Abstract: Objective To investigate the effect of human Galectin⁃4 (Gal⁃4) on CD8+T cells killing liver cells and its mechanism. Methods The human peripheral blood CD8+T cells and dendritic cells (DC) were extracted and identified. Normal human liver cells WRL68 and liver cancer cells including HepG2, SMMC⁃7721, Hep3B and Huh⁃7 were selected, and Western blot was used to detect the expression of Gal⁃4 protein. The liver cancer cells HepG2 and SMMC⁃7721 were transfected with Gal⁃4 siRNA and negative control (the si⁃Gal⁃4 group and si⁃NC group) using Lipofectamine 3000. The HepG2 and SMMC⁃7721 cells in the si⁃Gal⁃4 and si⁃NC groups were co⁃incubated with DC⁃activated CD8⁺T cells for 18 hours (the si⁃Gal⁃4/HepG2+CD8⁺T group, si⁃NC/HepG2+CD8⁺T group, si⁃Gal⁃4/SMMC⁃7721+CD8⁺T group, si⁃NC/SMMC⁃7721+CD8⁺T group). Cytotoxicity experiments were performed to evaluate the capability of CD8⁺T cells in killing the tumor cells in each group, and enzyme⁃linked immunosorbent assay (ELISA) was used to detect the levels of interferon ⁃γ (INF⁃ γ) and tumor necrosis factor⁃α (TNF⁃α) in the cell supernatants of each group. Western blot was used to detect the expression of PD⁃L1 protein in the tumor cells of each group. Results Compared with normal liver cell WRL68, the expression of Gal⁃4 protein were significantly up⁃regulated in liver cancer cells HepG2, SMMC⁃7721, Hep3B, and Huh⁃7 (all P<0.001). Compared with the si⁃NC/HepG2+CD8+T group or si⁃NC/SMMC⁃7721+CD8+T group, the killing ability of si⁃Gal⁃4/HepG2+CD8+T group and the si⁃Gal⁃4/SMMC⁃7721+CD8+T group were significant increased (all P<0.001). After inhibiting the expression of Gal⁃4, the concentrations of INF⁃γ and TNF⁃α in co⁃culture system were significantly increased (all P<0.001). Compared with the si⁃NC group, the expression of PD⁃L1 protein in HepG2 and SMMC⁃7721 cells of si⁃Gal⁃4 group were significantly down⁃regulated (P<0.001). Conclusions Gal⁃4 is highly expressed in liver cancer cells, and inhibition of Gal⁃4 may down⁃regulate the expression of PD⁃L1 in liver cancer cells and promote the killing ability of CD8⁺T cells.

Key words:  Liver cancer, Galectin-4, CD8⁺T cells, PD-L1