缺氧微环境中SIRT1参与肝癌干细胞可塑性调控的机制研究

Cancer stem cells (CSCs) plays an imprtant role，and hypoxic microenvironment prevalent in the occurrence and development of in tumors. The Important characteristics of CSCs to adapt to hypoxic microenvironment may be the plasticity of CSCs. In previous study, we found that hypoxia conduce to maintain the stemness of CD133+ CSCs and transform the stemness of CD133- CSCs, and SIRT1 may play an important role in these processes. However, the mechanism of SIRT1 to regulate the plasticity maintenance of CSCs is still unknow. In this study, we investigate the influnce of hypoxia on the expression of SIRT1 in CD133+ and CD133- cells in vitro and in vivo. We examine the effect of SIRT1 on cancer stem-like properties of CSCs, including expression of stem cell genes and CSC surface marker, colony formation and tumor-forming ability in vivo. The overexpression and siRNA technology are used to regulate the expression of SIRT1 in hepatoma cells in order to regulate the variation of hypoxia inducible factor, SIRT1 and stemness associated genes, to clarify the molecular mechanism of SIRT1 plasticity regulate CSCs in hypoxic microenvironment. Combined with clinical and pathological data, the correlation between the expression of HIF，SIRT1 and stemness associated genes and the prognosis of patient will be analysised. The results of this study will strengthen the understanding on the mechanism of liver cancer development, which provides a new target and theoretical basis for the hepatocellular carcinoma clinical therapy.

肿瘤干细胞是肿瘤发生发展的根源，缺氧微环境在肿瘤发生发展中普遍存在，肿瘤干细胞可塑性可能是其适应缺氧微环境的重要特征。我们前期发现缺氧条件有助于CD133+肝癌干细胞干性维持及CD133-肝癌细胞干性转化，SIRT1在上述过程中发挥重要作用。但缺氧微环境中SIRT1参与肝癌干细胞可塑性调控的机制尚不明确。本课题拟观察缺氧培养对CD133+和CD133-肝癌细胞SIRT1表达的影响及SIRT1对肝癌干细胞干性的影响；利用PCR芯片技术筛选SIRT1在肝癌干细胞可塑性调控中的关键分子靶点，通过过表达及shRNA技术调控缺氧诱导因子、SIRT1及干性调控分子表达，阐明缺氧微环境中SIRT1调控肝癌干细胞可塑性的分子机制；在肝癌临床标本中分析缺氧诱导因子、肝癌干细胞SIRT1及干性相关基因表达与患者预后的相关性。本课题有助于加深对肝癌发生发展机制的认识，为临床治疗研究提供新靶点和理论依据。

缺氧是肝癌恶劣微环境的重要特征，肝癌细胞干性的可塑性是肝癌细胞适应恶劣微环境的重要手段。然而，缺氧影响肝癌细胞可塑性的具体机制目前尚不够明确。我们研究发现肝癌细胞中缺氧诱导的低氧诱导因子Hif1α的累积与SIRT1的表达正相关。低氧条件下抑制SIRT1表达后，肝癌细胞增殖及集落形成能力明显下降。而在肝癌细胞中过表达SIRT1后，肝癌细胞适应缺氧恶劣微环境的能力显著增强，干性特征增加。肝癌干细胞中SIRT1表达也显著高于非干细胞。上述结果提示SIRT1可能参与了肝癌细胞和肝癌干细胞可塑性的调控。化疗抵抗是影响肝癌治疗效果的重要瓶颈，我们着重观察了低氧微环境对肝癌细胞化疗抵抗能力的影响及可能的机制。结果发现低氧条件下SIRT1介导的肝癌细胞干性增加可能是肝癌细胞在缺氧微环境中化疗抵抗能力增强的重要机制。相关研究结果为肝癌的临床治疗提供了新的理论依据。间充质干细胞（MSCs）在肿瘤发生发展中发挥重要作用，我们发现过表达SIRT1的MSCs可显著抑制肿瘤生长。在进一步的机制研究中发现高表达SIRT1的MSCs可募集CD8阳性T细胞至肿瘤局部，同时由于SIRT1抑制了MSCs中免疫抑制分子iNOS的表达，从而促进了抗肿瘤免疫应答的发生，抑制了肿瘤生长。上述结果为我们能够更好地将MSCs用于抗肿瘤免疫治疗提供了新思路。