低氧胁迫下团头鲂miR-462-731介导的Akt-FoxO通路在调控线粒体功能中的作用机制

One important reason for causing enormous economic losses in aquaculture is that a large amount of fish die due to hypoxia. Some regulation mechanisms about fish hypoxia adaptation have been reported, but it is limited about studies of miRNA function in fish hypoxia adaptation. Our previous studies showed that hypoxia induced the expression of teleost-specific miR-462 and miR-731 (located in the same gene cluster, namely miR-462-731). Function analysis revealed that the miRNA cluster was probably related with the Akt-FoxO pathway, and involved in regulation of mitochondrial function. Whether was miR-462-731 involved in mitochondrial function by the Akt-FoxO pathway to further affect oxygen utilization? Therefore, in the present study, effects of Megalobrama amblycephala miR-462-731 on mitochondrial biological functions will be investigated based on detection of mitochondrial oxidative phosphorylation, energy metabolism and morphological structure, etc. Function of the Akt-FoxO pathway mediated by miR-462-731 in mitochondrial responses will also be analyzed by miRNA target identification, gene overexpression and silencing etc. In addition, regulation mechanisms of the miRNA cluster in hypoxia response will be elucidated from the point of oxygen transportation and utilization using miR-462-731 knockout mutants as a model. Our results will not only contribute to systematically understand molecular regulatory mechanisms of fish hypoxia adaptation, but provide some theory evidences for breeding of hypoxia tolerant fish.

对水产业造成巨大损失的重要原因是缺氧导致的鱼类大量死亡，虽然人们对鱼类低氧适应机制进行了一些报道，但关于miRNAs的研究较少。在前期研究中我们发现miR-462-731簇为硬骨鱼类特有，受低氧诱导表达；功能分析初步显示该基因簇可能与Akt-FoxO通路有关，参与调节线粒体功能，那么miR-462-731是否通过Akt-FoxO通路调控线粒体功能进而参与氧的利用？本项目拟从线粒体氧化磷酸化、能量代谢及形态结构等方面研究团头鲂miR-462-731对线粒体功能的影响途径；通过靶标检测、基因过表达及沉默等实验分析Akt-FoxO通路在miR-462-731调节线粒体功能中的作用；在机体水平，利用该基因簇敲除模型从氧的运输和利用角度解析其在低氧调节中的分子机制。研究结果将有助于系统了解鱼类低氧适应的分子调控机制，还可为鱼类耐低氧品种的培育提供一定的理论基础。

低氧对水产业造成的巨大损失，而miRNAs作为“分子变阻器”可快速有效地调控基因的表达，在低氧应激中发挥重要作用，因此本项目在分子、细胞及机体水平分析了硬骨鱼类特有的miR-462-731簇的功能。结果显示miR-462和miR-731过表达可以提高ROS水平并降低线粒体膜电位，同时也会影响三羧酸循环及氧化应激关键酶的活性，说明miR-462-731可以影响线粒体功能。基于miR-462-731的MO敲降及CRISPR/Cas9敲除，研究发现敲除体miR-462-731-/-及miR-731-/-都具有较低的耐低氧能力；进一步分析发现该miRNA簇敲降不仅会影响血液血管母细胞发生，还会干扰红-髓系分化，导致红细胞缺失及血流障碍，髓细胞异常增多；基于双荧光素酶活性、Western blot及定量PCR进一步证实Pu.1与miR-462-731存在反馈调控。另外，miR-462-731敲降可导致FoxO通路显著富集，在细胞水平进一步研究证实低氧会导致ROS和线粒体膜电位发生变化，进而引发线粒体自噬；同时Akt的磷酸化水平及foxO1表达上升，而干扰foxO1会引起自噬相关基因的表达水平降低，推测Akt-FoxO1通路参与低氧介导的线粒体自噬。为了进一步研究FoxO功能，本项目利用CRISPR/Cas9技术构建了foxO1和foxO4斑马鱼敲除体，证实敲除foxO1会影响造血系统发育，导致红细胞数目减少；而敲除foxO4则不影响红细胞数目，但会降低机体对氧的需求进而表现出较强的耐低氧能力。本项目研究结果加深了对鱼类低氧适应分子机制的了解，也为鱼类耐低氧品种的培育提供了一定的数据支持。