Abstract
OBJECTIVE: The involvement of methyltransferase-like 3 (METTL3, an m6A methyltransferase) and the PI3K/AKT pathway in tumor progression and immune regulation remains poorly understood. This study investigates whether METTL3 influences the immune microenvironment of renal cell carcinoma (RCC) via m6A-dependent PI3K/AKT pathway activation and explores its translational potential. METHODS: METTL3 expression was assessed in tumor and adjacent tissues from 34 RCC patients using Western blot and immunohistochemistry. The clinical cohort comprised 24 male and 10 female patients with an age of 61-77 years. METTL3 was genetically silenced or overexpressed in 786-O cells, and the PI3K/AKT pathway was pharmacologically activated with 1,3-dicaffeoylquinic acid (1,3-diCQA, a PI3K/AKT pathway activator). Cellular proliferation (EdU assay), invasion (Transwell), and apoptosis (flow cytometry) were evaluated. Flow cytometry was employed to quantify PD-L1, HLA-I expression, and CD8+ T cell activity. Subcutaneous tumor xenografts in C57BL/6 nude mice were used to assess tumor growth and immune marker expression. METTL3, PI3K, and AKT co-localization was examined via immunofluorescence. RESULTS: METTL3 was elevated in RCC tissues (p < 0.05). Silencing METTL3 suppressed proliferation and invasion and promoted apoptosis in 786-O cells (p < 0.05), concomitant with reduced PI3K/AKT pathway phosphorylation (p-PI3K/PI3K, p-AKT/AKT). Conversely, METTL3 overexpression or PI3K/AKT pathway activation (via 1,3-diCQA) enhanced cell viability (p < 0.05). High METTL3 expression or PI3K/AKT pathway activation increased PD-L1 (p < 0.05), decreased HLA-I (p < 0.05), and impaired CD8(+) T cell function (p < 0.05), whereas METTL3 knockdown reversed these immune-evasion effects. Bioinformatic analysis further showed a positive association between METTL3 expression and neutrophil infiltration in TCGA-KIRC, supporting a link between METTL3 upregulation and an inflammatory, immunosuppressive microenvironment. In vivo, METTL3 knockdown significantly attenuated subcutaneous tumor growth in nude mice (p < 0.05) and reduced PD-L1 and CD163 expression (p < 0.05). However, cells pretreated with 1,3-diCQA prior to inoculation counteracted the tumor-suppressive effects of METTL3 silencing. Together, these findings support an m6A-dependent METTL3-YTHDF1-PI3K/AKT pathway regulatory axis in RCC, although direct m6A modification of individual PI3K/AKT pathway transcripts was not examined in the present study. CONCLUSION: METTL3 drives immune evasion and promotes RCC growth by activating the PI3K/AKT pathway through an m6A-dependent mechanism involving YTHDF1.