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长链非编码 RNA MEG3 通过调控 miR-195-5p/FGFR2 轴和抑制 NF-κB 通路参与雨蛙肽诱导的人胰腺细胞炎症损伤。

LncRNA MEG3 Participates in Caerulein-Induced Inflammatory Injury in Human Pancreatic Cells via Regulating miR-195-5p/FGFR2 Axis and Inactivating NF-κB Pathway.

机构信息

Department of Emergency and Critical Medicine, The Second Hospital of Jilin University, No. 218, Nanguan District, Ziqiang Street, Changchun, Jilin, 130041, China.

出版信息

Inflammation. 2021 Feb;44(1):160-173. doi: 10.1007/s10753-020-01318-6.

Abstract

Acute pancreatitis (AP) is a dysfunctional pancreas disease marked by severe inflammation. Long non-coding RNAs (lncRNAs) involving in the regulation of inflammatory responses have been frequently mentioned. The purpose of this study was to ensure the function and action mode of lncRNA maternally expressed gene 3 (MEG3) in caerulein-induced AP cell model. HPDE cells were treated with caerulein to establish an AP model in vitro. The expression of MEG3, miR-195-5p, and fibroblast growth factor receptor 2 (FGFR2) was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation and apoptosis were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry assay, respectively. The expression of CyclinD1, B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), FGFR2, P65, phosphorylated P65 (p-P65), alpha inhibitor of nuclear factor kappa beta (NF-κB) (IκB-α), and phosphorylated IκB-α (p-IκB-α) at the protein level was quantified by western blot. The concentrations of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were monitored by enzyme-linked immunosorbent assay (ELISA). The targeted relationship between miR-195-5p and MEG3 or FGFR2 was forecasted by the online software starBase v2.0 and verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. As a result, the expression of MEG3 and FGFR2 was decreased in caerulein-induced HPDE cells, while the expression of miR-195-5p was increased. MEG3 overexpression inhibited cell apoptosis and inflammatory responses that were induced by caerulein. Mechanically, miR-195-5p was targeted by MEG3 and abolished the effects of MEG3 overexpression. FGFR2 was a target of miR-195-5p, and MEG3 regulated the expression of FGFR2 by sponging miR-195-5p. FGFR2 overexpression abolished miR-195-5p enrichment-aggravated inflammatory injuries. Moreover, the NF-κB signaling pathway was involved in the MEG3/miR-195-5p/FGFR2 axis. Collectively, MEG3 participates in caerulein-induced inflammatory injuries by targeting the miR-195-5p/FGFR2 regulatory axis via mediating the NF-κB pathway in HPDE cells.

摘要

急性胰腺炎 (AP) 是一种以严重炎症为特征的胰腺功能障碍性疾病。长链非编码 RNA (lncRNA) 参与炎症反应的调节,已被频繁提及。本研究旨在确定母系表达基因 3 (MEG3) 在鹅膏蕈碱诱导的 AP 细胞模型中的功能和作用模式。体外采用鹅膏蕈碱处理 HPDE 细胞建立 AP 模型。采用实时定量聚合酶链反应 (qRT-PCR) 检测 MEG3、miR-195-5p 和成纤维细胞生长因子受体 2 (FGFR2) 的表达。通过 3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四唑溴盐 (MTT) 测定和流式细胞术分别检测细胞增殖和细胞凋亡。Western blot 定量检测细胞周期蛋白 D1 (CyclinD1)、B 细胞淋巴瘤/白血病-2 (Bcl-2)、Bcl-2 相关 X 蛋白 (Bax)、FGFR2、P65、磷酸化 P65 (p-P65)、核因子 kappa B 抑制剂α (IκB-α) 和磷酸化 IκB-α (p-IκB-α) 的蛋白水平。采用酶联免疫吸附试验 (ELISA) 监测肿瘤坏死因子 α (TNF-α)、白细胞介素 1β (IL-1β) 和白细胞介素 6 (IL-6) 的浓度。通过在线软件 starBase v2.0 预测 miR-195-5p 与 MEG3 或 FGFR2 的靶向关系,并通过双荧光素酶报告基因检测和 RNA 免疫沉淀 (RIP) 实验验证。结果显示,鹅膏蕈碱诱导的 HPDE 细胞中 MEG3 和 FGFR2 的表达下调,而 miR-195-5p 的表达上调。MEG3 过表达抑制了鹅膏蕈碱诱导的细胞凋亡和炎症反应。机制上,MEG3 靶向 miR-195-5p,并消除了 MEG3 过表达的作用。FGFR2 是 miR-195-5p 的靶标,MEG3 通过海绵吸附 miR-195-5p 调节 FGFR2 的表达。FGFR2 过表达消除了 miR-195-5p 富集加重的炎症损伤。此外,NF-κB 信号通路参与了 MEG3/miR-195-5p/FGFR2 轴。综上所述,MEG3 通过调节 NF-κB 通路,在 HPDE 细胞中介导 miR-195-5p/FGFR2 调控轴,靶向 miR-195-5p/FGFR2 调控轴参与鹅膏蕈碱诱导的炎症损伤。

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