Galectin-4调控PD-L1影响CD8^(+)T细胞杀伤肝癌细胞的功能  

Galectin-4 regulates PD-L1 and affects the function of CD8~+T cells in killing liver cancer cells

作　　者：魏晓环 单前前 刘媛媛[3] WEI Xiaohuan;SHAN Qianqian;LIU Yuanyuan(Cancer Department of Xuzhou Central Hospital,Xuzhou 221000,China;Pain Department of the Second Affiliated Hospital of Xuzhou Medical University,Xuzhou 221006,China;Respiratory Department of Xuzhou Central Hospital,Xuzhou 221000,China)

机构地区：[1]徐州市中心医院肿瘤科,徐州221000 [2]徐州医科大学第二附属医院疼痛科,徐州221006 [3]徐州市中心医院呼吸科,徐州221006

出　　处：《中国癌症防治杂志》2023年第5期532-537,共6页CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT

基　　金：江苏省研究生科研创新计划项目(KYCX20-2494)。

摘　　要：目的 探讨人半乳糖凝集素4(Galectin-4,Gal-4)影响CD8^(+)T细胞杀伤肝癌细胞的功能及机制。方法 提取并鉴定人外周血CD8^(+)T细胞及树突状细胞(dendritic cells,DC)。选取人正常肝细胞WRL68和肝癌细胞HepG2、SMMC-7721、Hep3B、Huh-7,并利用Western blot法检测Gal-4蛋白的表达。利用Lipofectamine 3000试剂在肝癌细胞HepG2和SMMC-7721中转染Gal-4 siRNA及其阴性对照(si-Gal-4组和si-NC组),si-Gal-4组和si-NC组HepG2和SMMC-7721细胞分别与被DC细胞活化的CD8^(+)T细胞共孵育18 h,依次记为si-Gal-4/HepG2+CD8^(+)T组、si-NC/HepG2+CD8^(+)T组、si-Gal-4/SMMC-7721+CD8^(+)T组、si-NC/SMMC-7721+CD8^(+)T组。采用细胞毒性实验检测CD8^(+)T细胞对各组肿瘤细胞的杀伤能力,酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)检测各组细胞上清液中干扰素(interferon-γ,INF-γ)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的浓度,Western blot法检测各组肿瘤细胞中PD-L1蛋白的表达。结果 相比于正常肝细胞WRL68,肝癌细胞HepG2、SMMC-7721、Hep3B、Huh-7中Gal-4蛋白表达水平均显著上调(均P<0.001);相比于si-NC/HepG2+CD8^(+)T组或si-NC/SMMC-7721+CD8^(+)T组,si-Gal-4/HepG2+CD8^(+)T组和si-Gal-4/SMMC-7721+CD8^(+)T组CD8^(+)T细胞杀伤能力均显著增加(均P<0.001),且抑制Gal-4表达后,共培养体系中INF-γ和TNF-α的浓度均显著增加(均P<0.001)。相比于si-NC组,si-Gal-4组HepG2和SMMC-7721细胞PD-L1蛋白表达显著下调(P<0.001)。结论 Gal-4在肝癌细胞中高表达,抑制Gal-4可能下调肝癌细胞PD-L1的表达,进而促进CD8^(+)T细胞的杀伤能力。Objective To investigate the effect of human Galectin-4 (Gal-4) on CD8~+T cells killing liver cells and its mechanism.MethodsThe 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.ResultsCompared with normal liver cell WRL68,the expression of Gal-4protein 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-γand TNF-α in co-culture system were significantly increased (all P<0.001).Compared with the si-NC group,the expression of PD-L1protein in HepG2 and SMMC-7721 cells of si-Gal-4 group were significantly down-regulated (P<0.001).ConclusionsGal-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 kill

关 键 词：肝癌 人半乳糖凝集素4 CD8^(+)T细胞 PD-L1 

分 类 号：R735.7[医药卫生—肿瘤]