ZEB1通过诱导NNMT的表达调控胃癌细胞的免疫逃逸

ZEB1 Regulates Immune Escape of Gastric Cancer Cells via NNMT

目的探索锌指结构E-box结合蛋白1(ZEB1)以及尼克酰胺-N-甲基转移酶(NNMT)在胃癌细胞的免疫逃逸调控中的作用和潜在分子机制。方法使用siRNAs或过表达载体对ZEB1和NNMT的表达进行干扰或上调,经不同处理的胃癌细胞与人外周血单个核细胞(PBMC)共培养。RT-qPCR和蛋白质印迹法(Western blotting)测定胃癌细胞中ZEB1、NNMT和程序性死亡配体1(PD-L1)的表达情况。流式细胞术和Elisa实验分别检测Treg细胞、NK细胞的比例以及细胞因子的含量。结果ZEB1和NNMT均在胃癌细胞中显著上调,干扰ZEB1表达引起了NNMT的下调。此外,干扰ZEB1或NNMT的表达显著抑制胃癌细胞中PD-L1的表达,降低共培养体系中Treg细胞的比例以及TGF-β和IL-10的含量,提高NK细胞占比和IFN-γ、TNF-α的含量。过表达NNMT可逆转干扰ZEB1的表达对免疫逃逸的抑制作用。结论干扰ZEB1表达通过诱导NNMT表达的下调阻碍胃癌细胞发生免疫逃逸。

Objective This study aimed to explore the role and potential mechanism of zinc finger structure e-box binding protein 1(ZEB1)and nicotinamide N-methyltransferase(NNMT)in the regulation of immune escape of gastric cancer(GC)cells.Methods SiRNAs or overexpression vector was used to perform loss-of-function assay on ZEB1 or gain-of-function assay on NNMT in GC cells.After various treatments,GC cells were co-cultured with normal peripheral blood mononuclear cells(PBMC).The expressions of ZEB1,NNMT and programmed death ligand 1(PD-L1)in GC cells were determined by RT-qPCR and Western blotting.Flow cytometry and ELISA were used to detect the distribution of Treg and NK cells and contents of pro-inflammatory cytokines in the co-culture system.Results Both ZEB1 and NNMT were up-regulated in GC cell,and silencing ZEB1 triggered the down-regulation of NNMT.Besides,in the co-culture system,silencing ZEB1 and NNMT inhibited PD-L1 and reduced the proportion of Treg cells and the contents of TGF-βand IL-10,while NK cell proportion and IFN-γand TNF-αcontents were increased.However,NNMT overexpression reversed the inhibitory effect of ZEB1 silencing on immune escape of GC cells.Conclusion Silencing ZEB1 inhibited immune escape of GC cells by inducing NNMT down-regulation.