microRNA-206 藉由抑制其標的基因調控斑馬魚體節邊界的生成

Abstract

MicroRNA-206 (miR-206)為骨骼肌專一表現之微型核醣核酸，調控肌肉纖維母細胞的增生與分化。藉由Labeled miRNA pull-down assay (LAMP)在斑馬魚胚胎發育早期骨骼肌增生與分化的過渡時期之16 hpf，篩選出miR-206的可能標的基因之一為reticulon (rtn)。在in vitro以及in vivo系統，發現 miR-206可經由rtn之三端非轉譯區抑制報導基因的表現，且於基因轉錄層次證實抑制miR-206會延緩rtn mRNA的降解使其表現量明顯提升。藉由全胚胎原位雜交實驗，抑制內生性miR-206以及過量表現rtn mRNA則可初步觀察到相似缺失的外表型。雖然已證實miR-206在mRNA層次上調控rtn mRNA，但其蛋白質層次上的調控以及在斑馬魚肌肉發育上的功能仍不清楚。因此首先藉由西方浸漬實驗，測定抑制內生性miR-206之胚胎，內生性Rtn蛋白質表現量具有顯著上升趨勢，證實內生性miR-206可在蛋白質層次調控內生性Rtn的表現。為了解miR-206調控rtn在斑馬魚肌肉發育上的功能，同時注射miR-206-MO以及三種rtn 亞型之mRNA至斑馬魚胚胎一細胞期，藉由phalloidin標定肌動蛋白絲，分析發育至48 hpf的胚胎肌肉細部結構，發現抑制內生性miR-206以及過量表現rtn皆會產生肌動蛋白絲橫跨體節的缺失，並且此缺失也會產生在rtn各亞型mRNA單獨過量表現之胚胎內。同時，我們也觀察到在肌動蛋白絲橫跨的部分，產生體節邊界 (somite boundary)結構的缺失，進一步統計其缺失比例，發現隨著發育時間的增加，此種體節邊界的缺失並不會在發育後期再被修復。若注射miR-206-MO以及rtn mRNA之細胞轉移至WT 的胚胎，發現轉移細胞在胚胎內發育成肌肉細胞，且轉移細胞之間的體節邊界會異常生長，使整體體節邊界結構產生缺失。總合上述研究結果顯示，rtn為miR-206的標的基因，並且miR-206會藉由抑制rtn的過度表現來調控斑馬魚胚胎發育初期之體節邊界的正常生成。

microRNA-206 is a muscle-specific miRNA that regulate myoblast proliferation and differentiation. According to Labeled miRNA pull-down assay (LAMP), the candidate gene rtn was screened from 16-hpf zebrafish embryo which at the transition state between proliferation and differentiation of muscle cell. Through in vitro and in vivo system, we found out that miR-206 was able to repress reporter activity and up-regulated the expression of post-transcription level through delayed the degradation of rtn mRNA. Using whole-mount in situ hybridization (WISH), the similar defect of morphology was observed by repression of endogenous miR-206 and over-expression of rtn mRNA. Although the regulation of rtn mRNA by miR-206 was confirmed, the effect of protein level and the function in zebrafish muscle development has not been study yet. First, we used western blot and found out that knockdown of endogenous miR-206 resulted in the increase of endogenous rtn protein expression. We proved that miR-206 was able to down-regulate the protein level of rtn. To verify the function of miR-206 in zebrafish muscle development through rtn, rtn mRNA and miR-206-MO were injected into one-cell stage embryos separately. According to phalloidin labeling, knockdown of miR-206 and over-expression rtn mRNA caused actin filament crossing-over between somites. The same defect was also observed by over-expression particular rtn isoform. Then, we found out that the defect of actin filament crossing-over between somite accompanied with defective somite boundary. Further quantified the ratio of defect, we discovered that the defective somite boundary were not regenerate through developmental time. Furthermore, we transplanted rtn mRNA injected cell into WT embryos. The transplanted cell developed to muscle and caused somite boundary growth abnormally. Taken together, these results suggested that rtn is one of target gene of miR-206. Through silencing rtn, miR-206 regulate somite and somite boundary formation in zebrafish embryo early development.