RBM15介导的RGS1 m6A修饰在IL-13诱导的鼻上皮细胞氧化应激损伤中的机制研究

文献信息

中文摘要

目的： 我们既往研究显示过敏性鼻炎（AR）中RGS1表达上调，而沉默RGS1可减轻白细胞介素-13（IL-13）诱导的人鼻上皮细胞（HNEpCs）上皮屏障破坏。然而，RGS1在AR相关氧化应激和炎症中的作用及其潜在调控机制仍很大程度上未被探索。本研究探讨了RGS1在AR氧化应激和炎症中的调控机制。
方法： 获取AR患者（N = 20）以及健康对照（N = 20）的鼻黏膜组织，检测RGS1和RBM15的表达。采用Pearson相关分析评估AR患者中RGS1与RBM15表达之间的关系。用IL-13诱导HNEpCs进行体外实验。通过qRT-PCR和Western blot评估RGS1和RBM15表达。分别使用Cell Counting Kit-8（CCK-8） assay、DCFH-DA和商业检测试剂盒评估活性氧（ROS）产生、超氧化物歧化酶（SOD）、丙二醛（MDA）和谷胱甘肽（GSH）水平以及细胞活力。通过酶联免疫吸附 assay（ELISA）测定肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）水平。使用甲基化RNA免疫沉淀（MeRIP）分析RGS1 mRNA的N6-甲基腺苷（m6A）修饰，使用RNA免疫沉淀（RIP）评估其与RBM15和YTHDF1/2的相互作用，并使用放线菌素D assay确定mRNA稳定性。
结果： RGS1和RBM15在AR样本中均上调，且二者表达水平呈显著正相关。在HNEpCs中，IL-13处理诱导氧化应激和炎症损伤，表现为细胞活力、SOD和GSH水平降低，同时ROS、MDA、TNF-α和IL-6水平升高。敲低RGS1或RBM15可减轻这些IL-13诱导的效应，而RGS1过表达逆转了RBM15沉默的保护作用。机制上，RBM15结合RGS1 mRNA，增强其整体m6A富集，并增加其稳定性和表达，可能通过YTHDF1识别实现。
结论： 总之，这些结果表明RBM15通过增强RGS1的m6A甲基化和稳定性（可能通过YTHDF1识别）促进RGS1表达，并且靶向RBM15或RGS1可减轻HNEpCs中IL-13诱导的氧化应激和炎症损伤。

英文摘要

AIM: Our previous study revealed upregulated RGS1 expression in allergic rhinitis (AR), while its silencing alleviates interleukin-13 (IL-13)-induced epithelial barrier disruption in human nasal epithelial cells (HNEpCs). However, the involvement of RGS1 in AR-related oxidative stress and inflammation, as well as its underlying regulatory mechanisms, remains largely unexplored. This study explored the regulatory mechanism of RGS1 in oxidative stress and inflammation in AR.
METHODS: Nasal mucosal tissues were obtained from patients with AR (N = 20) as well as healthy controls (N = 20) to detect the expression of RGS1 and RBM15. Pearson correlation analysis was used to assess the relationship between RGS1 and RBM15 expression in AR patients. HNEpCs were induced by IL-13 for in vitro experiments. RGS1 and RBM15 expression was evaluated by qRT-PCR and Western blot. Production of reactive oxygen species (ROS) and levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH), as well as cell viability were assessed using Cell Counting Kit-8 (CCK-8) assay, DCFH-DA, and commercial assay kits, respectively. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were measured by Enzyme-linked immunosorbent assay (ELISA). Methylated RNA immunoprecipitation (MeRIP) was used to analyze N6-methyladenosine (m6A) modification of RGS1 mRNA, RNA immunoprecipitation (RIP) to assess its interaction with RBM15 and YTHDF1/2, and actinomycin D assays to determine mRNA stability.
RESULTS: RGS1 and RBM15 were both upregulated in AR samples, with a significant positive correlation between their expression levels. In HNEpCs, IL-13 treatment induced oxidative stress and inflammatory injury, as indicated by reduced cell viability, SOD, and GSH levels, along with elevated ROS, MDA, TNF-α, and IL-6 levels. Knockdown of RGS1 or RBM15 alleviated these IL-13-induced effects, whereas RGS1 overexpression reversed the protective impact of RBM15 silencing. Mechanistically, RBM15 binds to RGS1 mRNA, enhances its overall m6A enrichment, and increases its stability and expression, potentially through YTHDF1 recognition.
CONCLUSION: Collectively, these results indicate that RBM15 promotes RGS1 expression by enhancing its m6A methylation and stability, potentially through YTHDF1 recognition, and that targeting RBM15 or RGS1 alleviates IL-13-induced oxidative stress and inflammatory injury in HNEpCs.