HOTAIR抑制肝细胞PTEN基因表达引起非酒精性脂肪肝的表观遗传学研究

Down-regulated expression of PTEN gene, a tumor suppressor,in hepatocytes triggers massive hepatomegaly, steatohepatitis and HCC with triglyceride accumulation, a phenotype similar to human non-alcohol fatty liver disease (NAFLD), However, the regulatory machanism of PTEN gene expression is unclear until now. Previous studies found that HOTAIR, a long non-coding RNA, is required for polycomb repressive complex 2 (PRC2) and lysine specific demethylase 1 (LSD1) to silence many tumor suppressor genes. Since overexpressed EZH2, an important constitute of PRC2, contributes to epigenetic inactivation of PTEN gene in B-cell acute lymphoblastic leukemia, and fatty heptocyte showed up-regulated HOTAIR and down-regulated PTEN mRNA in the preliminary experiment, it is speculated that HOTAIR may induce epigenetic suppression of PTEN gene expression in hepatocyte by recruiting PRC2 and LSD1 and it contribute NAFLD pathogenesis. As an aspect of long non-coding RNA regulating coding gene expression, this study will focus on identifying the epigenetic mechanism of HOTAIR regulating PTEN gene expression and it's roles on NAFLD pathogenesis by chromatin immunoprecipitation (ChiP) and RNA-ChiP on the basis of in vivo and in vitro experiments. The results of this study will contribute to afford the proof of clinical prevention and treatment of NAFLD.

肝脏肿瘤抑制基因PTEN表达降低是引起非酒精性脂肪肝（NAFLD）的关键靶点，但其表达调控机制尚未阐明。既往研究发现，长链非编码RNA HOTAIR通过招募多梳抑制复合物2（PRC2）与赖氨酸特异性去甲基化酶1（LSD1）抑制多个肿瘤抑制基因表达。鉴于B型急性淋巴细胞白血病PTEN基因表达降低与高水平EZH2（PRC2的一个关键成员）相关，因此结合预实验发现脂肪肝细胞存在PTEN低表达的同时伴HOTAIR表达增加，我们提出假设：HOTAIR通过招募PRC2与LSD1表观抑制肝细胞PTEN基因表达，促进NAFLD发生与演变。该研究将从长链非编码RNA调控编码基因表达的角度，在NAFLD动物与细胞层面，利用染色质免疫沉淀（ChiP）与RNA-ChiP技术，阐明HOTAIR表观调控肝细胞PTEN基因表达在NAFLD发病机制中的作用。研究结果将有助于揭示NAFLD的病理机制，为其临床防治提供依据。

非酒精性脂肪肝（NAFLD）与肝癌密切相关，肝脏脂肪变性是NAFLD早期特征之一。肿瘤抑制基因PTEN在NAFLD与肝癌患者或者啮齿动物模型肝组织表达降低，且肝特异性PTEN基因敲除小鼠脂肪性先后发生肝炎与肝癌比率增加。肝癌组织中长链非编码RNA HOTAIR表达增加，且HOTAIR表达水平与患者生存率负相关。在喉癌组织中，HOTAIR使PTEN基因启动子甲基化增加抑制PTEN基因表达。但是，目前NAFLD肝组织HOTAIR表达水平及与PTEN基因表达的关系尚不清楚。. 在研究中，我们利用不同种类、不同剂量的游离脂肪酸（FFA）构建了不同类型NAFLD细胞模型。在这些细胞模型中，我们发现在细胞内甘油三酯增加（即肝细胞脂肪变性）的基础上，PTEN表达降低，HOTAIR表达增加；siRNA干扰使HOTAIR表达降低后，肝细胞PTEN表达水平上升，且肝内甘油三酯水平降低；这提示FFA启动HOTAIR高表达，可能是引起肝细胞PTEN低表达及肝细胞脂肪变性的重要原因。进一步的研究发现，NFκBp65抑制剂CAPE抑制了FFA诱发的HOTAIR高表达，提示FFA通过 NFκBp65信号通路促进HOTAIR高表达。另外，我们发现FFA促进HOTAIR高表达与PTEN低表达不存在代谢记忆效应。. 结论：FFA通过NFκBp65信号通路促进HOTAIR高表达，抑制PTEN基因表达及诱导肝细胞脂肪变性。体内高水平FFA介导HOTAIR高表达可能是诱发NAFLD发病的关键环节之一。