细胞程式死亡-配体1通过糖基化修饰促进结直肠癌转移的机制

Mechanism of programmed cell death 1 ligand 1 promoting colorectal cancer metastasis by glycosylation modification

目的探讨细胞程式死亡-配体1(PD-L1)糖基化修饰在结直肠癌转移中的机制。方法对2014年至2015年中山大学附属第六医院收治的401例结直肠癌患者的临床资料进行总结,其中PD-L1低表达组321例,高表达组80例,并进行卡方检验及生存分析。应用免疫组织化学及免疫印迹及独立样本t检验检测PD-L1在发生转移与否的结直肠癌肿瘤组织中的表达水平及相对分子质量差异;构建PD-L1-Knock-out稳表达细胞株并采用Transwell检测并使用独立样本t检验验证其侵袭迁移能力变化;通过糖基酶实验明确PD-L1是否存在糖基化修饰并通过蛋白质谱分析鉴定糖基化位点。结果PD-L1在发生转移的结直肠癌患者中高表达(0.260±0.029比0.810±0.036),且PD-L1相对分子质量由33000处位移至约45000处;PD-L1-Knock-out的细胞株其侵袭迁移能力变弱[PD-L1-Knock-out细胞株(92.330±10.400)个比对照株(175.000±9.074)个,t=5.990,P<0.01],并证实PD-L1在结直肠癌中存在N-糖基化修饰且N192/N200为关键的糖基化位点。结论PD-L1在转移性结肠癌组织中高表达,其内在机制可能与PD-L1的N192及N200位点糖基化修饰相关。

Objective To investigate the role and mechanism of Programmed cell death 1 ligand 1(PD-L1)glycosylation in colorectal cancer metastasis.Methods The clinical data of 401 patients with colorectal cancer were summarized,including 321 patients in the low expression group and 80 patients in the high expression group with PD-L1.Chi-square test was performed on the clinical data of the patients and survival analysis was conducted on the prognosis.Immunohistochemistry,western blotting and independent sample t test were used to detect the expression level and molecular weight difference of PD-L1 in colorectal cancer tumor tissues with or without metastasis.The PD-L1-knock out cell line was constructed and Transwell assay and independent sample t test were used to detect the change of invasion and migration ability.The glycosylation sites of PD-L1 were identified by glycosylation experiments and glycosylation sites were identified by protein spectrum analysis.Results Western blotting showed that PD-L1 was highly expressed in colorectal cancer patients with metastasis(0.260±0.029 vs.0.810±0.036),and the molecular weight of PD-L1 shifted from 33000 to about 45000.The invasion and migration ability of PD-L1-knock out cell line was weakened[PD-L1-knock out cell line:(92.330±10.400)cells vs.The control group:(175.000±9.074)cells,t=5.990,P<0.01].Glycosylase and protein spectrum analysis confirmed the existence of n-glycosylation modification of PD-L1 in colorectal cancer and preliminarily confirmed that N192/N200 was the key glycosylation site.Conclusion PD-L1 is highly expressed in metastatic colorectal cancer tissues.The internal mechanism may be related to glycation modification at N192 and N200 sites of colorectal.