LOC554202-miR-31-c-MET调控枢纽对脊索瘤增殖和侵袭的影响及机制研究

Chordomas is aggressive and can’t remove completely due to its location, and it has high recurrence and disability rates because of its resistance to both chemotherapy and radiotherapy. Revealing molecular mechanisms of proliferation and invasion in chordomas is crucial for diagnosis and therapy. Our previous study showed that miR-31 modulates proliferation and invasion of chordoma cells, and that oncogene c-MET is a downstream target of miR-31 and regulated by miR-31, and that long non-coding RNA LOC554202 is the host gene of miR-31 and regulates expression of miR-31. We found LOC554202 bound to EZH2, the catalytic subunit of PCR2 complex, and exogenous expression of EZH2 caused decreased accumulation of miR-31. Based on these findings, we postulate that LOC554202 regulates expression of miR-31 through recruiting the PRC2 complex and miR-31 modulates proliferation and invasion of chordomas by targeting oncogene c-MET. The LOC554202-miR-31-c-MET axis is a complicated and multi-level axis and plays an important role in proliferation and invasion of chordomas. To verify our hypothesis, we plan to demonstrate the Effects and molecular mechanisms of LOC554202-miR-31-c-MET axis on proliferation and invasion of chordomas using in vitro and in vivo assays. The clinical significance of LOC554202-miR-31-c-MET axis is demonstrated by clinical analysis. Our project will provide detail information about the regulation mechanisms of the LOC554202-miR-31-c-MET axis on proliferation and invasion of chordomas, and help establishing novel strategy for diagnosis and therapy.

脊索瘤侵袭性强且不易完全切除，对放化疗均不敏感，复发率和致残率高，因此，阐明其增殖及侵袭的分子机制对攻克此顽疾十分重要。本课题组前期研究结果表明，miR-31和c-MET均可影响脊索瘤细胞的增殖和侵袭能力；进一步的研究显示，miR-31可以负性调节c-MET基因表达；而miR-31的宿主基因LOC554202以及PRC2复合物调节亚基EZH2均可调控miR-31的表达。由此我们推测LOC554202可能通过招募PRC2复合体调控miR-31的表达，进而调控c-MET的表达，LOC554202、miR-31和c-MET组成一个以miR-31为中心的调控枢纽，影响脊索瘤的增殖和侵袭的生物学行为。为验证这一假说，本项目通过细胞实验、动物实验并结合临床样本分析，逐步阐明LOC554202-miR-31-c-MET调控枢纽对脊索瘤增殖和侵袭的影响及具体机制，可为脊索瘤的诊断和治疗提供新的靶点及策略。

脊索瘤虽是低度恶性肿瘤，但手术难以全切，具有易复发、侵袭性强等特点。对该疾病从分子水平进行研究，找到新的分子治疗靶点，延长患者生存期具有重要的意义。本课题尝试从分子、细胞、组织、动物等实验进行研究；通过qPCR及FISH检测临床标本，发现脊索瘤组织中LOC554202较瘤旁组织高；LOC554202通过EZH2负性调节miR-31，过表达miR-31或敲降LOC554202后均能够显著抑制脊索瘤细胞的增殖和侵袭能力；并在WB实验中发现LOC554202和miR-31能够调节EMT相关分子的表达。再通过TargetScan6.2和miRanda等软件预测miR-31的下游靶基因为RNF144B，在过表达miR-31细胞系中进行验证，发现miR-31能够抑制RNF144B的表达；同时建立敲降RNF144B的脊索瘤细胞系，进行克隆形成、凋亡、侵袭和转移实验，发现其生物学效应与miR-31的相反，而过表达LOC554202后能够逆转，进而证实miR-31通过靶向负性调控RNF144B进而抑制脊索瘤的增殖和侵袭能力。因此，本研究从多层面进行LOC554202/EZH2/miR-31/RNF144B调控脊索瘤增殖和侵袭能力的生物学机制研究，可能为脊索瘤的治疗提供潜在的分子靶点。