氧感受因子CtBP1介导的上皮-间质转化在肝细胞肝癌肉瘤样转化中的作用及机制

C-terminal binding protein 1 (CtBP1) inhibited the expression of epithelial associated genes, tumor suppressor genes and antiapoptosis genes. NADH, which produce in hypoxic microenviroment, can promote the gene expression inhibtion of CtBP1. So CtBP1 mediated the malignant progress of tumor in hypoxic microenviroment. Classical hepatocelluar carcinoma (cHCC) can transform to sarcomatoid hepatocellular carcinoma (sHCC) after specific therapy, including transcatheter arterial embolization, percutaneous ethanol injection and radiofrequency ablation. We previously found CtBP1 upregulated in the sarcomatoid area of sHCC, and confirmed that CtBP1 promote epithelial-mesenchymal transition (EMT) in HCC cells. Moreover, hypoxia promoted CtBP1 binding with the promoter region of E-cadherin. Meanwhile, transcription factor p300, it's cofactor HIF1a and their target CtBP1 upregulated simultaneously. So we speculated that CtBP1 mediated sarcomatoid transformation of HCC through CtBP1's upreulation and recruitment to promoter region of target genes under hypoxic microenviroment. Through shRNA interference, ChIP and luciferase reporter system, this project aim to elucidate the mechanism in sarcomatoid transformation mediated by CtBP1. And verify this mechanism in animal model and human tissue samples. This study raises the significance in preventing malignant progression and target therapy of sHCC.

CtBP1能抑制上皮相关基因、抑癌基因、凋亡抑制基因的表达，缺氧微环境产生NADH促进其抑制作用，因而能介导低氧微环境下肿瘤的恶性进展。肉瘤样肝细胞肝癌由经典型肝细胞肝癌治疗后转化而来，包括经导管动脉栓塞、经皮乙醇注射治疗和射频消融治疗。我们前期发现CtBP1在肉瘤样肝细胞肝癌的肉瘤样区域高表达，并在肝癌细胞中证实其促进上皮-间质转化；同时发现低氧培养促进细胞中CtBP1与E-cadherin启动子区的结合，转录因子p300与HlF1a与其靶基因CtBP1表达同时增加。由此推测，经治疗导致的缺氧微环境通过增加CtBP1的表达及与靶基因启动子区结合介导了肝细胞肝癌的肉瘤样转化。本项目拟采用细胞缺氧培养，shRNA、ChIP、荧光素酶报告系统，阐明CtBP1介导肿瘤恶性进展及机制，并在荷瘤动物模型及人体组织标本验证该机制。对预防肝细胞肝癌的恶性进展及肉瘤样肝细胞肝癌的靶向治疗具有重要的意义。

肉瘤样肝细胞癌是肝细胞癌中侵袭能力最强、预后最差的一个组织学类型，由经典型的肝细胞癌转化而来，CtBP1是低氧感受因子，在肉瘤样肝细胞癌中表达上调，明确CtBP1是否介导了肝细胞癌的肉瘤样转化，有利于阐明肝癌的恶性进展机制并提供新的干预靶标。本课题采用体外细胞、人体组织标本和裸鼠移植瘤模型，阐明了低氧条件下CtBP1介导肝细胞癌肉瘤样转化的机制，并同时探讨了HIF1a在其中的调控作用。课题结果显示：肉瘤样肝细胞癌中CtBP1表达上调、与EMT表型相关；低氧培养条件下CtBP1表达上调、细胞发生EMT及肉瘤样细胞形态改变；在CtBP1低表达的肝癌细胞中过表达CtBP1，促进了EMT转变，模拟了低氧诱导的肉瘤样转化；而在CtBP1高表达的肝癌细胞中，敲低CtBP1抑制了低氧诱导的肉瘤样转化；CtBP1高表达的肝癌细胞构建的裸鼠移植瘤发生了自发性肉瘤样转化、敲低CtBP1后抑制了肉瘤样转化；同时肉瘤样转化周边的上皮样肝癌细胞中HIF1a表达上调，提示低氧可能参与了移植瘤肉瘤样转化的调控。该研究阐明了低氧感受CtBP1介导肝癌肉瘤样转化的分子机制，为肝癌的分子靶向治疗提供了新的依据；同时CtBP1是转录辅抑制因子，能结合多个癌相关信号通路的下游效应转录因子，提示靶向CtBP1是有效抑制低氧诱导肝癌的恶性进展，尤其是栓塞、射频治疗相关的肉瘤样转化。