MiR-27於肝臟中調控胰島素訊息傳遞所扮演的角色

Abstract

肝臟的代謝變化或在肝臟日夜週期中可以維持能量的恆定。在調控肝臟代謝變化的基因中，微RNA (microRNA)為主要且能調節許多代謝反應。先前的研究指出miR-27對肝臟脂質合成 (lipogenesis) 是重要的，且肝臟脂質合成對於日夜週期扮演重要的角色。而胰島素訊息傳遞 (Insulin/Akt signaling)也是調控肝臟代謝的重要因子，因此我們假設miR-27可以參與胰島素訊息傳遞，我們首先在細胞實驗中觀察胰島素對miR-27的影響，利用人類肝前驅細胞株 (HepaRG cell line)加入胰島素，並且使其胰島素訊息傳遞活化，會有效抑制miR-27表現。接著，利用文獻搜尋和生物資訊網站分析，預測miR-27a可能抑制胰島素訊息傳遞的下游基因，包含Pdpk1、Pik3和磷酸化Akt。事實上，利用人類肝前驅細胞株和分離出的小鼠肝細胞 (primary mouse hepatocytes)，透過轉染使其高度表現miR-27a，發現Pdpk1、Pik3ca和p-Akt表現量皆被抑制。之後利用螢光素酶報告分析方法（luciferase reporter assay）的結果，更進一步證實miR-27a可透過直接調控Pdpk1及Pik3r1的3’UTR促進其RNA之降解。為了更近一步找到胰島素調控miR-27的機制，我們搜尋文獻，發現CREB (cyclic AMP response element binding protein) 可以直接調控miR-23a~27a~24-2啟動子。在細胞及小鼠實驗中利用siRNA及Metformin兩種方式降低CREB表象，皆會抑制miR-27的表現。同時，當小鼠進行整夜禁食後或是高脂飲食餵食，使其肝臟高度表現CREB時，miR-27b會隨之顯著上升。我們進一步將禁食後之小鼠肝臟進行組織染色質免疫沈澱 (Tissue-ChIP)，結果確認CREB可與miR-23a~27a~24-2啟動子上之CRE 位點直接且高度的結合。綜上，我們的結果發現胰島素可透過抑制CREB去抑制miR-27的表現，進而促進以Akt為主之胰島素訊息傳遞。

Metabolic signaling pathways in livers are mostly under circadian control to maintain energy homeostasis. Among genes involved in hepatic metabolism, microRNAs (miR) function as a master regulator and orchestrate multiple metabolic signaling pathways. Previous studies have shown that circadian expression of miR-27 in liver contributes to hepatic lipogenesis. Here we test the hypothesis that miR-27 participates in insulin/Akt signaling, which is a critical regulator of liver metabolism. First, cell culture experiments showed that miR-27 was significantly downregulated when HepaRG cells, a human liver progenitor cells, were treated with insulin and insulin/Akt signaling was activated. Secondly, literature searches and bioinformatics analysis predicted that miR-27 may target Pdpk1, Pik3ca, Pik3r1, and Pik3cd, all of which are critical activators of insulin/Akt signaling. Indeed, over-expression of miR-27a significantly suppressed Pdpk1, Pik3r1, and Akt phosphorylation in primary mouse hepatocytes and HepaRG cells. Luciferase reporter assay demonstrated that miR-27a directly targeted Pdpk1 and Pik3r1 3’UTR. Next, we investigated the possible mechanism by which insulin regulates miR-27. Previous study showed that cyclic AMP-responsive element binding protein (CREB) transcriptionally regulated miR-23a~27a~24-2 cluster. Indeed, our data showed that both siRNA- and metformin-mediated silencing of CREB significantly decreased miR-27 expression in HepaRG cells and mice. Also, our animal studies showed that miR-27b is significantly increased when CREB is induced in mice livers by either overnight fast or high-fat-diet feeding. Furthermore, chromosome immunoprecipitation of fasted/non-fasted liver extract using CREB antibody determined that CREB directly binds to their previously verified CRE sites on the promoter area of miR-23a~27a~24-2 cluster. More experiments are required to determine the role of miR-27 in physiology homeostasis. Taken together, we concluded that insulin may negatively regulated miR-27 through inhibition of CREB and this insulin-mediated miR-27 suppression may promote insulin-induced Akt signaling in livers.