Abstract: Objective To investigate the regulatory effect of C⁃X⁃C motif chemokine ligand 8 (CXCL8) on the malignant biological behaviors of liver cancer cells, and to elucidate the underlying molecular mechanisms mediated by CKLF⁃like MARVEL transmembrane domain⁃containing protein 6 (CMTM6). The findings are expected to provide potential therapeutic targets and a theoretical foundation for the development of targeted therapy in liver cancer. Methods The expression levels of CXCL8 in the liver cancer cell lines HepG2, MHCC97H, Hep3B, and Huh7 were detected by real⁃time quantitative polymerase chain reaction (qPCR), and Hep3B cells which exhibited moderate expression levels, were selected for subsequent investigation. To establish Hep3B cell models, lentiviral and plasmid transfection were used to generate CXCL8 knockdown (CXCL8KD), CXCL8 overexpression (CXCL8OE), CMTM6 knockdown (CMTM6KD), and CMTM6KD combined with CXCL8OE cell lines. qPCR was performed to measure mRNA expression levels of CXCL8, CMTM6, and markers of epithelial⁃mesenchymal transition (EMT) , including E⁃cadherin, β⁃catenin, N⁃cadherin, and Vimentin. CCK⁃8, wound healing, and Transwell assays were used to detected the cell proliferation, migration, and invasion, respectively. Results CXCL8 knockdown significantly inhibited the proliferation, migration, and invasion of Hep3B cells (all P<0.01), which was associated with an upregulation of E⁃cadherin and β⁃catenin expression (all P<0.05), and a downregulation of N⁃cadherin and Vimentin expression (all P<0.01), whereas CXCL8 overexpression showed opposite effects (all P<0.05). CMTM6 overexpression significantly downregulated CXCL8 mRNA expression (P<0.001), while CMTM6 knockdown significantly upregulated CXCL8 mRNA expression (P<0.001). CMTM6 knockdown significantly inhibited Hep3B cell proliferation, migration, and invasion (all P<0.001), upregulated E⁃cadherin and β⁃catenin (all P<0.05), and downregulated N⁃cadherin and Vimentin (all P<0.05). Notably, simultaneous CXCL8 overexpression partially reversed these effects (all P<0.05). Conclusions CXCL8 has been identified as a downstream target gene of CMTM6 in liver cancer. CXCL8 may promote the proliferation, migration, and invasion of liver cancer cells by inducing EMT, and this mechanism may be associated with the negative regulation of CXCL8 expression by CMTM6 at the transcriptional level.This suggests that targeting the regulatory network involving CMTM6 and CXCL8 may offer a novel therapeutic strategy for the treatment of liver cancer.

Key words: Liver cancer, C?X?C motif chemokine ligand 8 （CXCL8）, Epithelial?mesenchymal transition, CKLF?like MARVEL transmembrane domain?containing ptotein 6 （CMTM6）