MicroRNA-802调控缺氧诱导肝细胞自噬的分子机制研究

Liver ischemic damage occurs in many clinically important scenarios, but the underlying mechanism remains unclear. Our preliminary study found that the expression of miR-802 and LC3-II, an indicator of autophagosome, were both increased in hepatocyte after 12h hypoxia. Meanwhile, over expression of miR-802 further enhanced the increased HIF-1α and LC3-II levels induced by hypoxia, and vice versa. Thus, we proposed that miR-802 regulated autophagy in hepatocytes induced by hypoxia through HIF-1α. With hepatocytes experienced hypoxia or hypoxia/reoxygenation, and mice undone hepatic ischemia or ischemia/reperfusion, this work was designed to demonstrate: 1) autophagic flux was impaired by hypoxia, which was positively related to the expression of miR-802; 2) miR-802 regulated hepatocyte autophagy induced by hypoxia via HIF-1α; 3) and to clarify the downstream targets of miR-802 regulating HIF-1α expression under hypoxia. Thus, to provide the potential targets pharmacologically in treatment of liver ischemic damage.

肝组织缺氧导致肝细胞损伤的机制仍不明确。我们预实验发现：肝细胞在缺氧12h后miR-802和自噬标志物LC3-II的表达均显著上调且呈正相关；过表达miR-802可进一步增加低氧诱导的HIF-1α和LC3-II表达水平，反之亦然。我们假设：miR-802经调节HIF-1α表达参与缺氧诱导的肝细胞自噬调节。从以下3方面验证：1)利用肝细胞及小鼠肝脏缺氧复氧模型证实肝细胞在缺氧时自噬流发生改变，miR-802表达与自噬状态相关；2)利用肝细胞及小鼠肝脏缺氧模型，证明miR-802通过调节缺氧诱导的肝细胞 HIF-1α的表达，参与肝细胞自噬调节；3)预测和筛选miR-802调控HIF-1α的靶基因，研究miR-802调节HIF-1α表达的分子机制。研究结果将为潜在的肝脏缺血再灌注损伤治疗药物和靶点提供理论基础。

肝组织缺氧在多种肝脏外科手术及病理情况下常见，但其损伤肝细胞机制不明。本项目明确了miR-802在肝细胞缺氧损伤过程的作用，并阐明miR-802对肝细胞缺氧诱导的自噬的调节机制。经研究我们发现：肝细胞缺氧后自噬标志物LC3-II的表达显著增加，肝细胞内自噬体数量显著增加，提示肝细胞在缺血缺氧后自噬流发生改变；而肝细胞在缺氧后miR-802的表达显著上调，过表达miR-802可进一步增加缺氧诱导的肝细胞内LC3-II及Beclin的高表达，同时增加肝细胞内自噬体数量，这提示miR-802 在缺氧诱导肝细胞自噬中具有调节作用。HIF-1α减轻缺氧损伤的机制已被证实与其调节缺氧诱导的自噬相关。我们的研究发现，肝细胞在经历缺氧后，HIF-1α表达量明显增加，上调 miR-802可进一步增加 HIF-1α的表达，而抑制miR-802表达后，HIF-1α的表达同样受到了抑制，这提示miR-802可调节缺氧诱导的HIF-1α表达改变。此外，HIF-1α的抑制剂YC-1预处理可显著减少缺氧诱导的LC3-II高表达。这说明miR-802可通过调节HIF-1α表达进而调节缺氧诱导的自噬改变。利用生物信息学手段，我们初步筛选出436个参与调控HIF-1α并与miR-802存在直接作用的靶基因，再利用基因沉默技术分别抑制肝细胞内靶基因的表达，检测其下游HIF-1α的表达改变情况，共筛选到6个靶基因参与调控HIF-1α，随后我们利用荧光素酶报告基因系统及western blot验证进一步明确TCF7L2与miR-802存在直接上下游关系。由此我们推测：miR-802经调节TCF7L2进而调节HIF-1α参与的缺氧诱导的肝细胞自噬。