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Targeting the miR-665-3p-ATG4B-autophagy axis relieves inflammation and apoptosis in intestinal ischemia/reperfusion.

ABSTRACT: Autophagy is an essential cytoprotective response against pathologic stresses that selectively degrades damaged cellular components. Impaired autophagy contributes to organ injury in multiple diseases, including ischemia/reperfusion (I/R), but the exact mechanism by which impaired autophagy is regulated remains unclear. Several researchers have demonstrated that microRNAs (miRNAs) negatively regulate autophagy by targeting autophagy-related genes (ATGs). Therefore, the effect of ATG-related miRNAs on I/R remains a promising research avenue. In our study, we found that autophagy flux is impaired during intestinal I/R. A miRNA microarray analysis showed that miR-665-3p was highly expressed in the I/R group, which was confirmed by qRT-PCR. Then, we predicted and proved that miR-665-3p negatively regulates ATG4B expression in Caco-2 and IEC-6 cells. In ileum biopsy samples from patients with intestinal infarction, there was an inverse correlation between miR-665-3p and ATG4B expression, which supports the in vitro findings. Moreover, based on miR-665-3p regulation of autophagy in response to hypoxia/reoxygenation in vitro, gain-of-function and loss-of-function approaches were used to investigate the therapeutic potential of miR-665-3p. Additionally, we provide evidence that ATG4B is indispensable for protection upon inhibition of miR-665-3p. Finally, we observed that locked nucleic acid-modified inhibition of miR-665-3p in vivo alleviates I/R-induced systemic inflammation and apoptosis via recovery of autophagic flux. Our study highlights miR-665-3p as a novel small molecule that regulates autophagy by targeting ATG4B, suggesting that miR-665-3p inhibition may be a potential therapeutic approach against inflammation and apoptosis for the clinical treatment of intestinal I/R.

SUBMITTER: Li Z

PROVIDER: S-EPMC5924757 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Targeting the miR-665-3p-ATG4B-autophagy axis relieves inflammation and apoptosis in intestinal ischemia/reperfusion.

Li Zhenlu Z Wang Guangzhi G Feng Dongcheng D Zu Guo G Li Yang Y Shi Xue X Zhao Yan Y Jing Huirong H Ning Shili S Le Weidong W Yao Jihong J Tian Xiaofeng X

Cell death & disease 20180501 5

Autophagy is an essential cytoprotective response against pathologic stresses that selectively degrades damaged cellular components. Impaired autophagy contributes to organ injury in multiple diseases, including ischemia/reperfusion (I/R), but the exact mechanism by which impaired autophagy is regulated remains unclear. Several researchers have demonstrated that microRNAs (miRNAs) negatively regulate autophagy by targeting autophagy-related genes (ATGs). Therefore, the effect of ATG-related miRN ...[more]

PMID: 29706629

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Project description:ObjectiveIntestinal ischemia-reperfusion (II/R) is a common pathological injury in clinic, and the systemic inflammatory response it causes will lead to multiple organ damage and functional failure. miR-185-5p has been reported to be a regulator of inflammatory response and autophagy, but whether it participates in the regulation of autophagy in II/R is still unclear. Therefore, we aimed to explore the mechanism of miR-185-5p regulating intestinal barrier injury in (II/R).MethodsCaco-2 cells was induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to establish II/R model. The superior mesenteric artery of C57BL/6 mice was clamped for 45 min and then subjected to reperfusion for 4 h for the establishment of II/R mice model. miR-185-5p mimic, miR-185-5p inhibitor, pcDNA-autophagy-related 101 (ATG101) were respectively transfected into Caco-2 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess miR-185-5p expression. Western blot detected the level of ATG101 and tight junction-associated proteins ZO1, Occludin, E-cadherin, β-catenin, as well as autophagy markers ATG5, ATG12, LC3Ⅰ/Ⅱ, Beclin1 and SQSTM1. Transepithelial electrical resistance (TEER) values was detected by a resistance meter. FITC-Dextran was performed to measure cell permeability. 5-ethynyl-2'-deoxyuridine (EDU) staining measured cell proliferation. Transmission electron microscope was conducted to observe autophagosomes. Hematoxylin & eosin (H&E) staining observed the damage of mice intestinal. Immunohistochemistry (IHC) measured the percentage of ki67 positive cells. TdT-mediated dUTP nick-end labeling (TUNEL) assay assessed cell apoptosis in intestinal tissues of II/R. Dual-luciferase assay verified the targeting relationship between miR-185-5p and ATG101.Results miR-185-5p was overexpressed in OGD/R-induced Caco-2 cells and intestinal tissues of II/R mice. Knocking down miR-185-5p markedly promoted autophagy and TEER values, reduced cell permeability, and alleviated intestinal barrier damage. ATG101 was a target of miR-185-5p, and overexpression of ATG101 promoted autophagy and dampened OGD/R-induced intestinal barrier damage. Overexpression of miR-185-5p reversed the effect of overexpressed ATG101 on OGD/R-induced Caco-2 cells.ConclusionKnockdown of miR-185-5p enhanced autophagy and alleviated II/R intestinal barrier damage by targeting ATG101.

Dataset Information

Targeting the miR-665-3p-ATG4B-autophagy axis relieves inflammation and apoptosis in intestinal ischemia/reperfusion.

Publications

Targeting the miR-665-3p-ATG4B-autophagy axis relieves inflammation and apoptosis in intestinal ischemia/reperfusion.

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