Title : The oncogenic role of KCNN4 in renal clear cell carcinoma: A mechanism involving immune regulation via Tregs and resting mast cells
Abstract:
Background: Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system, with clear cell RCC (ccRCC) representing its predominant subtype. The pathogenesis of ccRCC remains incompletely understood. KCNN4 (potassium calcium-activated channel subfamily N member 4) has recently emerged as a potential oncogene implicated in multiple malignancies. This study aimed to investigate the expression and functional role of KCNN4 in ccRCC and to elucidate its mechanism in modulating tumor immune responses through regulatory T cells (Tregs) and resting mast cells, with the goal of identifying novel therapeutic targets.
Methods: The GSE53757 dataset was obtained from the GEO database to analyze KCNN4 expression in ccRCC. Tissue samples were collected from 30 ccRCC patients (study group) and 30 patients with benign renal tumors (control group) at Jinling Hospital, Nanjing Medical University (April 2022–April 2023). KCNN4 mRNA and protein expression were validated using quantitative PCR and immunohistochemistry (IHC) in clinical samples and ccRCC cell lines (786-O and OS-RC-2). Functional assays, including CCK-8 proliferation, scratch wound healing, and Transwell migration/invasion, were performed following KCNN4 inhibition or siRNA knockdown. A xenograft model was established in BALB/c-nu mice using 786-O cells with or without KCNN4 knockdown to monitor tumor growth. Immune cell profiling was conducted via flow cytometry to assess CD4+Foxp3+ Tregs and resting mast cells. The expressions of KCNN4, Foxp3, TLR4, and GSK3β were detected by qPCR and IHC. Cytokine levels (IL-6, TNF-α, VEGF, TGF-β) in tumor tissues were measured by Western blot, and IL-4 and TGF-β in cell culture supernatants were quantified by ELISA.
Results: KCNN4 was significantly overexpressed in ccRCC tissues compared with normal adjacent tissues and benign controls (p < 0.05). Similarly, 786-O and OS-RC-2 cells showed higher KCNN4 levels than the normal renal cell line HK-2. KCNN4 knockdown significantly suppressed cell proliferation, migration, and invasion in vitro (p < 0.05). In vivo, KCNN4-silenced cells exhibited delayed tumor formation and reduced tumor volume compared with controls. Flow cytometry revealed increased Treg infiltration and decreased resting mast cells in ccRCC tissues. KCNN4 expression positively correlated with Foxp3 and negatively with TLR4 and GSK3β. Moreover, elevated levels of IL-6, TNF-α, VEGF, and TGF-β were observed in KCNN4-high tumors and were positively associated with KCNN4 expression. ELISA confirmed increased IL-4 and TGF-β in supernatants from KCNN4-expressing cells.
Conclusions: KCNN4 is highly expressed in ccRCC and promotes tumor progression, potentially through impairing antitumor immunity by enhancing Treg recruitment and suppressing resting mast cells, thereby facilitating immune escape. These findings identify KCNN4 as a novel oncogenic regulator and a promising therapeutic target in ccRCC.
Keywords: renal cell carcinoma; KCNN4; tumor microenvironment; regulatory T cells; resting mast cells; immune response.
