FOXJ3通过调控PTGS2影响低氧诱导肺动脉平滑肌细胞增殖和迁移

FOXJ3 regulates hypoxia-induced proliferation and migration of pulmonary artery smooth muscle cells by regulating PTGS2

摘要

目的 探讨叉头框蛋白J3（FOXJ3）对低氧诱导肺动脉平滑肌细胞（PASMCs）增殖和迁移的影响，并分析其与前列腺素-内过氧化物合酶2（PTGS2）的关系。方法 将PASMCs分别置于常氧及不同低氧浓度（10%、3%、1%）条件下培养48 h，采用定量聚合酶链式反应（qPCR）和蛋白质免疫印迹（Western blotting）检测FOXJ3表达。设常氧组、低氧组和低氧+pcDNA-FOXJ3组，采用qPCR和Western blotting检测FOXJ3表达，采用CCK-8法和跨室培养实验评估细胞增殖和迁移。采用qPCR和Western blotting筛选FOXJ3相关候选下游分子，并通过ChIP-qPCR与萤光素酶报告基因实验分析FOXJ3与PTGS2启动子区域的结合情况。另设阴性对照组、FOXJ3组、si-PTGS2组和FOXJ3+si-PTGS2组，观察PASMCs增殖和迁移的变化。结果 与常氧组相比，各低氧组PASMCs中FOXJ3表达均降低，且随氧浓度下降呈进一步下调趋势。低氧处理可增强PASMCs增殖和迁移能力，FOXJ3过表达后上述变化受到抑制。候选分子筛选结果显示，低氧条件下PTGS2表达下降，FOXJ3过表达后其表达回升。ChIP-qPCR结果显示，FOXJ3过表达后PTGS2启动子区域富集水平升高，萤光素酶报告基因实验进一步表明FOXJ3调控PTGS2启动子活性。PTGS2敲低可部分逆转FOXJ3过表达对PASMCs增殖和迁移的抑制作用。结论 低氧可下调PASMCs中FOXJ3的表达，FOXJ3过表达可抑制低氧诱导的PASMCs增殖和迁移，该作用是通过调控PTGS2表达实现。

Abstract

Objective To investigate the effect of forkhead box J3 (FOXJ3) on hypoxia-induced proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and to analyze its relationship with prostaglandin-endoperoxide synthase 2 (PTGS2). Methods PASMCs were cultured under normoxia or different hypoxic conditions (10%, 3%, and 1% O₂) for 48 h, and FOXJ3 expression was measured by quantitative polymerase chain reaction (qPCR) and Western blotting. Cells were divided into the normoxia, hypoxia, and hypoxia + pcDNA-FOXJ3 groups. FOXJ3 expression was assessed by qPCR and Western blotting, and cell proliferation and migration were evaluated by Cell Counting Kit-8 (CCK-8) assay and Transwell assay. Candidate downstream molecules related to FOXJ3 were screened by qPCR and Western blotting, and the binding of FOXJ3 to the PTGS2 promoter region was analyzed by chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assay. In addition, cells were divided into the negative control (NC), FOXJ3, si-PTGS2, and FOXJ3 + si-PTGS2 groups to observe the changes in proliferation and migration of PASMCs. Results Compared with the normoxia group, FOXJ3 expression in PASMCs was decreased in all hypoxia groups, with further downregulation as oxygen concentration decreased. Hypoxia treatment enhanced the proliferation and migration abilities of PASMCs, and these changes were inhibited after FOXJ3 overexpression. Candidate molecule screening showed that PTGS2 expression decreased under hypoxic conditions and increased after FOXJ3 overexpression. ChIP-qPCR results demonstrated that the enrichment level of the PTGS2 promoter region was elevated after FOXJ3 overexpression, and luciferase reporter gene assay further indicated that FOXJ3 regulated PTGS2 promoter activity. PTGS2 knockdown partially reversed the suppressive effects of FOXJ3 overexpression on PASMC proliferation and migration. Conclusion Hypoxia downregulates FOXJ3 expression in PASMCs. FOXJ3 overexpression inhibits hypoxia-induced proliferation and migration of PASMCs, and this effect is achieved through regulating PTGS2 expression.

关键词

低氧 / FOXJ3 / PTGS2 / 肺动脉平滑肌细胞 / 增殖 / 迁移

Key words

Hypoxia / FOXJ3 / PTGS2 / Pulmonary artery smooth muscle cells / Proliferation / Migration

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基金资助

巴彦淖尔市科技计划项目(k202534)

巴彦淖尔市科技计划项目(k202521)