缺氧诱导因子1α在缺氧诱导的肝癌上皮细胞间质化及索拉非尼耐药中的作用及其机制研究

Poor prognosis of hepatocllular carcinoma is due to recurence or metastasis and treatments resistance. As a transcription factor, HIF1α was over-expressed in a variety of tumors and was associated with high agressiveness, poor prognosis and resistance to chemotherapy and targeted drugs. Previous studies showed that hypoxia could induce HCC cell EMT and resistant to sorafenib, over-expression of HIF1α might be associated with poor prognosis after surgery and the patients with over-expression of HIF1α could exhibit resistant to sorafenib. It suggests that HIF1α should be a critical factor in hypoxia induced HCC EMT and resistant to sorafenib. The effects of HIF1α were investigated through following four fields, cellular experiments, animal models, moleculare mechanisms and clinical study in this study. In vitro and vivo, we transfer ad5-shHIF1α or ad5-HIF1α into HCC cells to upregulate or downregulate the expressions of HIF1α, and observe the changes of HCC proliferation, apoptosis, agressiveness and sensitivity to sorafenib. We will also study the effects of HIF1α on HCC EMT and its dependent signaling such as β-catenin and sorafenib targets kinases. Chip-on-chip and genome microarray will be employed to explore the possible molecular mechanisms of HIF1α-induced HCC metastasis and sorafenib resistance. Moreover, a clinical study will be performed to further verify the relationship among HIF1α, HCC metastasis and efficacy of sorafenib. It will contribute to improving efficacy of treatment, prolonging the survival, and reversing sorafenib resistance.

复发转移和治疗耐药是肝癌预后差的主要原因。转录因子HIF1α在肿瘤中高表达与高侵袭性预后差相关，并可致化疗和靶向药物耐药。研究证实缺氧可诱导肿瘤细胞EMT并对放化疗抵抗。前期研究发现低氧诱导肝癌对索拉非尼敏感性降低且出现EMT，HIF1α在肝癌中高表达与预后差有关，且索拉非尼无效患者中HIF1α表达显著增高。提示HIF1α介导了缺氧诱导的肝癌EMT和索拉非尼耐药。本研究从细胞实验、动物模型、分子机制和临床病例四个层面展开：体内外实验利用不同HIF1α表达载体上调/下调表达，观察肝癌增殖凋亡、侵袭性及索拉非尼抑制作用的变化；探讨HIF1α对βcatenin等EMT相关通路、索拉非尼靶点的影响；利用chip及表达谱芯片探索HIF1α靶基因及下游通路，阐明HIF1α促进肝癌转移及索拉非尼耐药的分子机制；进而结合临床病例分析HIF1α与肝癌转移、索拉非尼疗效间的关系。为肝癌治疗及逆转耐药提供新思路

背景：复发转移和治疗耐药是肝癌预后差的主要原因。转录因子HIF1α在肿瘤中高表达与高侵袭性预后差相关，并可导致化疗和靶向药物耐药。前期研究发现低氧诱导肝癌对索拉非尼敏感性降低且出现EMT，HIF1α在肝癌中高表达与预后差有关，并且索拉非尼无效患者中HIF1α表达显著增高，提示HIF1α介导了缺氧诱导的肝癌EMT和索拉非尼耐药。本研究重点探讨HIF1α在索拉非尼耐药中的作用，及可能的分子机制，为肝癌治疗及逆转耐药提供新思路。.主要研究内容：（1）观察缺氧条件下，肝癌细胞EMT和侵袭力的变化，并初步探讨其可能机制；（2）研究HIF1α、YAP在肝癌EMT和索拉非尼耐药中的作用和机制；（3）结合临床病例分析HIF1α与索拉非尼疗效间的关系。.重要结果及关键数据：（1）缺氧环境下，HIF-1α的蛋白水平升高，细胞表面蛋白E-cadherin表达下降，N-cadherin增加，转录因子Twist，Snail随缺氧时间增加表达增强。TGF-β信号通路相关蛋白表达随缺氧时间增加表达增强。Transwell实验证实肝癌细胞在缺氧条件下迁移和侵袭能力均较常氧增强。（2）肝癌细胞在常氧环境下加用甲苯磺酸索拉非尼，HIF1α及YAP主要集中在细胞质，在低氧环境下，HIF1α及YAP共同向核内迁移，细胞核阳性率升高，提示低氧环境可能促成肝癌细胞对甲苯磺酸索拉非尼产生抵抗力，促使其产生耐药；当YAP主要集中在肝癌细胞核时，EMT相关指标E-钙粘附减少，N-钙粘附素、波形蛋白表达水平相应增多。（3）HIF1α通过降低索拉非尼抑制肝癌细胞增殖和促进细胞凋亡的作用，可以诱导索拉非尼耐药，其机制可能是HIF1α上调了索拉非尼作用靶点的磷酸化水平。HIF1α蛋白阳性表达是影响总体生存时间和无疾病进展生存时间的不良预后因素。.科学意义：阐明了低氧环境下HIF1α促进肝癌侵袭转移、诱导索拉非尼耐药的作用及可能机制，为肝癌治疗及逆转耐药提供新思路。