臺灣大學: 分子與細胞生物學研究所蔡懷楨丁郁芸Ding, Yu-YunYu-YunDing2013-03-202018-07-062013-03-202018-07-062012http://ntur.lib.ntu.edu.tw//handle/246246/247348微型核醣核酸 microRNA-1 (miR-1) 為一段 22 nt 之非轉譯核醣核酸，是肌肉專一型微型核醣核酸 (muscle-specific miRNA)。雖然已知 miR-1 可專一地表現於心肌與骨骼肌，但參與在軀幹部骨骼肌的分子調控機制尚不清楚。我們利用 Labeled microRNA pull-down (LAMP) assay system 篩選，並配合 Microarray assay 分析，先找到在斑馬魚中 miR-1 會與 seryl-tRNA synthetase (sars) mRNA 的 3’ 端非轉譯區 (3’UTR) 結合。再利用 Luciferase reporter assay system 於斑馬魚胚胎及小鼠肌肉纖維母細胞株 C2C12 中證實了 miR-1 會透過 sars-3’UTR抑制報導基因的表現；並且由西方浸漬法證明當 miR-1 被抑制時斑馬魚內生性 Sars 蛋白質的表現量會增加。若於胚胎中降低 miR-1 表現以及過量表現 sars mRNA皆造成斑馬魚胚胎體節末端異常，並且軀幹部快肌的肌動蛋白絲 (actin filament) 彎曲、排列混亂，α-sarcomeric G-actin/F-actin 比率增加，顯示肌動蛋白絲傾向去聚合化，形成游離型肌動蛋白。進一步發現 Sars 蛋白質可表現於快肌肌小節的 Z-disc，並且由 GST pull-down assay 證明 Sars 與肌肉特異性的 α-橫紋肌肌動蛋白 (α-sarcomeric actin) 具有交互作用；此外，於胚胎中血管發育缺失，其體節間血管 (ISV) 生長遲緩，無法正常向上延伸至背部。經定量 RT-PCR (quantitative real-time PCR) 檢測，降低 miR-1 表現以及過量表現 sars 之胚胎，會造成血管內皮生長因子 (vegfa) mRNA 表現量降低，經由西方浸漬法證實 Vegfa 蛋白質表現也下降，並且於C2C12 細胞中證實過度表現 miR-1 則會抑制 Sars 蛋白質，並促進 Vegfa 蛋白質表現。以 Cytochalasin D 浸泡斑馬魚胚胎使其肌動蛋白絲去聚合化，發現體節間血管生長緩慢，並於 C2C12 細胞中偵測到 Vegfa 蛋白質表現量下降。綜合以上結果，我們證實 miR-1 藉著微量調降目標基因 sars 之蛋白質轉譯以維持軀幹部體節常態發育及快肌肌動蛋白絲的正常聚合化，進而促使 vegfa mRNA 及蛋白質適當表現以維繫血管正常生長之發育途徑模式。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.2894071 bytesapplication/pdfen-US微型核醣核酸血管新生胚胎發育MicroRNA-1seryl-tRNA synthetaseangiogenesisembryogenesisthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/247348/1/ntu-101-R99b43022-1.pdf