Integrated bioinformatics and experimental validation identifies CLIC6 as a novel tumor suppressor regulating NF-κB signaling and immune microenvironment in nasopharyngeal carcinoma

• M1 macrophages enriched in NPC, correlate with cilia genes. • CLIC6 downregulated in NPC, inhibits NF-κB signaling. • CLIC6 suppresses tumor growth, invasion, and M1 polarization. • CLIC6-NF-κB axis as diagnostic and therapeutic target for NPC. Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus–associated malignancy. Tumor-associated macrophages play a pivotal role in NPC development, but molecular mechanisms remain unclear. This study aimed to identify M1 macrophage-associated hub genes and investigate their biological functions in NPC via bioinformatics and experimental validation. GSE12452 and GSE53819 datasets were integrated to assess immune cell infiltration using CIBERSORT. WGCNA identified gene modules correlated with M1 macrophages. Hub genes were identified by intersecting differentially expressed genes with module genes, followed by LASSO regression and clinical specimen validation. CCK-8, Transwell assays, and macrophage co-culture systems were used to evaluate CLIC6 effects on NPC cell proliferation, invasion, and M1 polarization. RNA-seq and Western blot were performed to explore downstream mechanisms, with rescue experiments using NF-κB activator or inhibitor. M1 macrophages were significantly enriched in NPC tissues and negatively correlated with cilia-related gene modules. Six hub genes (MUC16, MS4A8, MUC20, AMIGO1, PHYHD1, CLIC6) were identified, with CLIC6 showing significant downregulation in NPC tissues and cell lines and negative correlation with M1 macrophages. CLIC6 recombinant protein promoted NPC cell proliferation and invasion while inhibiting M1 polarization, whereas CLIC6 silencing produced opposite effects. Mechanistically, CLIC6 suppressed NF-κB signaling by inhibiting IκBα and p65 phosphorylation, confirmed by RNA-seq and Western blot. Functional rescue experiments demonstrated that NF-κB modulation reversed CLIC6-mediated effects. CLIC6 functions as a tumor suppressor in NPC by inhibiting NF-κB signaling, thereby regulating tumor cell proliferation, invasion, and macrophage polarization. The CLIC6-NF-κB axis represents a potential diagnostic biomarker and therapeutic target for NPC.