基于Notch1信号活化探讨胃癌细胞诱导肿瘤免疫逃逸机制研究

Study on the mechanism of gastric cancer cells inducing tumor immune escape based on Notch1 signal activation

目的探讨胃癌标本中Notch1表达与程序性死亡配体1(PD-L1)表达的相关性,并通过体外实验探讨了这种关系在胃癌细胞对T细胞介导的杀伤敏感性中的作用。方法2020年5月至2021年5月,95例胃癌组织标本(男55例和女40例)取自盘锦辽油宝石花医院和锦州医科大学附属第三医院病理科。采用免疫组织化学方法检测标本中PD-L1和Notch1的表达。体外实验中,采用sgRNA-慢病毒处理敲低MGC-803细胞中Notch1的表达,并分为MGC-803对照组(Con组)或MGC-803 Notch1敲低组(KD组)。流式细胞仪细胞分选法(FACS)分析细胞表面PD-L1表达。通过免疫沉淀分析PD-L1和Notch1相互作用,以及PD-L1泛素化情况,免疫荧光测定PD-L1/程序性死亡1(PD-1)结合情况。将慢病毒处理的MGC-803细胞与活化的外周血单个核细胞(PBMC)(1∶1)共培养72 h以考察胃癌细胞对T细胞介导的杀伤敏感性。结果胃癌组织中PD-L1与Notch1表达之间存在高度相关性,PD-L1阳性胃癌组织样本中Notch1高表达的比例高于PD-L1阴性胃癌组织样本(P<0.05)。胃癌细胞系中Notch1表达量均高于人胃黏膜细胞(GES-1)。KD组细胞中和细胞表面PD-L1表达较Con组细胞显著降低(P<0.05)。免疫沉淀证实Notch1与PD-L1可以相互作用并使PD-L1去泛素化。敲低Notch1导致PD-1与MGC-803细胞表面上的PD-1结合降低。与Con组细胞相比,KD组细胞与活化的PBMC细胞(1∶1)共培养72 h的细胞计数显著降低(P<0.001)。结论胃癌组织中Notch1与PD-L1呈正相关,并且Notch1在胃癌组织中可充当PD-L1的上游去泛素化酶,其敲低通过下调PD-L1相关的免疫抑制来抑制肿瘤生长。

Objective To explore the correlation between Notch1 expression and PD-L1 expression in gastric cancer(GC)specimens,and to explore the role of this relationship in the sensitivity of GC cells to T cell-mediated killing through in vitro experiments.Method From May 2020 to May 2021,95 gastric cancer tissue specimens(55 males and 40 females)were collected from Panjin Liaoyou Baoshihua Hospital and the Department of Pathology of the Third Affiliated Hospital of Jinzhou Medical University.The expressions of PD-L1 and Notch1 in 95 cases of gastric cancer were detected by immunohistochemistry.In vitro,sgRNA lentivirus treatment was used to knock down Notch1 expression in MGC-803 cells,and they were divided into MGC-803 control group(Con)or MGC-803 Notch1 knockdown group(KD).FACS was used to analyze PD-L1 expression on cell surface.The interaction between PD-L1 and Notch1 and the ubiquitination of PD-L1 were analyzed by immunoprecipitation,and the binding of PD-L1/PD-1 was determined by immunofluorescence.The lentivirus treated MGC-803 cells were co cultured with activated PBMC cells(1:1)for 72 h to investigate the sensitivity of GC cells to T cell-mediated cytotoxicity.Result There was a high correlation between the expression of PD-L1 and Notch-1 in gastric cancer tissues,and the proportion of high expression of Notch-1 cells in PD-L1-positive GC samples was much higher than that in PD-L1-negative GC samples(P<0.05).The expression of Notch1 in GC cells was higher than that in human gastric mucosal cells(GES-1)cells.When Notch1 was knocked down in KD group cells,the expression of PD-L1 in cell and cell surface was significantly reduced compared to Con group cells(P<0.05).Immunoprecipitation confirmed that Notch1 interacted with PD-L1 and deubiquitinates PD-L1.Notch1 knockdown resulted in decreased PD-1 binding to MGC-803 cells.In addition,compared with Con group cells,the cell count of KD group cells co cultured with activated PBMC cells(1:1)for 72 h was significantly lower(P<0.001).Conclusion Notch1 can act as an upstream deubiquitinase of PD-L1 in GC,and its knockdown inhibits tumor growth by down-regulating PD-L1-associated immunosuppressionn.