卵黄样羧肽酶CPVL调控IFN-γ信号通路在胶质瘤细胞凋亡中的分子机制研究

Glioma is one of the most common intracranial malignant tumors. At present, traditional therapy such as surgical resection, radiotherapy and chemotherapy has not significantly improved the prognosis of glioma patients. The current clinical practice of glioma has found that the method of inducing apoptosis of glioma cells is better in treatment effect and safety. In the early stage, the new functional gene CPVL, which was closely related to the development of glioma, was selected through human gene expression spectrum chip technology and HCS technology. Through the previous experiment, it was found that CPVL was highly expressed in human glioma cells and clinical glioma tissue samples, and was significantly correlated with the pathological stage classification of glioma. Combined with in vitro functional experiments, it was found that CPVL knockdown could significantly inhibit the proliferation of glioma cells and promote apoptosis of glioma cells, and was closely related to the glioma cell cycle. Meanwhile, CPVL was found to be involved in the regulation of apoptosis of glioma cells through IFN- gamma signaling pathway through IPA software analysis. However, the specific molecular mechanism needs to be further studied. This study based on the previous research, we explored the effect of CPVL regulating IFN- gamma signaling pathway on the apoptosis of glioma cells by gene blocking or overexpressing combined with animal experiments. These studies will attempt to elucidate the role and molecular mechanism of CPVL in the development of glioma, thus providing a new theoretical basis and therapeutic target for the treatment of glioma.

申请人前期通过人基因表达谱芯片技术结合HCS技术筛选出与胶质瘤发生发展密切相关的新功能基因CPVL；通过前期实验发现，CPVL基因在人胶质瘤细胞及临床胶质瘤组织样本中高表达，并且与胶质瘤病理分期分级显著相关；结合体外功能实验，发现CPVL敲减能够明显抑制胶质瘤细胞的增殖和促进胶质瘤细胞的凋亡，并且与胶质瘤细胞周期密切相关；同时通过IPA软件分析发现CPVL通过IFN-γ信号通路参与了胶质瘤细胞凋亡的调控。然而，其中的具体分子机制有待于进一步阐明。本研究在明确CPVL可通过IFN-γ信号通路参与胶质瘤细胞凋亡的基础上深入开展机制性研究，拟通过阻断或过表达等方法结合体内动物实验，探讨CPVL调节IFN-γ信号通路在胶质瘤细胞凋亡过程中的影响。这些研究将试图阐明CPVL在胶质瘤细胞凋亡过程中的作用及分子机制，从而为胶质瘤的治疗提供新的理论基础和治疗靶点。

在本项目中，我们通过人基因表达谱芯片技术结合HCS技术筛选出与胶质瘤发生发展密切相关的新功能基因CPVL，CPVL（卵黄样羧肽酶）是一种最早在人类巨噬细胞中发现的丝氨酸羧肽酶。然而CPVL在多种肿瘤中的作用尚不清楚。我们通过qPCR、Western blotting及IHC技术分析发现，与正常脑细胞和脑组织相比，CPVL在胶质瘤细胞和组织中显著上调。此外，CPVL高表达与胶质瘤临床分级和不良预后密切相关。结合体内外功能实验发现CPVL敲减能够明显抑制胶质瘤细胞增殖和促进胶质瘤细胞凋亡，并且与胶质瘤细胞周期密切相关。同时通过IPA软件分析发现CPVL敲减激活了IFN-γ/STAT1信号通路，从而诱导胶质瘤细胞凋亡。机制上，我们通过免疫纯化、质谱、IP及GST pull-down实验发现CPVL与BTK相互作用，并通过促进p300介导的STAT1乙酰化来抑制STAT1的磷酸化，从而促进胶质瘤进展。以上这些结果表明CPVL可作为胶质瘤治疗的潜在预后生物标志物和治疗靶点。