摘要： 目的 通过大数据挖掘与实验相结合，探讨CDK5对胃癌PD-L1表达及生物学特性的影响。方法 通过癌症基因组图谱（TCGA）数据库下载胃癌FPKM数据，通过R软件及其相关软件包依次进行CDK5与PD-L1表达及其相关性分析、CDK5高低表达分组的差异基因的基因本体（GO）和京都基因和基因组数据库（KEGG）通路富集分析，以及CDK5与免疫检查点、免疫细胞间相关性等生物信息学分析；通过CCK-8、Transwell、实时PCR、Western blotting、流式细胞术等检测CDK5抑制剂20-223对胃癌BGC-823细胞增殖、迁移、PD-L1表达、细胞周期及凋亡等的影响。结果 通过大数据发现CDK5在多种癌症中均高表达；CDK5与PD-L1在胃癌中高表达，且呈正相关。CDK5抑制剂抑制CDK5后，胃癌BGC-823细胞PD-L1蛋白的表达下降（P=0.013 4），PD-L1 mRNA表达水平也下降（P=0.000 4）；过表达CDK5上调PD-L1 mRNA表达（P=0.005 9）。CDK5抑制剂可显著抑制IFN-γ促进的PD-L1转录（P < 0.000 1）。抑制CDK5后，细胞周期阻滞于G₂/M期；细胞凋亡增加，40 μmol/L组总凋亡细胞比例高于对照组（P=0.037 7），80 μmol/L组总凋亡较40 μmol/L组进一步增加（P=0.000 5）；细胞迁移减少，Transwell实验结果显示，CDK5抑制剂组细胞穿过数量明显低于对照组（P=0.002 5）；划痕实验结果显示，CDK5抑制剂组迁移率明显低于对照组（4.39% vs 24.37%，P=0.002 6）。CDK5抑制剂浓度0.04 μmol/L组细胞克隆形成能力明显低于对照组（克隆形成率32.60% vs 80.87%，P=0.000 4）。结论 CDK5与PD-L1在胃癌中均高表达且呈正相关。CDK5通过IFN-γ通路促进胃癌BGC-823细胞PD-L1表达。CDK5抑制剂可促进胃癌细胞凋亡，抑制细胞周期、细胞迁移能力及克隆形成能力。

CDK5对胃癌PD-L1表达及生物学特性的影响

黄谊强¹, 王子明¹, 罗杨¹, 罗水妹¹, 谢贤和^1,2,3, 张帆^1,2,3

1. 福建医科大学附属第一医院肿瘤内科, 福建医科大学分子肿瘤研究所, 福州 350005;
2. 福建医科大学附属第一医院滨海院区国家区域医疗中心肿瘤内科, 福州 350212;
3. 福建省肿瘤精准诊疗重点实验室, 福州 350005

收稿日期:2023-04-12出版日期:2023-09-30发布日期:2023-09-02
通讯作者:张帆E-mail:zhangfan@fjmu.edu.cn
作者简介:黄谊强(1993-),男,硕士研究生.
基金资助:福建省卫生健康科技计划项目医学创新课题（2021CXB004）


关键词: 胃癌, CDK5, PD-L1
Abstract: Objective To investigate the effect of cyclin-dependent kinase 5 (CDK5) on PD-L1 expression and biological characteristics of gastric cancer using big data mining combined with experiments. Methods The FPKM data of gastric cancer were obtained from the TCGA database, and the expression and correlation of CDK5 and PD-L1 were analyzed. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway-enrichment analyses of the differentially expressed genes grouped by CDK5 expression levels were sequentially conducted using R and other related software. CDK5 and immune checkpoints, immune cell correlation, and other parameters were analyzed using bioinformatics. The effects of CDK5 inhibitor 20-223 on the cell proliferation and migration, cell cycle, apoptosis, and PD-L1 expression were analyzed in BGC-823 gastric cancer cells using various techniques, including CCK-8 and Transwell assays, real-time PCR, Western blotting, and flow cytometry. Results Big data mining revealed that CDK5 is highly expressed in various cancers, and both CDK5 and PD-L1 are highly expressed and positively correlated in gastric cancer. The mRNA level and protein expression of PD-L1 decreased after CDK5 inhibition in BGC-823 gastric cancer cells. Overexpressed CDK5 up-regulated PD-L1 mRNA (P=0.005 9);however, CDK5 inhibition significantly rescued the transcription of PD-L1 promoted by IFN-γ (P < 0.000 1). Inhibition of CDK5 caused cell-cycle arrest in the G₂/M phase and increased cell apoptosis than that in the control group. The proportion of total apoptotic cells increased in the 40 μmol/Lgroup (P=0.037 7);further, the proportion in the 80 μmol/L group was higher than that in the 40 μmol/L group (P=0.000 5). Moreover, cell migration was reduced. The Transwell assay detected that the number of migrating cells in the CDK5 inhibitor group, with 15.20 cells/field of view (20 times objective lens), was significantly lower than that in the control group, with 65.87 cells/field of view (P=0.002 5). The scratch test revealed that the cell mobility after CDK5 inhibition (4.39%) was significantly lower than that of the control group (24.37%) without CDK5 inhibition (P=0.002 6). The clonal formation rate reduced from 80.87% in the control cells to 32.60% in the cells exposed to 0.04 μmol/L of CDK5 inhibitor (P=0.000 4). Conclusion CDK5 and PD-L1 are highly expressed and positively correlated in gastric cancer. CDK5 promotes the expression of PD-L1 through the IFN-γ pathway in BGC-823 cells. Moreover, in BGC-823 cells, the CDK5 inhibitor promotes cell apoptosis, whereas it inhibits cell cycle, cell migration, and colony formation.
Key words: gastric cancer, cyclin-dependent kinase 5, PD-L1
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