PD-L1/PD-L2对食管鳞癌细胞恶性表型的影响及耐药研究

Effect of PD-L1/PD-L2 on malignant phenotype and drug resistance of esophageal squamous cell carcinoma cells

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20μmol/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40μmol/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。

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 subsequent 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 viability 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 proliferation(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 proliferation 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 20μmol/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 40μmol/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 between the 2 groups was statistically significant(P<0.05).Compared with ctrl group,the cell proliferation activity of OE-PD-L2 group was significantly decreased(P<0.01).Comparing the differentially expressed 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.