The Mechanism of microRNA -In300 regulates myogenesis through silencing the target gene homer-1 in zebrafish embryos

• Main Authors: CHUNG-CHING HSIAO, 蕭崇景
• Other Authors: 蔡懷禎
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/54512512363860331963

碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 100 === microRNA miR-In300 is an intronic miRNA which is located within the first intron (intron-1) of the zebrafish myogenic factor 5 (myf5) gene. When the transcription of myf5 is decreased in 32 hours post-fertilization (32 hpf), miR-In300 is still detectable in brain and muscle until 7 days post-fertilization. We suspeat whether the remnant miR-In300 is able to regulate gene expression and to affect zebrafish myogenesis. To address this issue, we choose the 32 hpf zebrafish embryos to perform Labeled microRNA pull-down assay system and microarray analysis to obtain candidates of miR-In300 target genes. And then, we constructed the 3’-untranslated translated region (3’ UTR) of trunk muscle-expressed candidates, including homer-1、col1a-2、trmt-2a、six-1b and dnajc-10, into a vector to create a fusion to the luciferase reporter gene (luc). Amoung the five candidates, by using of dual-luciferase reporter system, we found that only homer1 3’UTR could be bound by exogenous miR-In300, so that inhibiting luc expression in HEK-293T cell line and zebrafish embryo. The luc expression would be effectively by the endogenous miR-In300. Moreover, miR-In300 mopholino (MO) was co-injected to block the dicing of mature miR-In300, the luc expression increased significantly. We demostrated that the expression of homer-1 3’UTR-fused luc gene incersely correlated with the miR-In300 content. Furthermore, by using whole mount in situ hybridization (WISH) and western blot, the inhibition of endogenous miR-In300 resulted in increasing of homer-1 mRNA and Homer-1 protein expression. Next, we observed that homer-1 and miR-In300 co-localized in fast muscle of zebrafish embryo by WISH. Besides, we observed that overexpression of miR-In300 could result in curved axis and short tail of zebrafish embryo. When we knock down of homer-1 expression by injection with homer-1 MO, the phenotype was similar to the embryos overexpressing miR-In300. And the miR-In300-induced defect could be partially rescued by co-injection of homer-1 mRNA. Taken together, we concluded that homer-1 is one of miR-In300 target genes, and miR-In300 could inhibit the protein synthesis of Homer-1 by binding to homer-1 3’UTR to affect zebrafish myogenesis.