Transgenic liver-specific overexpression of miR-122 could promote lipid accumulation in zebrafish

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 previou...

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Main Authors: Ya-Wen Lee, 李雅雯
Other Authors: Jen-Leih Wu
Format: Others
Language:en_US
Published: 2009
Online Access:http://ndltd.ncl.edu.tw/handle/38177004240377405824
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spelling ndltd-TW-097NTOU51110172016-04-27T04:11:49Z http://ndltd.ncl.edu.tw/handle/38177004240377405824 Transgenic liver-specific overexpression of miR-122 could promote lipid accumulation in zebrafish 斑馬魚肝臟專一過量表現miR-122促進肝臟的脂肪累積 Ya-Wen Lee 李雅雯 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. Jen-Leih Wu 吳金洌 2009 學位論文 ; thesis 43 en_US
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language en_US
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description 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.
author2 Jen-Leih Wu
author_facet Jen-Leih Wu
Ya-Wen Lee
李雅雯
author Ya-Wen Lee
李雅雯
spellingShingle Ya-Wen Lee
李雅雯
Transgenic liver-specific overexpression of miR-122 could promote lipid accumulation in zebrafish
author_sort Ya-Wen Lee
title Transgenic liver-specific overexpression of miR-122 could promote lipid accumulation in zebrafish
title_short Transgenic liver-specific overexpression of miR-122 could promote lipid accumulation in zebrafish
title_full Transgenic liver-specific overexpression of miR-122 could promote lipid accumulation in zebrafish
title_fullStr Transgenic liver-specific overexpression of miR-122 could promote lipid accumulation in zebrafish
title_full_unstemmed Transgenic liver-specific overexpression of miR-122 could promote lipid accumulation in zebrafish
title_sort transgenic liver-specific overexpression of mir-122 could promote lipid accumulation in zebrafish
publishDate 2009
url http://ndltd.ncl.edu.tw/handle/38177004240377405824
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