miR-15b-5p通过USP9X影响PIK3CA/AKT1通路缓解哮喘气道炎症  

miR-15b-5p affects PIK3CA/AKT1 pathway through USP9X to alleviate airway inflammation in asthma

作　　者：周宇洋 王知广 朴艺花 韩雪[1,2] 宋艺兰 延光海 朴红梅 ZHOU Yuyang;WANG Zhiguang;PIAO Yihua;HAN Xue;SONG Yilan;YAN Guanghai;PIAO Hongmei(Department of Respiratory Medicine,Affiliated Hospital of Yanbian University,Yanji 133000;Jilin Provincial Key Laboratory of Immunity and Targeting of Common Allergic Diseases,Yanji 133002;Department of Critical Care Medicine,Affiliated Hospital of Yanbian University,Yanji 133000;Department of Anatomy,School of Medicine,Yanbian University,Yanji 133002,China)

机构地区：[1]延边大学附属医院呼吸内科,吉林延吉133000 [2]吉林省过敏性常见疾病免疫与靶向研究重点实验室,吉林延吉133002 [3]延边大学附属医院重症医学科,吉林延吉133000 [4]延边大学解剖学教研室,吉林延吉133002

出　　处：《细胞与分子免疫学杂志》2025年第3期193-203,共11页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(82060004,82160004);吉林省科技厅项目(YDZJ202401083ZYTS,20240404025ZP);吉林省教育厅项目(JJKH20240690KJ)。

摘　　要：目的 研究miR-15b-5p是否能通过负调控泛素特异性肽酶9X(USP9X)下调磷脂酰肌醇4, 5-二磷酸3-激酶催化亚基α/AKT丝氨酸/苏氨酸激酶1(PIK3CA/AKT1)通路的表达,从而缓解哮喘气道炎症。方法 通过在线数据库(miRWalk)预测USP9X可能是miR-15b-5p的直接靶点,并进行了荧光素酶报告基因检测验证。通过免疫共沉淀(CO-IP)验证了USP9X与PIK3CA之间能否直接结合以及USP9X和其小分子抑制剂WP1130在PIK3CA的去泛素化中作用。取C57小鼠,随机分为Control组、 OVA组、 OVA联合NC组以及miR-15b-5p agomir治疗组,每组10只。BEAS-2B细胞用白细胞介素13(IL-13)诱导,并用miR-15b-5p mimic治疗。进行了HE、 Masson、 PAS、免疫组织化学、免疫荧光染色及流式细胞术、 Western blot法、实时定量PCR检测。结果 发现给予miR-15b-5p agomir和mimic后能够减少支气管周围炎性细胞,改善气道炎症,并且miR-15b-5p能靶向负调控USP9X。USP9X能够与PIK3CA直接结合,以蛋白酶体依赖性方式调节PIK3CA水平,并且USP9X对K29连接的PIK3CA蛋白起去泛素化作用,下调USP9X会使PIK3CA的泛素化水平增加。USP9X的小分子抑制剂WP1130与敲低USP9X作用一致,均能够增加PIK3CA的泛素化水平,使PIK3CA的蛋白水平降低。结论 miR-15b-5p/USP9X/PIK3CA/AKT1信号通路可能为哮喘提供潜在的治疗靶点。Objective To investigate whether miR-15b-5p can alleviate airway inflammation in asthma by negatively regulating ubiquitin specific peptidase 9X(USP9X)to down-regulate the expression of phosphatidylinositol 4,5-diphosphate 3-kinase catalytic subunitα/AKT serine/threonine kinase 1(PIK3CA/AKT1)pathway.Methods USP9X was predicted to be a direct target of miR-15b-5p by using an online database(miRWalk),and the luciferase reporter gene assay was performed to verify it.Co-immunoprecipitation(CO-IP)was used to verify the direct binding between USP9X and PIK3CA and the role of USP9X and its small molecule inhibitor WP1130 in the deubiquitination of PIK3CA.C57 mice were randomly divided into Control group,OVA group,OVA combined with NC group and miR-15b-5p agomir group,with 10 mice in each group.BEAS-2B cells were induced with interleukin 13(IL-13)and treated with miR-15b-5p mimic.HE,Masson,PAS,immunohistochemistry, immunofluorescence staining, flow cytometry, Western blot and quantitative real-time PCR (qRT-PCR) were performed. Results It was found that the administration of miR-15b-5p agomir and mimic could reduce peribronchial inflammatory cells and improve airway inflammation, and miR-15b-5p could target negative regulation of USP9X. USP9X could directly bind to PIK3CA and regulate PIK3CA level in a proteasome-dependent manner, and USP9X could deubiquitinate K29-linked PIK3CA protein. Down-regulation of USP9X could increase PIK3CA ubiquitination level. WP1130, a small molecule inhibitor of USP9X, has the same effect as knockdown of USP9X, both of which could increase the ubiquitination level of PIK3CA and reduce the protein level of PIK3CA. Conclusion The miR-15b-5p/USP9X/PIK3CA/AKT1 signaling pathway may provide potential therapeutic targets for asthma.

关 键 词：miR-15b-5p USP9X PIK3CA AKT1 泛素化 哮喘 

分 类 号：R392[医药卫生—免疫学] R562.25[医药卫生—基础医学] R364.5