RNA‐Binding Protein NOVA1 Restrains Immune Escape and Liver Metastasis in Colorectal Cancer via Stabilizing NRXN1 mRNA

ABSTRACT Background Colorectal cancer (CRC) liver metastasis is a primary cause of cancer‐related mortality. The occurrence of colorectal liver metastasis (CRLM) is closely associated with tumor immune escape, through which cancer cells evade immune surveillance via multiple mechanisms, thereby promoting metastatic dissemination. RNA‐binding proteins (RBPs) play crucial roles in gene expression regulation. Neuro‐oncological ventral antigen 1 (NOVA1), an important RBP, is often dysregulated in cancer progression. This study aimed to investigate the molecular mechanisms underlying the role of NOVA1 in CRLM. Methods The expression levels of NOVA1 and NRXN1 in CRC tissues and cell lines were detected by quantitative real‐time polymerase chain reaction (RT‐qPCR) and Western blot. Cell proliferation, migration, and invasion were evaluated using Cell Counting Kit‐8 (CCK‐8), colony formation assays, Transwell assays and wound healing assays. Flow cytometry was used to analyze CRC cell apoptosis. Subsequently, CRC cells were co‐cultured with CD8 + T cells. Flow cytometry, enzyme‐linked immunosorbent assay (ELISA), and lactate dehydrogenase (LDH) release assay were used to measure CD8 + T‐cell proliferation, apoptosis, cytokine secretion (tumor necrosis factor‐α [TNF‐α] and interferon‐γ [IFN‐γ]), and cytotoxic activity. RNA immunoprecipitation (RIP), RNA pull‐down, and actinomycin D assays were performed to verify the binding of NOVA1 to NRXN1 mRNA and its regulatory effect on mRNA stability. In vivo validation was performed using a mouse liver metastasis model. Liver metastasis was observed monitored by live imaging, and liver metastatic foci were quantified using hematoxylin and eosin (H&E) staining. The expression of Ki‐67, CD8, and programmed death‐ligand 1 (PD‐L1) in tumor tissues was detected by immunohistochemistry (IHC) and immunofluorescence (IF). Results NOVA1 expression was downregulated in CRC tissues and cell lines. Functional assays demonstrated that NOVA1 overexpression inhibited CRC cell proliferation (colony formation reduced by approximately 50%), migration (migrated cells reduced by approximately 40%), and immune escape (CD8 + T cell proliferation increased by approximately 45%) in vitro, whereas NOVA1 knockdown promoted these processes. RIP and mRNA stability assays revealed that NOVA1 binds to NRXN1 mRNA (5.16‐fold enrichment over IgG) and enhances its stability (a 30% increase relative to the LV‐NC group), thereby upregulating NRXN1 expression. Rescue experiments showed that NRXN1 knockdown reversed the inhibitory effects of NOVA1 overexpression on CRC cell immune escape (evidenced by a 55% decrease in CD8 + T cell proliferation) and malignant progression (colony formation and migrated cells increased by approximately 40%–50%). In vivo experiments confirmed that compared with the control group, NOVA1 overexpression reduced the number of liver metastatic foci by 50%; decreased percentages of Ki‐67‐positive and PD‐L1‐positive cells by 40% and 30%, respectively; and increased CD8 + T cell infiltration by 65%. Conversely, NRXN1 knockdown increased liver metastatic foci by 30%; increased percentages of Ki‐67‐positive and PD‐L1‐positive cells by 40% and 30%, respectively; and reduced CD8 + T cell infiltration by 45%. Conclusion NOVA1 influences the immune escape, proliferation, and migration of CRC cells by directly binding to NRXN1 mRNA and regulating its mRNA stability. This study reveals the key regulatory role of the NOVA1‐NRXN1 axis in CRLM, suggesting that this axis may serve as a potential therapeutic target and prognostic biomarker. Targeting the NOVA1‐NRXN1 signaling pathway may provide a novel strategy for clinical intervention in CRLM.