miR-125a表达形成正循环，促进肿瘤侵袭性能的提高。本课题结果揭示了IL-17a调控骨巨细胞瘤生长和侵袭的具体作用机制，为靶向治疗提供了可能的潜在靶标。