目的 探讨程序性死亡配体1/2(PD-L1/PD-L2)对食管鳞癌细胞增殖、迁移和侵袭的影响及对化疗药物的敏感性.方法 在体外食管鳞癌KYSE150细胞系水平上,分别构建PD-L1杂合子敲除细胞系,慢病毒转染PD-L2过表达,将细胞分为WT组(PD-L1野生型)和PD-L1+/-组(PD-L1杂合子敲除)、Ctrl组(对照)和OE-PD-L2组(PD-L2过表达),用RT-qPCR和 Western blot验证PD-L1杂合子敲除和慢病毒转染PD-L2 过表达效果以用于后续实验.采用EdU、细胞克隆形成实验、Transwell实验检测各组细胞增殖、迁移和侵袭能力.通过CCK8法检测细胞增殖能力并计算抑制率来评估细胞对化疗药物的敏感性.结果 与野生型WT组相比,PD-L1+/-组细胞增殖(P<0.001)、迁移(P<0.01)和侵袭能力(P<0.05)明显降低,差异有统计学意义.与Ctrl组相比,OE-PD-L2组细胞增殖和迁移能力增加(P<0.05),而侵袭能力无明显差异(P>0.05).在顺铂药物梯度浓度干预下,与WT组相比较,PD-L1+/-组细胞增殖活力明显下降,差异具有统计学意义(P<0.05).当顺铂药物浓度为20 μmol/L时,与Ctrl组相比,OE-PD-L2组细胞增殖活力无明显变化(P>0.05),而在40 μmol/L药物浓度作用下,与Ctrl组相比,OE-PD-L2组细胞增殖活力显著上升,差异有统计学意义(P<0.001).加入不同浓度的五氟尿嘧啶后发现,与WT组相比,PD-L1+/-组细胞增殖活力显著升高,差异有统计学意义(P<0.05).与Ctrl组相比,OE-PD-L2组细胞增殖活力显著下降(P<0.01).比较WT组与PD-L1+/-组特异性表达差异的蛋白,共获得25个差异表达蛋白质,其中差异倍数最为显著的上调蛋白为EGFR(Ser1070),下调蛋白为Smad4.结论 PD-L1能够促进食管鳞癌细胞的恶性表型;PD-L2可以促进食管鳞癌细胞(ESCC)的增殖和迁移;PD-L1/PD-L2可以影响肿瘤细胞对顺铂和五氟尿嘧啶化疗药物的敏感性.
Effect of PD-L1/PD-L2 on malignant phenotype and drug resistance of esophageal squamous cell carcinoma cells
Objective To investigate the effect of programmed death-ligand 1/2(PD-L1/PD-L2)on the proliferation,migration and invasion of esophageal squamous cell carcinoma cells and its sensitivity to chemotherapy drugs.Methods PD-L1 heterozygous knockout cell lines were constructed at the level of KYSE150 cell lines in esophageal squamous cell carcinoma in vitro,lentivirus transfection with PD-L2 overexpression,and the cells were divided into WT group(PD-L1 wild type),PD-L1+/-group(PD-L1 heterozygous knockout),ctrl group and OE-PD-L2 group,and the PD-L1 heterozygous knockout and lentivirus transfection PD-L2 overexpression effects were verified by RT-qPCR and Western blot for subse-quent experiments.EdU,clone formation assay and Transwell assay were used to detect the proliferation,migration and invasion of the cells in each group.The effect of chemotherapy drugs on cell proliferation vi-ability was evaluated by CCK8 method to detect cell proliferation capacity and calculate the inhibition rate.Results Compared with the wild-type WT group,the PD-L1+/-group had significantly reduced cell pro-liferation(P<0.001),migration(P<0.01)and invasion ability(P<0.05).Compared with the ctrl group,the OE-PD-L2 group had increased cell proliferation and migration ability(P<0.05),but there was no significant difference in invasion ability(P>0.05).Compared with the WT group,the cell prolif-eration activity of PD-L1+/-group was decreased significantly,and the difference between the 2 groups was statistically significant(P<0.05).When the concentration of cisplatin was 20 pmol/L,there was no significant change in the cell proliferation activity of OE-PD-L2 group compared with ctrl group(P>0.05),while the cell proliferation activity of OE-PD-L2 group was significantly increased compared with ctrl group under the effect of 40 pmol/L drug concentration,and the difference was statistically significant(P<0.001).After adding different concentrations of fluouracil,it was found that compared with WT group,the cell proliferation activity of PD-L1+/-group was significantly increased,and the difference be-tween the 2 groups was statistically significant(P<0.05).Compared with ctrl group,the cell prolifera-tion activity of OE-PD-L2 group was significantly decreased(P<0.01).Comparing the differentially ex-pressed proteins between WT group and PD-L1+/-group,a total of 25 differentially expressed proteins were obtained.Among them,the up-regulated protein with the most significant multiple of differences was EGFR(Ser1070),and the down-regulated protein was Smad4.Conclusion PD-L1 can promote the malignant phenotype of esophageal squamous cell carcinoma cells.PD-L2 can promote the proliferation and migration of esophageal squamous cell carcinoma cells.PD-L1/PD-L2 can affect the sensitivity of tumor cells to cisplatin and fluorouracil chemotherapy drugs.