PBLD直接或通过ERK/MAPK信号通路调控HIF-1α与肝癌侵袭转移的机制研究

Primary hepatocellular carcinoma ranks third among the causes of death from cancer because of its high malignance, metastasis and recurrence tendency. Hypoxia caused by the rapid growth of cancer cells is closely related to its advanced progression and metastasis. Hypoxia-inducible factor-1α (HIF-1α) is the core transcription factor of hypoxia response which can promote angiogenesis, proliferation, invasion and metastasis of the tumor. Our study found that loss expression of serine/threonine kinase receptor-binding protein PBLD in HCC is significantly associated with the prognosis of patients. PBLD overexpression inhibits the proliferation and migration of liver cancer cells, while PBLD silence activate HIF-1α. It is a potential tumor suppressor gene. In this study, confocal laser, site-directed mutagenesis, in vitro kinase assay, LC-MS/MS, luciferase reporter gene, ChIP will be performed to explore the mechanism of HIF-1α regulated by PBLD as well as the impact on liver cancer cell proliferation, invasion and metastasis. The relationship between PBLD and HIF-1α will be assessed according to clinical data. Mechanism between PBLD and HIF-1α will be further elaborated with PBLD knockout mice. Outcomes of this research may provide certain novel approach for diagnosis, prognosis and treatment of liver cancer.

原发性肝癌为第三大致死肿瘤，恶性程度高，易转移复发，肿瘤细胞失控生长而导致的缺氧与肝癌的恶性进展和远处转移密切相关。缺氧诱导因子-1α（HIF-1α）是缺氧应答的核心转录因子，HIF-1α的激活可促进肿瘤的血管生成、增殖及侵袭和转移能力。我们前期研究发现，丝氨酸/苏氨酸受体激酶结合蛋白PBLD在肝癌组织中表达缺失，与患者预后显著相关；PBLD过表达可抑制肝癌细胞增殖和迁移，PBLD沉默可激活HIF-1α，是一个潜在的抑癌基因。本研究拟通过激光共聚焦、定点诱变、体外激酶活性实验、LC-MS/MS、荧光素酶报告基因、ChIP等探求候选抑癌基因PBLD调控HIF-1α与肝癌侵袭转移的相关分子机制；结合临床资料评估PBLD和HIF-1α的相关性及与肝癌预后的关系；并在动物体内进一步验证PBLD和HIF-1α的上述作用和机制。本项目的顺利实施有望为肝癌预后的预测和靶向基因治疗提供新的科学依据。

原发性肝癌为第三大致死肿瘤，恶性程度高，肿瘤细胞失控生长而导致的缺氧与肝癌的恶性进展和远处转移密切相关。HIF-1α是缺氧应答的核心转录因子，可促进肿瘤的血管生成、增殖及侵袭能力。我们前期发现，PBLD在肝癌组织表达缺失，与患者预后显著相关；PBLD可抑制肝癌细胞增殖和迁移，PBLD沉默可激活HIF-1α及ERK/MAPK信号通路，是一个潜在抑癌基因。因此本研究提出了“PBLD可通过 ERK/MAPK 通路或直接调控 HIF-1α活性，影响肝癌细胞侵袭和转移等生物学行为”的研究假设。为验证假设，本研究利用组织检测及临床资料分析PBLD和HIF-1α的相关性及与患者预后的关系、利用CCK-8、transwell、体外血管形成、裸鼠成瘤等实验明确PBLD在肝癌增殖、侵袭转移及血管形成中的作用。利用激光共聚焦、免疫共沉淀、PBLD基因敲除小鼠组织检测、WB等实验进一步分析PBLD与HIF-1α的关系及其对HIF-1α的调控机制。组织芯片的IHC检测发现PBLD与HIF-1α及VEGFA的表达呈负相关，PBLD+/HIF-1α-组病人相较于PBLD-/HIF-1α+的病人预后较好。体外细胞学实验结果显示，PBLD可抑制肝癌细胞的增殖、侵袭及血管形成能力。裸鼠成瘤组织及PBLD敲除鼠肝脏IHC检测均显示PBLD与HIF-1α的表达呈负相关。另外，PBLD可抑制CoCl2诱导的HIF-1α的表达。关于PBLD调控HIF-1α的机制：1）激光共聚焦检测发现PBLD可以抑制HIF-1a的核转位过程，进一步CO-IP验证了二者之间存在结合。2）裸鼠成瘤组织及PBLD敲除鼠肝脏组织的检测均提示PBLD可抑制ERK/MAPK通路的激活，同时，ERK抑制剂U1026可抑制HIF-1α的表达。因此，PBLD还可通过抑制ERK/MAPK抑制HIF-1α的激活。综上，PBLD在肝癌中可通过抑制HIF-1α的活性进而在体内外抑制肝癌细胞的增殖和血管形成，是一个抑癌基因。PBLD通过抑制ERK/MAPK信号通路抑制重要缺氧调控因子HIF-1α的表达，同时还可与HIF-1α直接结合抑制其核转位，进而抑制HIF-1α下游靶分子VEGFA及snail的表达，最终抑制肝癌细胞的生长、血管形成及侵袭转移能力。本研究为肝癌预后的预测和靶向基因治疗提供新的科学依据。