急性腎臟損傷的自噬機轉研究

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Title:	急性腎臟損傷的自噬機轉研究 Autophagy in Acute Kidney Injury
Authors:	蔡宗訓;Tsung-Hsun Tsai
Contributors:	臨床醫學研究所博士班
Keywords:	急性腎損傷;自噬作用相關蛋白;自噬體;acute kidney injury;autophagy?related proteins;autophagosome;autophagy;HuR;RNA binding protein
Date:	2019-05-17
Issue Date:	2019-11-11 09:20:12 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	急性腎損傷會導致腎功能突然惡化，而缺血性腎損傷的機制目前尚未清楚。儘管改善血液透析和病患護理方法，但急性腎衰竭還是常發生在患有嚴重疾病的病患，並且極有可能因併發症產生而導致死亡。自噬作用是一種細胞促進生存的機制，在急性腎損傷期間提供細胞保護作用。自噬作用是細胞內將存在較久和折疊異常的蛋白質或受損細胞胞器予以分解，來維持細胞穩定狀態。當細胞處於壓力狀況下(例如飢餓，缺氧或感染狀況)時，常會誘發自噬作用發生。非編碼RNA（non-coding ncRNA, ncRNA）在基因組中扮演著調控角色。最近研究顯示，ncRNA會透過標靶自噬相關蛋白來調控自噬作用。在研究中，我們證明ncRNA（miR-20a和EGOT）會調節缺氧誘導的腎小管細胞的自噬作用。當DNA模板轉錄成RNA乃至蛋白質時，這中間的調節機制包含了修飾序列，調節穩定性，轉運信使RNA(mRNA)和轉譯蛋白質。該過程稱為轉錄後基因調控，而RNA結合蛋白(RNA binding protein, RBP)參與其中的調控作用。Human antigen R(HuR)是36-kDa的RBP。HuR主要位於細胞核中，可透過與轉運因子結合而易位至細胞質，進一步影響標靶mRNA的穩定和轉譯。研究結果發現，HuR在人類腎臟-2(human kidney-2, HK-2)細胞缺氧狀況下，會誘導自噬作用的功能表現。HK-2細胞在缺氧條件下，顯示HuR與自噬作用相關蛋白(autophagy-related proteins, ATG)的表現上升相關，如ATG7，ATG16L1和LC3，並且可從在缺氧細胞中LC3螢光點(LC3 puncta)表現，來顯示自噬體形成的增加。此外，利用short hairpin-RNA將HK-2細胞中HuR功能降低調解下，明顯降低了ATG7和ATG16L1的蛋白表達。利用生物信息方法預測顯示，在編碼區上ATG7和富含AU的元件的3’UTR ATG16L1的mRNA能與HuR基序結合。RNA免疫沉澱研究顯示，HuR在缺氧條件下，與ATG7和ATG16L1 mRNA有著顯著相關。此外，HuR透過調節LC3表達來顯示自噬體形成有增加現象。這些結果顯示，HuR會調節ATG7和ATG16L1表達，進一步調節HK-2細胞中的自噬作用。另外，透過terminal deoxynucleotidyl transferase dUTP nick end labeling 檢測的研究發現，knockdwon HuR的細胞在缺氧期間會經歷凋亡情形。這些發現顯示了HuR在缺氧條件下與抑制腎小管細胞凋亡有著關鍵作用。 Acute kidney injury refers to the sudden deterioration of renal function. The mechanism of ischemic renal injury is currently unclear. Despite the use of renal replacement therapy and improved patient care, acute renal failure usually occurs in patients with severe illness and is highly likely to cause death. Autophagy, a prosurvival mechanism offers a protective role during acute kidney injury. Autophagy is the breakdown of long-lived and abnormally folded proteins in cells or damaged organelles to maintain cell homeostasis. Autophagy occurs when cells are under stress conditions (e.g. starvation, hypoxic, or infection conditions). Noncoding RNAs (ncRNAs) are emerging as an integral part of the regulatory information encoded in the genome. Recent studies have shown that ncRNAs regulate autophagy by targeting autophagy-related proteins. In this study, we demonstrate that ncRNA(miR-20a and EGOT) regulates hypoxia-induced autophagy in renal tubular cells (HK-2). When the DNA template is transcribed into RNA, regulation mechanisms are involved in modifying the sequence, adjusting stability, transporting mRNA, and translating proteins. This process is called post-transcriptional gene regulation (PTGR), in which RNA-binding proteins (RBPs) participate. Human antigen R (HuR) is a 36-kDa RNA-binding protein. HuR, which is mainly located in the cell nuclei, can be translocated to the cytoplasm by binding to transport factors, thereby influencing the stability and translation of target mRNA. The study show novel findings on the functional role of RNA binding protein, HuR during hypoxia?induced autophagy in renal proximal tubular cells?2 (HK?2). HK?2 cells showed upregulated expressions of HuR and autophagy?related proteins such as autophagy related 7 (ATG7), autophagy related 16 like 1 (ATG16L1), and LC3 under hypoxia. Increased autophagosome formation was visualized as LC3 puncta in hypoxic cells. Further, short hairpin?RNA?mediated loss of HuR function in HK?2 cells significantly decreased ATG7 and ATG16L1 protein expressions. Bioinformatics prediction revealed HuR motif binding on the coding region of ATG7 and AU?rich element at 3’UTR ATG16L1 messnger RNA (mRNA). The RNA immunoprecipitation study showed that HuR was predominantly associated with ATG7 and ATG16L1 mRNAs under hypoxia. In addition, HuR enhanced autophagosome formation by regulating LC3II expressions. These results show that HuR regulates ATG7 and ATG16L1 expressions and thereby mediate autophagy in HK?2 cells. Importantly, HuR knockdown cells underwent apoptosis during hypoxia as observed through the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Collectively, these findings show the crucial role of HuR under hypoxia by regulating autophagy and suppressing apoptosis in renal tubular cells.
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