指導教授：吳金洌臺灣大學：漁業科學研究所張巧宜Chang, Chiao-YiChiao-YiChang2014-11-302018-07-062014-11-302018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/263810微核糖核酸為短序列、不具蛋白編碼，且可調控轉錄後基因表現之RNA分子，其中肌肉專一性表現之微核糖核酸在肌肉發育與成長上扮演重要角色。已知miR-1和miR-133的表現量下降會導致骨骼肌肥大，但其如何調控下游訊息路徑尚未清楚。已知肌肉質量受肌細胞內蛋白質的合成與降解兩者間之平衡所調節，主要由IGF-1/-Akt/mTOR訊息傳遞路徑的活化所調控。AP-1轉錄因子所屬JunB轉錄因子可有效維持肌肉質量並且快速促進肌肉肥大，可透過抑制下游兩個肌肉萎縮基因Atrogin-1和MuRF-1的表現而不影響類胰島素生長因子的作用和衛星細胞的活化。在本研究中，以生物資訊軟體分析miR-133可能調控的目標基因，不論在人類或斑馬魚均發現JunB轉錄因子的3端非編碼區有一結合區域，利用斑馬魚 (Danio rerio)為模式動物，顯微注射miRIDIAN microRNA-133 mimic 或inhibitor 發現斑馬魚肌肉會出現萎縮和肥大現象。利用冷光酵素檢測分析法證實miR-133會透過結合JunB 訊息核糖核酸而抑制基因之表現。再以西方點墨法之結果證實，miR-133可控制下游轉錄因子JunB蛋白質之表現。透過JunB拯救實驗顯示miR-133所造成的肌肉萎縮可透過JunB 訊息核糖核酸之添加來回復。本研究證實miR-133透過調控JunB影響骨骼肌肉之肥大。未來在肌肉萎縮疾病也許可提供一個新的疾病治療方法。MicroRNAs (miRNAs) are short non-coding RNA molecules that post-transcriptionally inhibit gene expression. Muscle specific miRNAs, myomiRs, have been shown to play an essential role in regulating muscle development and growth. A previous study showed that downregulated miR-1 and miR-133 expression results in skeletal muscle hypertrophy after functional overload. However, the muscle hypertrophy regulatory roles of miRNAs are still unclear. It has been known that muscle mass is regulated by the balance between the synthesis and degradation of proteins in myofibers. Intracellular protein synthesis is mainly regulated by the activation of IGF-1/-Akt/-mammalian target of rapamycin (mTOR) pathway. The AP-1 transcription factor JunB is another efficient factor that induces rapid adult muscle hypertrophy without affecting satellite cell activation and Akt/mTOR pathway by inhibiting the downstream atrophy-related genes, Atrogin-1 and MuRF-1 expression. Our research utilized a bioinformatic software to predict the miRNA binding sites of target genes and has identified a putative miR-133 binding site on the 3’UTR of JunB in humans and zebrafish. Here we identify JunB as one of the direct regulatory targets of miR-133 by a luciferase assay, real-time PCR, and western blot in vitro. The JunB mRNA rescue assay successfully reversed the miR-133-induced muscle atrophy in zebrafish larvae. miR-133 modulates skeletal muscle hypertrophy by regulatiing JunB translation in zebrafish. The novel mechanism may pave the way for new therapies for disease-induced muscle atrophy.Table of contents 誌謝 III 摘要 IV Abstract V List of figures IX List of tables X Chapter 1: Introduction 1 1.1 Background and significance 1 1.2 MicroRNA biogenesis and function. 5 1.3 Muscle specific microRNAs in skeletal muscle development and growth 5 1.4 Signaling pathways involve in skeletal muscle hypertrophy and atrophy 7 1.5 Transcription factor JunB in physiology function and skeletal muscle hypertrophy 9 Chapter 2: Materials and Methods 10 2.1 Fish strains 10 2.2 Microinjection 10 2.3 RNA isolation, stem loop RT-PCR and real-time PCR analysis 11 2.4 Muscle histological analysis 12 2.5 Luciferase assay plasmids construction 12 2.6 Cell culture and transfection 13 2.7 Dual-luciferase reporter assays 14 2.8 Western Blot 15 Chapter 3: Results 17 3.1 miRIDIAN miR-133a mimic and inhibitor regulate miR-133 expression profiles during zebrafish embryo development 17 3.2 Modulation of endogenous miR-133 affects skeletal muscle hypertrophy and atrophy in zebrafish larvae 18 3.3 miR-133 directly targets JunB 19 3.4 miR-133 suppresses JunB at the level of transcriptional in C2C12 cell line 20 3.5 Supplement of JunB mRNA could recover the phenomenon of muscle atrophy induced by miR-133 21 Chapter 4: Discussions 22 References 25   List of figures Fig 1. Time expression profiling of miR-133 during zebrafish embryonic development 30 Fig 2. Time expression profiling of miR-133 is delimited by injecting miR-133a mimic and inhibitor into zebrafish embryos 31 Fig 3. miR-133 regulates skeletal muscle atrophy and hypertrophy in zebrafish larvae. 32 Fig 4. Constructs designed for psiCHECK-2 vector to perform Dual Luciferase Assay system (4A). The sequence of two miR-133 binding site mutation (4B) 34 Fig 5. Graphical representations of JunB luciferase activity under different concentrations of miR-133a mimic transfection in HEK293T cell line 35 Fig 6. Graphical representations of JunB luciferase activity under 200nM of mimic negative control, miR-133a mimic and miR-133 inhibitor transfection in HEK293T cell line 36 Fig 7. miR-133 regulates JunB and downstream atrophy related gene expression in C2C12 cell line (7A, 7B) 37 Fig 8. miR-133 regulates JunB protein expression in C2C12 cell line. 38 Fig 9. Supplement of JunB mRNA could prevent miR-133 from inducing muscle atrophy. 39   List of tables Table 1. Tabular representation of cross-sectional area of muscle fibers in 6 days wild type and 6 days post miR-133a mimic and inhibitor microinjected zebrafish larvae. 40 Table 2.Primer list 413029342 bytesapplication/pdf論文使用權限：不同意授權斑馬魚骨骼肌肥大微核糖核酸-133JunBAtrogin-1MuRF-1[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/263810/1/ntu-103-R00b45012-1.pdf