饥饿诱导黄鳝肌肉萎缩因子atrogin-1激活表达的分子机制研究——上游信号通路的鉴定及整合

Atrogin-1是肌肉萎缩的关键因子，Atrogin-1的缺失能使骨骼肌抵抗肌肉萎缩，而过度表达Atrogin-1则导致骨骼肌肌管的萎缩。鱼类中饥饿诱导的肌肉萎缩也与Atrogin-1相关，然而人们对鱼类中Atrogin-1的信号调控机制却缺乏足够的了解，与之相关的信号途径也不清楚。基于以上原因，我们以黄鳝为研究对象，采用从转录因子倒推信号通路的方法，首先对Atrogin-1基因的 5’上游序列进行启动子活性分析，鉴定其结合的转录因子，并对这些转录因子进行转录调控活性分析，随后通过生物信息学方法发掘与这些转录因子关联的信号通路，并在细胞和活体水平对这些信号通路进行抑制、激活研究，同时对信号通路中的关键分子进行过表达或敲减研究，从而鉴定出饥饿条件下调控Atrogin-1表达的信号通路，并研究饥饿诱导对这些通路的影响。从而揭示饥饿状态下鱼类通过Atrogin-1调控肌肉萎缩的分子机制。

The natural life cycle of many fish species often includes long periods of low winter temperatures and restricted feeding opportunities or prey availability that lead to a depletion of energy reserves and a reduction in growth rate. Long-term fasting will cause the loss of weight and release of amino acids from muscle fibers, leading to muscle atrophy. Atrogin-1 gene has been thought to involved in the starvation-induced muscle atrophy. Atrogin-1(also called Muscle Atrophy F-box (MAFbx), which has been identified as important enzymes in ubiquitin-mediated proteolysis and muscle atrophy. Overexpression of Atrogin-1 in myotubes produced atrophy, whereas mice deficient in Atrogin-1 were found to be resistant to atrophy. However, in fish, the knowledge concerning the molecular mechanisms that modulate muscle atrophy by Atrogin-1 has been scarce and limited, mainly focusing on the cloning and evaluation of expression patterns of this gene. Therefore, it is really necessary in fish to uncover the signal pathways regulating and activating the Atrogin-1 in starvation. .In this study, the regulation pathways about Atrogin-1 gene will be studied in rice-field eel (Monopterus albus), a commercially important fish species which hibernate and eat nothing for 3-4 months in winter, causing the muscle atrophy. Here the strategy of inferring the signal pathway from the transcription factors are used. First, the transcription factors binding to Atrogin-1 promoter will be identified by analysing the 5 'upstream sequence of the Atrogin-1 gene. Then the transcription regulation activity of these transcription factors will be analysed using Dual-Luciferase® Reporter (DLR™) Assay System. After that, the bioinformatics methods will be used to explore the signal pathways associated with these transcription factors. The agonist and inhibitor of these pathways, and the overexpress-plasmid or knockdown siRNA of the key molecules of these signal pathways will be used to evaluate the regulatory expression of atrogin-1 by these signal pathways in starvation conditions, in vitro and in vivo. Finally, we will analyse all these regulation pathways and try to integrate these relevant pathways so as to deeply understand the atrogin-1 regulation pathways.