胃癌间充质干细胞培养上清液上调退变样蛋白4(VGLL4)表达促进胃癌细胞PD-L1表达及肿瘤生长  

Conditional medium of gastric cancer mesenchymal stem cells promotes PD-L1 expression in gastric cancer cells and tumor growth via upregulating VGLL4

作　　者：丁颖 朱伟[1] 王倩倩 陈斌 王梅[1] 赵媛媛[1] DING Ying;ZHU Wei;WANG Qianqian;CHEN Bin;WANG Mei;ZHAO Yuanyuan(School of Medicine,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]江苏大学医学院,江苏镇江212013

出　　处：《细胞与分子免疫学杂志》2022年第4期321-327,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81972313)。

摘　　要：目的探讨胃癌间充质干细胞(GCMSC)上清液(GCMSC-CM)是否通过调节退变样蛋白4(VGLL4)上调胃癌细胞程序性死亡蛋白1配体1(PD-L1)表达并促进胃癌进展。方法首先采用Western blot法检测SGC-7901、HGC-27和MGC-803胃癌细胞VGLL4表达;使用VGLL4特异小干涉RNA(siRNA)抑制HGC-27和SGC-7901细胞VGLL4的表达,转染质粒过表达SGC-7901胃癌细胞VGLL4,将胃癌细胞分为对照组、GCMSC上清处理组、VGLL4干扰或过表达组以及VGLL4干扰联合GCMSC上清处理组。实时定量PCR和Western blot法分别检测SGC-7901胃癌细胞PD-L1的mRNA和蛋白表达,平板克隆实验检测细胞的增殖能力,Transwell^(TM)迁移实验检测细胞迁移能力。构建人外周血单个核细胞过继的NCG免疫缺陷小鼠(PBMC-NCG)皮下瘤模型观察小鼠肿瘤生长情况,将小鼠分为对照组、GCMSC上清处理组、VGLL4敲低组、VGLL4敲低后联合GCMSC上清处理组。实时定量PCR检测肿瘤组织VGLL4和PD-L1的mRNA水平,免疫组织化学染色检测VGLL4、PD-L1、KI-67抗原(ki67)、CD8和颗粒酶B(GZMB)的表达。结果VGLL4在SGC-7901、HGC-27和MGC-803细胞均有表达。干扰SGC-7901和HGC-27细胞VGLL4后PD-L1 mRNA和蛋白表达明显下降。过表达SGC-7901细胞VGLL4后PD-L1 mRNA和蛋白表达上调。GCMSC上清处理HGC-27和MGC-803细胞后VGLL4表达升高,干扰HGC-27细胞VGLL4表达能够逆转上清对PD-L1的上调。干扰SGC-7901细胞VGLL4表达使GCMSC上清促细胞增殖和迁移的能力减弱。敲低VGLL4抑制了GCMSC上清促小鼠肿瘤生长作用并增强PBMC-NCG小鼠抗肿瘤免疫作用。结论GCMSC上清液通过上调胃癌细胞VGLL4表达促进PD-L1表达及肿瘤生长。Objective To investigate whether conditional medium of gastric cancer mesenchymal stem cells(GCMSCs-CM)could up-regulate the expression of programmed death 1 ligand 1(PD-L1)and promote gastric cancer progression by vestigial-like protein 4(VGLL4).Methods Western blot was used to detect the expression of VGLL4 in SGC-7901,HGC-27 and MGC-803 gastric cancer cells.The expression of VGLL4 in HGC-27 and SGC-7901 cells was inhibited by being transfected with VGLL4 specific siRNA and plasmid was used to overexpress VGLL4 in SGC-7901 cells.The gastric cancer cells were divided into control group,GCMSCs-CM treatment group,VGLL4 interference or overexpression group,and VGLL4 interference followed by GCMSCs-CM treatment group.The expression of PD-L1 in each group was detected by Western blot and real time quantitative PCR(qRT-PCR).The proliferation of SGC-7901 cells was detected by colony formation assay.The migration of SGC-7901 cells was detected by Transwell^(TM) migration assay.Human peripheral blood mononuclear cells adoptive NCG immunodeficiency mouse(PBMCs-NCG)subcutaneous tumor model was constructed to observe the tumor growth in mice.The mice were divided into control group,GCMSCs-CM treatment group,VGLL4 knockdown group,and VGLL4 knockdown followed by GCMSCs-CM treatment group.VGLL4 and PD-L1 mRNA levels of tumor tissue were detected by qRT-PCR.Immunohistochemical staining was used to detect the expression of VGLL4,PD-L1,ki67,CD8 and granzyme B(GZMB).Results VGLL4 is expressed in SGC-7901,HGC-27 and MGC-803 cells.The expression ofPD-L1 mRNA and protein decreased significantly in SGC-7901 and HGC-27 cells transfected with siVGLL4.The expression of PD-L1 mRNA and protein was up-regulated after overexpression of VGLL4 in SGC-7901 cells.The expression of VGLL4 increased after GCMSCs-CM treatment of HGC-27 and MGC-803 cells.Inhibition of VGLL4 in HGC-27 could reverse the up-regulation of PD-L1 by GCMSCs-CM.Inhibition of VGLL4 in SGC-7901 weakened the ability of GCMSCs-CM to promote cell proliferation and migration.Knock

关 键 词：间充质干细胞(MSC) 胃癌 退变样蛋白4(VGLL4) 程序性死亡蛋白1配体1(PD-L1) 

分 类 号：Q279[生物学—细胞生物学] R735.2[医药卫生—肿瘤] R392-33[医药卫生—临床医学]