WTAP-mediated N6-methyladenosine modification promotes the inflammation, mitochondrial damage and ferroptosis of kidney tubular epithelial cells in acute kidney injury by regulating LMNB1 expression and activating NF-κB and JAK2/STAT3 pathways.

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS
ACS Applied Bio Materials Pub Date : 2024-06-01 Epub Date: 2024-03-22 DOI:10.1007/s10863-024-10015-0
Fan Huang, Yuchen Wang, XiaoLi Lv, Chenda Huang
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引用次数: 0

Abstract

Acute kidney injury (AKI) is a serious complication of sepsis patients, but the pathogenic mechanisms underlying AKI are still largely unclear. In this view, the roles of the key component of N6-methyladenosine (m6A)-wilms tumor 1 associated protein (WTAP) in AKI progression were investigated. AKI mice model was established by using cecal ligation and puncture (CLP). AKI cell model was established by treating HK-2 cells with LPS. Cell apoptosis was analyzed by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining and flow cytometry analysis. Cell viability was analyzed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The concentrations of inflammatory factors were examined with ELISA kits. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and Fe2+ levels were detected with related kits. Gene expression was detected by western blot assay or quantitative real-time polymerase chain reaction (qRT-PCR) assay. The relation between WTAP and lamin B1 (LMNB1) was verified by Methylated RNA Immunoprecipitation (meRIP) assay, RIP assay, dual-luciferase reporter assay and Actinomycin D assay. CLP induced significant pathological changes in kidney tissues in mice and promoted inflammation, mitochondrial damage and ferroptosis. LMNB1 level was induced in HK-2 cells by LPS. LMNB1 knockdown promoted LPS-mediated HK-2 cell viability and inhibited LPS-mediated HK-2 cell apoptosis, inflammation, mitochondrial damage and ferroptosis. Then, WTAP was demonstrated to promote LMNB1 expression by m6A Methylation modification. Moreover, WTAP knockdown repressed LPS-treated HK-2 cell apoptosis, inflammation, mitochondrial damage and ferroptosis, while LMNB1 overexpression reversed the effects. Additionally, WTAP affected the pathways of NF-κB and JAK2/STAT3 by LMNB1. WTAP-mediated m6A promoted the inflammation, mitochondrial damage and ferroptosis in LPS-induced HK-2 cells by regulating LMNB1 expression and activating NF-κB and JAK2/STAT3 pathways.

Abstract Image

WTAP介导的N6-甲基腺苷修饰通过调节LMNB1的表达和激活NF-κB和JAK2/STAT3通路,促进急性肾损伤中肾小管上皮细胞的炎症、线粒体损伤和铁变态反应。
急性肾损伤(AKI)是脓毒症患者的一种严重并发症,但 AKI 的致病机制在很大程度上仍不清楚。有鉴于此,本研究对 N6-甲基腺苷(m6A)的关键成分--WTAP(肿瘤 1 相关蛋白)在 AKI 进展中的作用进行了研究。通过盲肠结扎和穿刺(CLP)建立了 AKI 小鼠模型。用 LPS 处理 HK-2 细胞,建立 AKI 细胞模型。通过TdT介导的dUTP镍末端标记(TUNEL)染色和流式细胞仪分析细胞凋亡。细胞活力通过 3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-溴化四氮唑(MTT)检测法进行分析。用 ELISA 试剂盒检测炎症因子的浓度。用相关试剂盒检测活性氧(ROS)、丙二醛(MDA)、谷胱甘肽(GSH)和 Fe2+ 的水平。基因表达通过 Western 印迹或定量实时聚合酶链反应(qRT-PCR)检测。甲基化 RNA 免疫沉淀(meRIP)试验、RIP 试验、双荧光素酶报告器试验和放线菌素 D 试验验证了 WTAP 与层粘连 B1(LMNB1)的关系。中电蛋白诱导小鼠肾脏组织发生明显病理变化,并促进炎症、线粒体损伤和铁变态反应。LPS 诱导了 HK-2 细胞中 LMNB1 的水平。LMNB1 基因敲除可促进 LPS 介导的 HK-2 细胞活力,并抑制 LPS 介导的 HK-2 细胞凋亡、炎症、线粒体损伤和铁凋亡。WTAP通过m6A甲基化修饰促进LMNB1的表达。此外,敲除 WTAP 可抑制 LPS 处理的 HK-2 细胞凋亡、炎症、线粒体损伤和铁凋亡,而过表达 LMNB1 则可逆转这些影响。此外,WTAP 通过 LMNB1 影响了 NF-κB 和 JAK2/STAT3 的通路。WTAP 介导的 m6A 通过调节 LMNB1 的表达、激活 NF-κB 和 JAK2/STAT3 通路,促进了 LPS 诱导的 HK-2 细胞的炎症、线粒体损伤和铁变态反应。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
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