探索日夜節律微型核醣核酸在哺乳類動物醣類代謝的角色

Abstract

為了適應外在環境的變化，生物體內存在著一個內化的生理時鐘，精細調控生物體一天的生理變化，使生物更能適應外在環境變化。哺乳類動物的中央節律時鐘位於上視交叉神經核，驅動生物體的晝夜節律、整合生理與行為模式，如睡眠周期。中央節律時鐘可以自發性的產生，然而環境線索，如光線、溫度等，也可以重置中央節律時鐘。大部分哺乳動物的周邊組織如肝臟、胰臟等器官，亦存在著節律時鐘，其可透過血液訊號，如荷爾蒙，達成與中央節律時鐘同步的生理狀態。節律時鐘的失調會影響生物體生理行為並產生代謝疾病甚至是癌症的產生，所以節律時鐘的調控對於生物體的健康是相當重要。 從分子的角度來探討，晝夜節律的現象是源自細胞中存在一個自動的轉錄與後轉譯回饋路徑，許多轉錄因子參與其中的核心部分，其表現量呈現晝夜節律變化且下游的基因表現也呈現節律變化。其中一個下游基因產物稱做Nocturnin，具有去腺嘧啶酶(deadenylase)的活性，藉由去除標的mRNA尾巴上的聚腺嘧啶(poly (A) tail)影響下游基因的表現。許多文獻指Nocturnin會影響生物體內葡萄糖與脂肪的代謝。 微型核醣核酸(miRNA)，一群大小約22個鹼基且無法被轉譯成蛋白質的核糖核酸。微型核醣核酸扮演調控基因與蛋白質表現的重要角色，並且近年來亦被發現參與在晝夜節律的調控。釐清這些詳細的分子機制有助於我們獲得更多的訊息，期望對於預防或治療代謝相關疾病能有更多突破性的發展。 此篇論文中有兩個目的：一個是想要探討Nocturnin對於下游基因的調控機制。Nocturnin調控標的mRNA尾巴上的聚腺嘧啶(poly(A) tail)的機制尚未明瞭。Nocturnin與酵母菌轉錄因子yCCR4很相像，且yCCR4可以藉由結合RNA沉默複合體(RISC)去調控下游基因表現，所以我們假設Nocturnin同樣可藉由結合RNA沉默複合體(RISC)去調控下游基因表現，但在我們共同免疫沉澱的實驗結果裡，Nocturnin與AGO2是無法結合形成複合物。另一個主要的目的是希望在老鼠的肝臟中找到具有晝夜節律變化的微型核醣核酸，並且進一步想了解此微型核醣核酸於肝臟中所扮演的調控角色為何。首先我們利用了微型核醣核酸微陣列的方法分析了約700個微型核醣核酸，找到日夜變化大於一點五倍變化的微型核醣核酸，並進一步利用及時定量聚合酶連鎖反應做確認。在此我們找到了miR-494，其晚上的表現比白天的表現來的高。後續並找到miR-494的標的基因「PCK1、G6PC、G6PC2」亦呈現日夜節律的變化。這些標的基因分別參與在肝臟與胰臟的醣類代謝中。實驗結果發現，不同的醣類濃度會影響miR-494以及PCK1、G6PC、G6PC2的表現。穩定表現miR-494的細胞株，細胞產生葡萄糖的濃度相較於對照組來得低，以及降低老鼠肝臟中CREB的表現量會使miR-494表現量增加。我們發現miR-494可能藉由影響PCK1、G6PC、G6PC2的表現進而調控體內的醣類代謝。未來可以在老鼠體內加以應證具有日夜節律變化的miR-494對於糖類代謝的影響

Circadian rhythm is an endogenously driven cycle with a roughly 24-hour period in various biochemical, physiological, and behavioral processes. The rhythm is generated by a transcriptional feedback loop existing in cells. There are two major types of circadian clocks, one is the “central clock” that is expressed within the pacemaker neurons in the suprachiasmatic nucleus (SCN), and the other is the “peripheral clock” that exists in almost all other tissue types. Central clock provides “standard time” to maintain proper phase alignment of peripheral tissue clocks. Nocturnin (Noc), a proteinwith the circadian pattern, is abundant in liver and has deadenylase activity. Moreover, there are studies indicating thatNoc KO mice have abnormal glucose regulations and insulin sensitivity etc. Hence,Noc is thought to link circadian clock and metabolism by affectingtarget mRNA stability through post-transcriptional regulations. MicroRNAs (miRNAs) are a group of about 22 nucleotides-long non-coding small RNA molecules that play significant roles in regulating gene and protein expressions. Recently, many studies suggested that miRNA might also be important regulators of circadian rhythm. In this study, we have two goals. One is to figure out the mechanism Noc regulates target mRNA stability. We hypothesize that Noc regulates target mRNA stability through binding to RISC complex, but we couldn’t detect such binding using co-immunoprecititation. The other one is to identify miRNAswith circadian rhythm and to investigate the functions of these rhythmic miRNAsin the mammalian liver. We identified several rhythmic miRNAs by microarray experiments, and focused on mmu-miR-494 because its predicted targets, PCK1, G6PC and G6PC2, also display rhythmic expression patterns with a phase opposite to mmu-miR-494. G6PC and PCK1areboth critical enzymesin gluconeogenesis.We further proved that G6PC and PCK1 expression level can be suppressed by overexpression of mmu-miR-494 in BNLCL2 cells, and also confirmed the post-transcriptional regulation of mmu-miR-494 on the two genes by luciferase reporter assays. Furthermore, glucose production rate is lower in stable miR-494 overexpression BNLCL2 cell. To identify upstream transcription factors controlling the rhythmic expression of mmu-miR-494, we used the transcription binding site prediction website, TFSEARCH, and searched out ttwo nearly perfect CREB binding sites. Knockdown of CREB actually increased miR-494 expression, we hypothesized that CREB might directly inhibits miR-494 expression or indirectly by promoting expressions of other repressors. According to these data, we found a rhythmic miR-494 in mouse liver and also identified PCK1, G6PC, and G6PC2 might to be the targets of miR-494. We also found that miR-494 expression would be up-regulated after knockdown CREB. We consider that this rhythmic miR-494 may play an important role in regulating glucose metabolisms. In the future, we can perform in vivo experiments to further confirmthe role of rhythmic miR-494 in glucose metabolisms.