| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 97 === Liver performs vital role on metabolic functions. MicroRNAs (miRNAs) are small regulatory RNAs that open a new level of fine-tuning regulation of gene expression and have been shown involve in metabolic homeostasis. MiR-122 is a liver-specific miRNA and previous studies revealed that miR-122 might regulate lipid metabolism by in vivo antisense targeting in mouse system. However, due to the system limitation, whether lipid metabolic deficient disease is caused by miR-122 in liver and the lipid metabolic regulatory mechanism is still unclear. In this study, we aim to establish transgenic zebrafish with liver-specific overexpression of sense and antisense miR-122 to study the regulatory mechanism of miR-122 on lipid metabolism. First, we applied miRNA-based siRNA expression vectors and liver specific L-FABP (liver-type fatty acid binding protein) promoter to generate a miR-122 expression construct. And then screened the vector-integrated transgenic fish by red fluorescent in liver and detected the expression level of exogenous miR-122. According to Q-PCR data, we confirmed the transgenic zebrafish with mature miR-122 which was spliced from miRNA-based siRNA expression vector. Additionally, liver section stained by Oil-Red O showed lipid accumulation in liver could be leaded in 3-month old F2 overexpression of miR-122 transgenic zebrafish. And further analysis showed overexpression of miR122 could up-regulate lipid and cholesterol biosynthesis related enzyme and transcription factor, including ACC、FAS、SREBP1 and HMG-CoA Reductase. Our result showed overexpression of miR122 can induced lipid accumulation of liver through promotes lipid biosynthesis.
AMPK (AMP-activated protein Kinase) is a central metabolic sensor that acts to balance fatty-acid oxidation and fatty-acid synthesis. It had been proposed that AMPK might involve in the miR-122 regulated lipid metabolism. In this study, compared with wild type zebrafish, the expression level of miR-122 is negatively correlated with expression level of phorsphorylated-AMPK. Additionally, downstream factors regulated by AMPK (ACC, FAS) were also responding to expression level of miR-122. It is important to identify what gene can be targeted by miR-122 in AMPK pathway. And base on algorithm prediction of miR-122 target gene showed miR-122 might target calcium-binding protein anxa11a and CaM. Firstly, we confirmed the miR-122 can target anxa11a and CaM by luciferase assay. The result showed luciferase activity of anxa11a is inhibited about 40% and renilla/firefly ratio of CaM is inhibited about 20%. The importance for further examine whether miR-122 really target these two calcium-binding proteins and involve in AMPK pathway will be highly emphasized.
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