Single-nucleotide genome editing uncovers the biological impact of rs16944 in the IL1B promoter

Abstract

Interleukin-1β (IL-1β) exerts context-dependent effects in cancer immunity, promoting both inflammatory tumor progression and antitumor immune responses. The IL1B promoter single-nucleotide polymorphism rs16944 has been associated with poor prognosis in hepatocellular carcinoma, yet its functional impact on immune signaling remains unclear. Here, we used prime editing to generate LSEC-like SK-Hep-1 cells enriched for the rs16944 A allele through precise conversion from the endogenous G/G genotype. The edited cell population exhibited a marked reduction in IL1B mRNA expression and IL-1β secretion without detectable changes in basal endothelial or innate immune surface marker expression. Transcriptomic profiling by RNA sequencing revealed coordinated suppression of cytokine- and chemokine-related genes, including IL1B , CCL2 , CXCL2 , CXCL8 , and IL6 , accompanied by increased ITGA5 expression. Cytometric bead array analysis further demonstrated significantly reduced secretion of IL-8/CXCL8, MCP-1/CCL2, RANTES/CCL5, and MIG/CXCL9, providing protein-level evidence of an altered immune-related secretory profile. Restoration of IL-1β by transient overexpression selectively rescued cytokine and chemokine expression. These findings demonstrate that rs16944 A-allele enrichment is associated with reduced IL-1β expression and broad alterations in immune-related transcriptional and secretory programs, emphasizing the importance of the IL1B genotype in tumor immunology research.