基于单细胞测序技术分析Osterix在脊索瘤恶性生物学行为中的调控机制研究

Chordoma, which seriously affects the quality of life of patients, is a common malignant spinal tumor with strong invasiveness, but so far there is no clear pathogenesis and treatment targets. Using single cell sequencing technique, we found that Osterix, a transcription factor related to bone metabolism, was significantly distributed in chordoma cell subsets, and the prognosis of chordoma patients with high expression of Osterix was poor. We further found that Osterix knockdown significantly inhibited chordoma cell proliferation, migration, invasion and induced G1 phase arrest. In addition, Brachyury, Galectin-3, Survivin and other chordoma-related tumor factors were also inhibited after Osterix knockdown. Therefore, we preliminarily believe that Osterix, as an upstream transcription factor, plays an important role in the malignant behavior of chordoma. On this basis, our project intends to further explore the mechanism of Osterix in regulating the biological function of chordoma at the molecular and cellular levels. The effects of Osterix on the proliferation and metastasis of chordoma are verified in situ and metastatic tumor models, so as to reveal its key role in malignant biological behavior of chordoma and provide new theoretical basis and potential drug targets for the treatment of chordoma.

脊索瘤是一种常见的具有强侵袭性的恶性脊柱肿瘤，严重影响患者生存质量，但迄今尚缺乏明确的发病机制及治疗靶点。本项目组前期利用单细胞测序技术发现，一种调控骨代谢的转录因子Osterix在脊索瘤细胞中分布显著，并且高表达Osterix的脊索瘤患者预后较差。我们还发现，干扰Osterix表达可显著抑制脊索瘤细胞增殖、周期、侵袭迁移，而自噬现象增加。此外，Brachyury、Galectin-3、Survivin等脊索瘤相关肿瘤因子的表达也受到不同程度抑制。因此，我们初步认为Osterix作为转录因子在脊索瘤恶性生物学行为中发挥重要作用。在此基础上，本项目拟从分子、细胞水平进一步探索Osterix调控脊索瘤恶性生物学表现的作用机制，构建脊索瘤荷瘤和肺转移模型，从体内水平验证Osterix对脊索瘤增殖和转移的影响，以揭示其在脊索瘤恶性生物学行为中的关键作用，为脊索瘤的治疗提供新的理论基础和药物靶点。

脊索瘤是一种常见的具有强侵袭性的恶性脊柱肿瘤，严重影响患者生存质量，但迄今尚缺乏明确的发病机制及治疗靶点。结合课题组前期的脊柱和骶骨脊索瘤的临床样本信息和患者临床数据信息，通过该部分内容从临床患者资料和宏观实际角度证实，进一步证实过量表达的Osterix提示患者肿瘤局部侵袭性强、恶性程度高、预示较差预后结局。利用 CRISPR/Cas9 基因编辑系统成功构建OSX敲除稳转脊索瘤细胞株，表明Osterix可以促进细胞迁移能力和侵袭能力，并且促进脊索瘤肿瘤细胞的自噬形成现象。课题组通过脊索瘤组织、细胞和脊索来源髓核细胞在基因和蛋白层面证明了Osterix在脊索瘤中的高表达。在脊索瘤U-CH1细胞中，抑制Osterix可显著降低脊索瘤细胞的增殖、迁移和成瘤。鉴于OSX在骨微环境重塑中的重要作用，课题组进一步探寻骨微环境对脊索瘤发生发展的影响。为进一步解析OSX的调控机制，课题组利用Tubor-ID技术和质谱分析，发现Osterix可与MMP-2、 MMP-9、 SOX9 和 PDGFR相互作用。通过免疫共沉淀技术证明外界的炎性信号可促进Osterix调控SOX9-NLRP3蛋白复合体的形成。抑制OSX的蛋白表达及活性可抑制SOX9与NLRP3的相互作用和下游成熟IL-6的分泌。课题组通过上述研究内容揭示Osterix在脊索瘤恶性生物学行为中的关键作用，为脊索瘤的治疗提供新的理论基础和药物靶点。