microRNA-206藉由抑制其新型專一標的基因調控斑馬魚胚胎之肌肉發育

MicroRNA-206 Controls Muscle Development in Zebrafish Embryos through Silencing Novel MicroRNA-206-Specific Target Genes

張文彥(Wen-Yen Chang)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10096

摘要

微型核醣核酸microRNAs (miRNAs)為小片段非轉譯之單股核醣核酸，其藉由保守性種子序列(conserved seed sequence)與標的基因mRNA之3’端非轉譯區(3’-untranslated region，3’UTR)以非完全互補的方式結合，於轉錄後層次(post-transcriptional level)調控標的基因的表現。MicroRNA-206(miR-206)為肌肉專一微型核醣核酸，雖然研究已知其專一地表達於骨骼肌調控肌肉纖維母細胞的細胞增生和分化，但miR-206於早期胚胎肌肉發育參與在軀幹部骨骼肌的分子機制尚不清楚。我們選擇斑馬魚胚胎發育早期骨骼肌細胞增生和細胞分化的過渡時期16 hpf進行Labeled miRNA pull-down assay system，並配合microarray進行比對分析進而獲得斑馬魚miR-206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cited3、gadd45ab、rtn4a和znf142進行研究。我們利用Dual-lucuferase assay於HEK293T、C2C12和斑馬魚胚胎中證實in vitro和in vivo的系統中，miR-206均可透過cited3及rtn4a之3’UTR序列抑制報導基因的表現。並而藉由WISH和冷凍切片，我們瞭解在16 hpf，miR-206、cited3和rtn4a三者會共同表現於軀幹部快肌的肌肉組織中。進一步探討內生性miR-206和cited3及rtn4a mRNA表現量的相關性，則發現若注射miR-206 morpholino (MO)到斑馬魚胚胎抑制內生性mature miR-206生成，cited3 mRNA和rtn4a mRNA表現量分別於36和48 hpf有延緩降解而表現明顯增加的現象。另一方面，當抑制內生性miR-206與cited3或rtn4a mRNA過量表現的情況下皆會影響斑馬魚胚胎肌肉發育異常，尤其是抑制內生性miR-206和過量表現rtn4a mRNA均會造成胚胎體軸延長、胚體些微上拱彎曲和尾端上翹的異常外表型，藉由IF肌肉細部結構功能性的探討則發現，miR-206缺失與cited3或rtn4a mRNA過量表現三者確實會造成斑馬魚快肌之肌動蛋白和肌凝蛋白排列混亂的缺失。此外，WISH證實myod於斑馬魚胚胎會調控miR-206的表現。綜合以上實驗結果，顯示myod可活化miR-206，而miR-206可專一地透過cited3和rtn4a之3’UTR序列上的結合位抑制下游標的基因cited3和rtn4a mRNA表現影響快肌之肌動蛋白和肌凝蛋白其排列，進而影響斑馬魚軀幹部快肌的生成與維持。

關鍵字

miR-206 ； miR-206專一標的基因 ； cited3 ； rtn4a ； 肌肉發育

並列摘要

MicroRNAs (miRNAs) are short, endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level by targeting the 3’-untranslated region (3’UTR) of mRNAs through a conserved seed sequence. MiR-206 is a muscle-specific miRNA and is highly expressed in skeletal muscle. Although miR-206 plays a key regulatory role in myoblast proliferation and differentiation, the detailed molecular regulatory mechanism of miR-206 in early myogenesis is still unknown. Particularly, miR-206 and miR-1 share an identical seed sequence, making it difficult to differentiate their target genes and functions. To address this issue, we employed our labeled miRNA pull-down assay (LAMP) (Hsu et al., 2009) to screen the target mRNAs specific for miR-206 from cell extracts of 16-hpf zebrafish embryos. After microarray analysis, we selected 117 high-scoring genes for further study. Using whole-mount in situ hybridization, we observed that the expression patterns of cbp/p300-interacting transactivator, Glu/Asp-rich carboxy-terminal domain 3 (cited3), and reticulon 4a (rtn4a) were co-localized with miR-206 in the fast muscle of trunk somites. Then, we demonstrated that miR-206 was able to greatly repress luciferase activity through binding to the 3’UTRs of cited3 and rtn4a mRNAs in HEK293T cells, C2C12 cells, and even zebrafish embryos. On the other hand, knockdown of endogenous miR-206 by injecting miR-206 morpholino (MO) resulted in the increase of cited3 and rtn4a transcripts. Furthermore, injection of excessive cited3 mRNA and rtn4a mRNA resulted in shorter and longer of trunk somites, respectively. Interestingly, when knockdown of endogenous miR-206, the defect was similar to the embryos injected with rtn4a mRNA, suggesting miR-206 may play a role on normal development of somites. In addition, loss-of-miR-206, overexpression-of-cited3 and overexpression-of-rtn4a disrupted the organization of actin and myosin in the fast muscle. Importantly, the regulation of miR-206 to cited3 and rtn4a is myod mRNA expression-dependent. Evidence thus far accumulated leads us to hypothesize that miR-206 controls muscle development in zebrafish embryos through silencing cited3 and rtn4a, two novel miR-206-specific target genes unrelated to miR-1.

並列關鍵字

miR-206 ； microRNA-206-Specific Target Genes ； cited3 ； rtn4a ； Muscle Development

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