microRNA-In300抑制其標的基因 homer-1 而影響斑馬魚胚胎肌肉發育之分子機制

Abstract

microRNA-In300為一種intronic microRNA （miRNA），其位於斑馬魚肌肉專一表現基因myf5 intron-1序列中，且在斑馬魚胚胎myf5 mRNA表現開始消退時期，miR-In300仍可持續在腦部以及肌肉組織中被偵測到其表現。因此，我們欲探討在myf5不表現時期，miR-In300是否仍具有經由調控標的基因表現，而影響斑馬魚胚胎肌肉發育之功能。首先，我們選擇myf5表現消退時期32 hpf之斑馬魚胚胎進行Labeled microRNA pull-down assay system，並配合microarray分析，進而獲得斑馬魚miR-In300可能標的基因之資料庫，並篩選出會表現於斑馬魚胚胎軀幹部肌肉組織基因homer-1、col1a-2、trmt-2a、six-1b以及dnajc-10進行研究。接著，我們將上述五個miR-In300可能標的基因之3’-untranslated translated region（3’UTR）構築入報導基因luciferase（luc）下游，進行Dual-luciferase reporter system分析。綜合於HEK-293T細胞株以及斑馬魚胚胎之實驗結果顯示，在給予外源性miR-In300狀況下，miR-In300僅可透過homer-1 3’UTR序列抑制報導基因luc的表現。且在斑馬魚胚胎不外加miR-In300，僅具有內生性miR-In300情況下，報導基因luc表現即可受到抑制。若注射miR-In300 mopholino （MO）到斑馬魚胚胎以抑制內生性mature miR-In300之生成，報導基因luc表現量則會顯著增加。顯示，在斑馬魚胚胎中，隨著miR-In300表現量的改變，含有homer-1 3’UTR之報導基因luc表現程度也會隨之受到調控。進一步，在全胚胎原位雜合反應（whole mount in situ hybridization，WISH）以及western blot實驗中，我們可藉由抑制內生性miR-In300表現，發現homer-1 mRNA以及蛋白質表現量均較野生型（wild-type）斑馬魚胚胎增加。另一方面，我們利用WISH實驗顯示，miR-In300以及homer-1均可表現於軀幹部肌肉之快肌中，顯示兩者的表現區域具有同位性。而在斑馬魚胚胎進行miR-In300以及homer-1 MO注射實驗，在分別過量表現miR-In300以及抑制內生性Homer-1蛋白質的轉譯時，會造成斑馬魚胚胎體軸彎曲以及尾巴變短之相似缺失。並且，在斑馬魚胚胎共同注射homer-1 mRNA與miR-In300狀況下，則可降低miR-In300過量表現造成之缺失比例。由以上實驗結果顯示，homer-1為miR-In300的一個標的基因，且miR-In300可經由homer-1 3’UTR序列而抑制Homer-1蛋白質的產生，並影響斑馬魚之肌肉發育。

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.