MiR-20a-5p在缺血腎損傷透過ATG16L1調節低氧誘導細胞的自噬作用

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Title:	MiR-20a-5p在缺血腎損傷透過ATG16L1調節低氧誘導細胞的自噬作用 MiR-20a-5p mediates hypoxia-induced autophagy by targeting ATG16L1 in ischemic kidney injury
Authors:	王怡寬;I-Kuan Wang
Contributors:	臨床醫學研究所博士班
Keywords:	細胞自噬;急性腎損傷;微核醣核酸;Autophagy;acute kidney injury;miroRNA
Date:	2015-08-06
Issue Date:	2015-11-04 17:03:08 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	背景與目的：急性腎損傷合併高罹病率和死亡率。缺血是急性腎損傷主要原因之一。然而，缺血性的急性腎損傷發病機制尚不完全清楚，了解相關的機制對預防和治療急性腎損傷是非常重要的。基礎細胞自噬透過去除壽命長、錯誤折疊或聚集的蛋白質和受損胞器對細胞內恆定很重要。細胞自噬能在壓力下被誘發，並提供細胞保護反應。微RNA的調控網絡在細胞自噬的調節有重要的角色。本研究的目的是評估在缺血性急性腎損傷和缺氧的腎近端小管細胞，微RNA是否調控細胞自噬並探討相關機制。材料和方法：使用雄性,6-8週齡的C57BL/6小鼠。兩側腎動脈用止血鉗夾60分鐘。HK2細胞置入0.3％的O 2低氧艙。即時聚合?鏈反應測定微RNA的表達變化。報告基因分析用來證實的miRNA作用於自噬相關基因和HIF-1α的結合到的miRNA的啟動子。染色質免疫沉澱測定法用於證實HIF-1α結合到的微RNA的啟動子。結果：細胞自噬在腎缺血再灌注腎損傷的小鼠被誘導，細胞自噬也在缺氧的HK2細胞被誘導。ATG16L1對自噬體的形成有重要角色。藉著生物信息學分析，選擇候選的微RNA為miR-20a-5p，他的潛在的目標基因為ATG16L1。即時聚合?鏈反應顯示miR-20a-5p的表現在6小時和24小時的小鼠腎缺血再灌注損傷後下降。同樣地，與未處理的細胞相比，miR-20a-5p的在低氧條件下6小時和24小時的 HK2細胞表現下降。為了驗證了miR-20a-5p預測的微RNA結合序列， ATG16L1的3'UTR含miR-20a-5p結合序列被轉殖到pmirGLO-ATG16L1。Luciferase活性在miR-20a-5p mimic轉殖的24小時低氧HK2細胞大幅度降低。相反地，Luciferase活性在HK2細胞帶有ATG16L1突變的3'UTR是不被miR-20a-5p mimic抑制。過度表現的mi-20a-5p在低氧HK2抑制LC3-II和 ATG16L1的表現, 然而antagomir-20a翻轉此抑制。總結，在缺氧條件下miR-20a-5p的表達下降，且透過抑制ATG16L1，負調控細胞自噬。即時聚合?鏈反應證實了miR-20a-5p在shHIF-1α細胞的表現增加。Luciferase活性在24小時低氧轉殖miR-20a-5 promoter-pmirGLO 的shHIF-1α HK2細胞表現增加。此外， ChIP分析表明，HIF-1α與miR-20a-5p啟動子區域的結合和對照組相比（shLacZ細胞）增加，而HIF-1α在相同條件下在shHIF-1α細胞和miR-20a-5p啟動子區域的結合降低。綜上所述，HIF-1α，在缺氧表現增加，透過結合到啟動子抑制了miR-20a-5p表現。結論： miR-20a-5p在缺血腎損傷表達下降，且透過抑制ATG16L1，調控細胞自噬。HIF-1α透過抑制了miR-20a-5p表現來調控細胞自噬。我們的結論是HIF-1α, miR-20a-5p, ATG16L1此細胞自噬路徑, 在缺血腎損傷為重要的調控機制。需要進一步活體的研究去探索miR-20a -5P在缺血再灌注急性腎損傷的發病機制和治療的角色。 Background and purpose: Acute kidney injury (AKI) is associated with high morbidity and mortality, and ischemia is one of its major causes. The pathogenesis of ischemic AKI is not fully understood, and understanding the relevant mechanisms is crucial to prevent and treat AKI. Autophagy is a cellular homeostatic mechanism activated under stress conditions and might act as protective response for cell survival in ischemic kidney injury. The micro RNA (miRNA) network may be critically involved in the regulation of autophagy. The aim of this study was to evaluate whether miRNA regulates autophagy in ischemic kidney injury and renal proximal tubular cells under hypoxic conditions. Material and methods: In vivo studies: Male C57BL/6 mice, 6–8 weeks old, were used. The bilateral pedicles were clamped using microaneurysm clips for 60 min. Sham mice underwent the same surgical procedure, without the use of clamping. In vitro studies: Human kidney proximal tubular (HK2) cells were cultured in RPMI 1640 (Gibco, Invitrogen) supplemented with 10% FBS (Gibco, Invitrogen) and 1% penicillin/streptomycin (Gibco, Invitrogen) at 37℃ and 5% CO2. The HK2 cells were grown until confluence and were subsequently serum- and glucose-deprived for 24 h. HK2 cells are then incubated in a hypoxia chamber with 0.3% O2. ATG16L1 is essential for autophagosome formation. Bioinformatics analyses were used to select the candidate miRNA, miR-20a-5p, which potentially targets ATG16L1. Gain-of-function and loss-of-function methods were employed to evaluate the effects of miRNA on autophagy. Chromatin immunoprecipitation analysis and promoter luciferase reporter assays were used to evaluate the interaction of transcriptional factors with miRNA. Results: Increased expression of punctate LC3 and ATG16L1, autophagy-related proteins, and down-expression of miR-20a-5p were detected in kidneys at 6 h and 12 h after ischemic injury and in HK-2 cells under hypoxic conditions. To validate the predicted miR-binding sequence for miR-20a-5p, the 3’UTR of ATG16L1 with miR-20a-5p binding sequences was cloned into pmirGLO-ATG16L1, and the luciferase reporter assay results confirmed the binding. Normalized luciferase activity considerably decreased in HK2 cells transfected with miR-20a-5p mimic under hypoxic conditions at 24 h. By contrast, normalized luciferase activity in HK2 cells constructed in the mutant 3’UTR region of ATG16L1 was refractory to the inhibition of miR-20a-5p mimic. In addition, over-expression of miR-20a-5p reduced the expression of LC3-II and ATG16L1 in HK-2 cells under hypoxic conditions, whereas antagomiR-20a reversed the inhibition. These results suggest that miR-20a-5p could negatively regulate autophagy through suppressing ATG16L1 by binding to its 3’UTR in HK-2 cells under hypoxic conditions. Real-time PCR demonstrated the upregulation of miR-20a-5p in shHIF-1α cells. Reporter assay indicated an increase in luciferase activity in shHIF-1α cells transfected with miR-20a-5p promoter-pmirGLO under hypoxic conditions at 24 h. In addition, ChIP assays demonstrated increased HIF-1α binding to the promoter regions of miR-20a-5p in the control samples (shLacZ cells), whereas HIF-1α binding in the shHIF-1α cells decreased under the same conditions. Taken together, these results indicated that HIF-1α, up-regulated under hypoxia, is an important regulator of autophagy by suppressing miR-20a-5p. Conclusion: MiR-20a-5p, down-regulated in kidneys after ischemic injury, could modulate autophagy by suppressing ATG16L1. HIF-1α is an important regulator of autophagy by suppressing miR-20a-5p. The signaling axis of HIF-1α, miR-20a-5p, and ATG16L1 in autophagic process might be a critical adapting mechanism for ischemic kidney injury. Further studies are required to explore the role of miR20a-5p in the pathogenesis and treatment of ischemia-reperfusion AKI.
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