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MicroRNA-1 modulates angiogenesis through silencing the seryl-tRNA synthetase gene during zebrafish embryogenesis
Date Issued
2012
Date
2012
Author(s)
Ding, Yu-Yun
Abstract
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.
Subjects
MicroRNA-1
seryl-tRNA synthetase
angiogenesis
embryogenesis
Type
thesis
File(s)
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Name
ntu-101-R99b43022-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):9e46020bc761477ab9ea0aa76953e168