血根碱对HIF-1α/TGF-β正反馈通路诱导的肝癌上皮间质转化的作用研究

Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide and the control of metastasis is a key factor affecting the clinical outcome. Hypoxia-induced epithelial-mesenchymal trasition (EMT) plays an important role in tumor invasion and metastasis, which indicates that studies on the mechanisms of hypoxia-induced EMT in HCC may bring insights to the development of targeted drugs. Our previous study has found that sanguinarine, a natural compound, could inhibit EMT in transforming growth factor beta (TGF-β) induced cells. Sanguinarine could also reverse the expression of E-cadherin and N-cadherin in HepG2 and SMCC-7721 cell subcutaneous xenograft HCC models, respectively. In this study, the EMT inhibiting effect of sanguinarine in a cobalt chloride hypoxia model and a hypoxic chamber model will be evaluated by western blotting and immunohistochemistry. To demonstrate hypoxia inducible factor-1α (HIF-1α) and transforming growth factor beta (TGF-β) feed-forward loop in EMT, further experiments will be performed to investigate the interaction of HIF-1α and TGF-β by chromatin immunoprecipitation (ChIP) assay, luciferase assay and alteration of their levels using HIF-1α small interfering RNA, TGF-β recombinant protein and a small molecular inhibitor. HIF-1α overexpressing and silencing plasmids hydrodynamics transfected mice model will be used to confirm the interaction. This project aims to study the effect of sanguinarine in EMT models with high level of HIF-1α expression and the addition of TGF-β mimicking the tumor hypoxia microenvironment in vitro. The hypothesized mechanism of the effect of sanguinarine that is to target both the HIF-1α and TGF-β signaling will be demonstrated. To visualize the activity of HIF-1a, MHCC-97H cells will be stably transfected with p5HRE-EGFP, which has five hypoxia responsive elements upstream of the reporter gene. In vivo HCC orthotopic and metastasis models that employ orthotopic implantation and tail vein injection of MHCC-97H /p5HRE-EGFP cells into nude mice will be established to confirm the effect of sanguinarine on HCC EMT and pulmonary metastasis as well as the underlying HIF-1α and TGF-β targeted mechanism. This work may bring insights to the mechanism of hypoxia induced EMT and have significant clinical relevance to the development of drugs inhibiting HCC metastasis from natural sources.

控制复发转移是提高肝癌患者生存率的关键。缺氧诱导的EMT是肿瘤侵袭转移的主要原因之一，针对缺氧诱导因子HIF-1α引发EMT的研究可作为肝癌转移药物发现的重要途径。前期实验发现血根碱在TGF-β诱导和裸鼠移植瘤模型中可以逆转EMT标志物的表达。本项目将用化学和物理缺氧细胞模型，考察血根碱对HIF-1α诱导肝癌EMT的作用；通过ChIP和荧光素酶报告基因实验、siRNA、重组蛋白和抑制剂刺激的细胞和HIF-1α转基因小鼠，体内体外阐明EMT中HIF-1α/TGF-β正反馈通路的形成，考察血根碱是否可以通过该通路抑制肝癌细胞EMT、迁移和侵袭；构建HRE调控GFP表达的肝癌原位和转移动物模型，明确血根碱具有干预HIF-1α/TGF-β正反馈通路及抑制EMT和肺转移的作用。本研究将有助于阐明肝癌EMT中HIF-1α/TGF-β正反馈通路，并为揭示血根碱抑制肝癌EMT的作用和机理提供实验依据。

肝细胞癌是一种生长快、高转移、复发率高、预后差的消化系统实体恶性肿瘤，控制复发转移是提高患者生存率的关键。EMT可由多种肿瘤微环境因素诱导发生，如缺氧和转化生长因子TGF-β。寻找针对肝癌缺氧微环境诱导EMT中的一些关键分子的靶向药物是抑制肝癌转移的核心。本课题研究了缺氧在肝癌细胞中诱导的HIF-1α/TGF-β正反馈通路和EMT，并考察了血根碱通过该通路对肝癌细胞生长和EMT的抑制作用关系。在CoCl2诱导的化学缺氧模型和1%O2缺氧培养箱模型中证实了缺氧可诱导肝癌细胞TGF-β的基因和蛋白表达和释放；TGF-β可以上调HIF-1α和HIF-1α靶基因的mRNA和蛋白表达；TGF-βR抑制剂可以抑制肝癌细胞中存在的缺氧微环境诱导的HIF-1α/TGF-β正反馈通路。肝癌临床样本中HIF-1α/TGF-β正反馈通路与肝癌患者的生存及肝癌组织的增殖和EMT均密切相关。血根碱可以抑制肝癌细胞体外增殖、平板克隆形成，诱导肝癌细胞HepG2和SMMC-7721的细胞凋亡，细胞周期阻滞于S期；血根碱能够明显抑制HepG2和SMMC-7721移植瘤的生长，且对裸鼠体重及脏器重量无明显影响，证明血根碱在体外及体内均具有良好的抗肿瘤活性。TGF-β诱导的EMT肝癌细胞模型中Smad2/3通路和PI3K-AKT通路的激活；缺氧可以激活PI3K-AKT通路并诱导细胞EMT；血根碱可以抑制缺氧激活的HIF-1α通路和EMT，也可以逆转TGF-β诱导的EMT，并对PI3K-AKT和HIF-1α/TGF-β正反馈通路有抑制作用；血根碱可以有效抑制TGF-β诱导的肝癌细胞迁移。本研究围绕缺氧诱导的HIF-1α/TGF-β正反馈通路，通过体内外药理学、生物信息学、分子生物学实验对血根碱的抗肝癌EMT作用进行分析，并对其调控Smad和PI3K-AKT通路的作用机制进行了研究。本课题的完成将有助于阐明血根碱抑制肝癌EMT的作用和机理，为抗肝癌EMT药物的筛选和发现提供了新的研究思路。