- 學位論文
異位性表達微核糖核酸let-7h干擾斑馬魚心臟發育過程
Ectopic expression of let-7h disturbs heart development in zebrafish
摘要
先天性心臟病(CHD)是世界上最常見的新生兒缺陷，同時伴隨著異常的心臟發育過程以及形態。因此，更深入地了解潛在的致病調節機制對於精確診斷和開發潛在療法至關重要。微核糖核酸(MicroRNAs，miRNA)藉由轉錄後修飾進而調節基因表現以調控器官發育過程中不同的細胞訊息傳導路徑，其中也包括心臟發育。儘管已知某些miRNA會介導心臟發育過程，但許多其他miRNA的心臟相關功能仍然尚未明瞭。先前研究指出，Let-7 miRNA家族其中之一的miR-98在各種類型的心血管疾病皆發現到有表現失調的現象。然而，對於miR-98是否參與心臟發育過程知之甚少。Let-7 miRNA家族已知是早期胚胎發育過程中的關鍵因子，並且與心臟病相關。在斑馬魚中，let-7的過度表達已被證明會延遲胚胎發育並甚至導致死亡。我已經通過注射相似劑量的let-7h模擬物證實了先前報告的發現，而let-7h即是人類miR-98的斑馬魚同源基因。我們進一步發現到注入亞致死劑量的let-7h模擬物並不會導致明顯的早期胚胎缺陷，並且經過let-7h過量表達的胚胎可以在受精後存活長達 72 小時。在這種條件下，我們觀察到let-7h過量表達的斑馬魚胚胎的心室縮小了，但相反的，心房體積卻擴大了。而let-7h的過量表達也降低了各種心臟標誌基因的表現量。此外，通過共同注射幾種預測的let-7h目標基因的mRNA，包括wnt9b、bmp7a和fgf4，可以在某種程度上的挽救這種let-7h所引起的心臟發育缺陷。此外，我們發現let-7h誘導的心臟缺陷是經由lin28ab所調控，同時過量表達lin28ab也對let-7h所引起的心臟缺陷產生顯著的挽救效果。最後，為了闡明let-7h在心臟發生過程中的特定功能，我們建立並驗證了可以在特定時間點以及空間經由tamoxifen誘導的let-7h過量表達基因轉殖魚，以供後續在活體分析let-7h對心臟發育之影響。總結來說，適量的let-7h表現會通過調節許多下游心臟基因表現量維持正常心臟發育過程，同時此等調控機制是經由lin28ab的作用途徑進行調節。
並列摘要
Congenital heart diseases (CHDs) are the most common newborn defects with abnormal heart development and morphogenesis. It is crucial for accurate diagnosis and development of potential therapy to have a better understanding of the underlying pathogenic regulatory mechanisms. MicroRNAs (miRNAs) post-transcriptionally regulate gene expression to control different cellular pathways during organogenesis, including heart development. Although some miRNAs have been known to mediate cardiac development the heart-related functions of many other miRNAs remain largely elusive. The expression of miR-98, a member of Let-7 family, had been reported to be dysregulated in various types of cardiovascular diseases. However, little is known whether miR-98 is engaged in the heart developmental processes. The Let-7 miRNA family is critical factor during early embryogenesis and are correlated to heart diseases. In zebrafish, overexpression of let-7 had been shown to retard development and result in embryonic death. I have confirmed the reported effects by similar dosages of mimics for let-7h, a zebrafish ortholog of human miR-98. Sublethal dosages of let-7h mimics did not cause obvious early embryonic defects and the treated-embryos could survive up to 72 hours post fertilization. In this circumstance, I found the heart ventricle is reduced but the atrium is enlarged in size in let-7h mimic-injected zebrafish embryos compared to that of untreated controls. The overexpression of let-7h also reduced the gene expression levels of various cardiac marker genes. Moreover, this phenotype could be partially rescued by co-injecting mRNAs of several predicted let-7h target genes, including wnt9b, bmp7a, and fgf4. Furthermore, I further demonstrated that the let-7h-induced heart defects are lin28ab-dependent as evident by its significant rescue effect. Finally, to elucidate the specific function of let-7h during cardiogenesis, the temporally and spatially controlled tamoxifen-inducible let-7h overexpression transgenic fish were generated, validated and awaited for further investigation. Collectively, the proper expression of let-7h is necessary for heart development by modulating cardiac gene expression and is regulated through lin28ab-dependent pathway.