探討微型核醣核酸 miR-277 在果蠅細胞凋亡所扮演的角色

The Roles of miR-277 During Apoptosis in Drosophila

10.6342/NTU.2014.10983

陳昭儀

黑腹果蠅 ； 微型核醣核酸miR-277 ； 細胞凋亡 ； 支鏈胺基酸代謝 ； X光照射 ； Drosophila melanogaster ； miR-277 ； apoptosis ； BCAA catabolism ； X-ray

臺灣大學分子與細胞生物學研究所學位論文

2014年

碩士

陳俊宏

英文

在動物發育過程中，細胞死亡、生長和存活是彼此互相協調的。許多控制細胞生長和死亡的基因已經被確認，然而這些效應是如何連接在一起仍是未知的。在本篇研究中，藉由基因篩選，我們發現一個微型核醣核酸，miR-277，能夠抑制 hid誘導的細胞凋亡。異位表現miR-277能抑制在眼睛發育過程並且延緩在果蠅翅膀發展中產生的細胞死亡。我們預測了四個促進細胞凋亡基因為miR-277之標靶，包含 hid， dronc， dark和drice。 經由螢光素酶檢測和生物活體分析，我們證明過量表現miR-277能藉由靶擊 hid 和 dronc 進而專一性地抑制細胞凋亡。為了進一步確認這項發現，我們以TALEN的技術產生miR-277突變的果蠅品系。在異結合型或是同結合型miR-277突變的基因背景下，hid誘導的果蠅眼睛因凋亡而產生消融現象有被增強，進一步證實hid是miR-277的標靶基因。在電離輻射誘導的DNA損傷中，hid的mRNA增幅量在同結合型miR-277突變果蠅的三齡幼蟲中時程上有被延長的現象。hid下游的caspase 3的訊號因此增加，這意味著在照射電離輻射後，同結合型miR-277突變會促進細胞死亡。對於調節miR-277表現的上游訊號，我們證明過量表現yki 或是p53 並不會影響miR-277的表現，意味著miR-277很可能不是Hippo訊號路徑和p53的直接下游基因。前人研究證明miR-277在調控支鏈胺基酸 (branched chain amino acid, BCAA) 代謝路徑的開關中扮演重要角色。從生物資訊的搜索發現，在果蠅中幾乎所有參與支鏈胺基酸代謝的酵素都是miR-277可能的標靶基因。在本篇研究中，我們顯示在飢餓的情況下，與控制組相比，同結合型 miR-277 突變的果蠅增加平均存活率，這意味著miR-277 可能透過控制BCAA代謝路徑來參與生存的調節。在本篇研究中，我們也顯示以miRNA或是RNA干擾的方式下調在BCAA代謝中最主要的三個酵素或是CG5599 (其中一個主要的酵素) 能部份抑制 hid 但是不能抑制 rpr 和 grim 誘導的果蠅眼睛消融現象，這意味著 BCAA代謝路徑可能參與hid誘導的死亡。綜合上述，miR-277藉由調控hid、dronc和BCAA代謝路徑中的酵素進而在細胞死亡和存活中扮演重要的角色。這暗示著在對能量平衡的反應中，miR-277可能是連接細胞死亡與存活的橋樑。

Cell death, cell growth and survival are coordinated processes during development in animals. Although many genes that control cell growth and apoptosis have been identified, how these effectors are link the two cellular processes together remains unknown. In this study, we examined the roles of the microRNA, miR-277, in hid-induced cell death and in BCAA catabolism. Using a genetic screen, miR-277 was found to be able to regulate hid-induced cell death in D. melanogaster. Ectopic expression of miR-277 inhibits cell death during eye development and delays apoptosis during wing maturation. Overexpression of miR-277 in S2 cells and Drosophila eyes were found to inhibit apoptosis by targeting the putative apoptotic genes, hid and dronc, while TALEN-generated heterozygous and homozygous miR-277 mutants enhances hid-induced eye ablation as identified through a luciferase activity assay. Cell death is exacerbated when miR-277 homozygous mutant is exposed to ionization radiation (IR), as marked by prolonged hid transcript levels in third instar larvae and caspase 3 signals in wing imaginal discs. qPCR results indicate that yorkie and p53 are not upstream of miR-277 when the dominant active form of yorkie and p53 was overexpressed, suggesting miR-277 may not be involved in the Hippo or p53 pathway. In addition to the regulatory role of apoptosis, miR-277 has been identified that function importantly in activating branched chain amino acid (BCAA) catabolism. Bioinformatics analysis revealed that almost all enzymes within the BCAA catabolic pathway are predicted targets of miR-277 in Drosophila. In this study, we show that miR-277 homozygous mutant flies increase mean survival rate in both male and female compared to wild type under starvation, suggesting that miR-277 may participate importantly in the regulation of survival through controlling BCAA catabolism. Additionally, the knockdown of three major enzymes or CG5599 within BCAA catabolism using RNA interference partially inhibited hid but not rpr and grim-induced eye ablation, suggesting BCAA catabolism may involve in hid-induced death. In summary, miR-277 functions as an essential role in cell death and survival through regulating hid, dronc and enzymes within BCAA catabolism. Our results imply that miR-277 may function to link cell death and survival in response to energy homeostasis.