碩士 / 國立臺灣大學 / 分子與細胞生物學研究所 / 100 / MicroRNA-1 (miR-1), a 22-nucleotide, endogenous non-coding RNA, is a muscle-specific miRNA significantly expressed in cardiac and skeletal muscle. However, the detailed molecular regulatory mechanism of miR-1 in the skeletal muscle is still unknown. Therefore, using both miRNA pull-down assay and microarray analysis, we screened putative mRNA targets of miR-1 from whole-cell extracts of zebrafish embryos at 48-hpf, and seryl-tRNA synthetase (sars) was obtained. When miR-1 bound the 3’-untranslated region (3’UTR) of sars mRNA (sars-3’UTR) in zebrafish embryos, luciferase activity was repressed, unless the miR-1 binding site of sars-3’UTR was mutated. As validated by western blot analysis, knockdown of miR-1 increased the protein level of endogenous Sars. Both overexpression of sars mRNA and knockdown of miR-1 in zebrafish embryos caused the twisting tail of embryo body, the disruption of fast-twitch muscle actin organization and the winding of actin filaments. We found that the ratio of α-sarcomeric G-actin / F-actin was also increased, indicating that α-sarcomeric actin filements were depolymerized. Furthermore, Sars was interacted with α-sarcomeric actin and located at Z-disc. Besides, we observed the disorganized vessels and abnormal delay of established intersegmental vessels in embryos which either gained sars or lost miR-1. Moreover, vegfa mRNA and its protein level were all decreased. When we disrupted the actin filaments in embryos which was treated with Cytochalasin D, a depolymerized actin filaments drug, the intersegmental vessels was disorganized and abnormal delay. And the Vegfa protein of C2C12 cells that were treated with Cytochalasin D was decreased. These findings led to the conclusion that miR-1 contributes to normal angiogenesis in zebrafish embryos by reducing the amount of Sars protein to maintain actin organization of trunk fast-twitch fibrils that keeps the appropriate expression of vegfa mRNA and protein.
| Identifer | oai:union.ndltd.org:TW/100NTU05061065 |
| Date | January 2012 |
| Creators | Yu-Yun Ding, 丁郁芸 |
| Contributors | Huai-Jen Tsai, 蔡懷楨 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 82 |
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