关键词: 刚地弓形虫, 棒状体蛋白16, 肺腺癌, 程序性死亡配体?1, 程序性死亡蛋白1

Abstract: Objective　To investigate the impact of Toxoplasma gondii type I, II and II rhoptry protein 16 (ROP16) on programmed cell death ligand 1 (PD⁃L1) expression in lung adenocarcinoma cells, and to examine the effects of T. gondii type IROP16 protein on the relative PD⁃L1 expression, the relative PD⁃L1 distribution on the cell membrane surface, and the binding of programmed cell death 1 (PD⁃1) to PD⁃L1 in lung adenocarcinoma cells. Methods　Lentiviral vectors overexpressing T. gondii type I, II and III ROP16 proteins were generated, and transfected into the human lung adenocarcinoma A549 cell line. A549 cells were used as a blank control group, and A549 cells transfected with an empty lentiviral expression vector were used as a negative control group, while A549 cells transfected with lentiviral vectors overexpressing T. gondii type I, II and III ROP16 proteins served as experimental groups. Stably transfected cells were selected with puromycin and verified using Western blotting, quantitative real⁃time PCR (RT⁃qPCR), and immunofluorescence assays. The PD⁃L1 expression was quantified at translational and transcriptional levels using Western blotting and RT⁃qPCR assays in A549 cells in the five groups, and the relative PD⁃L1 distribution was detected on the A549 cell membrane surface using flow cytometry. In addition, the effect of T. gondii type I ROP16 protein on the PD⁃1/PD⁃L1 binding was measured in A549 cells enzyme⁃linked immunosorbent assay (ELISA).   Results　The relative ROP16 protein expression was 0, 0, (1.546 ± 0.091), (1.822 ± 0.047) and (2.334 ± 0.089) in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 1 339.00, P < 0.001), and the relative ROP16 mRNA expression was (2.153 ± 0.949), (2.436 ± 1.614), (14.343 ± 0.020), (12.577 ± 0.285) and (15.090 ± 0.420) in the blank control group, negative control group and the T. gondii type I, II and III ROP16 protein overexpression groups, respectively (F = 483.50, P < 0.001). The ROP16 expression was higher in the T. gondii type I, II and III ROP16 protein overexpression groups than in the blank control group at both translational and transcriptional levels (all P  values < 0.001). Immunofluorescence assay revealed that T. gondii type I, II and III ROP16 proteins were predominantly localized in A549 cell nuclei. Western blotting showed that the relative PD⁃L1 protein expression was (0.685 ± 0.109), (0.589 ± 0.114), (1.007 ± 0.117), (0.572 ± 0.151), and (0.426 ± 0.116) in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 9.46, P < 0.05), and RT⁃qPCR assay quantified that the relative PD⁃L1 mRNA expression was (1.012 ± 0.190), (1.281 ± 0.465), (1.950 ± 0.175), (0.889 ± 0.251), and (0.230 ± 0.192) in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 14.18, P < 0.05). The PD⁃L1 expression was higher in the T. gondii type I ROP16 protein overexpression group than in the blank control group at both translational and transcriptional levels (both P values < 0.05). Flow cytometry detected that the relative distributions of PD⁃L1 protein were (10.83 ± 0.60)%, (11.23 ± 0.20)%, and (14.61 ± 0.50)% on the A549 cell membrane surface (F = 28.31, P < 0.05), and the relative distribution of PD⁃L1 protein was higher in the T. gondii type I ROP16 protein overexpression group than in the blank control group and negative control group (both P values < 0.001). ELISA measured significant differences in the absorbance (A value) among the T. gondii type I ROP16 protein overexpression group, the blank control group and the negative control group if the concentrations of the recombinant PD⁃1 protein were 0.04 (F = 10.45, P < 0.05), 0.08 μg/mL (F = 11.68, P < 0.05) and 0.12 μg/mL (F = 52.68, P < 0.05), and the A value was higher in the T. gondii type I ROP16 protein overexpression group than in the blank control group and the negative control group (both P values < 0.05), indicating that T. gondii type I ROP16 protein promoted the PD⁃L1/PD⁃1 binding in A549 cells in a concentration⁃dose manner.   Conclusions　T. gondii type I ROP16 protein overexpression may up⁃regulate PD⁃L1 expression in A549 cells at both transcriptional and translational levels and the relative PD⁃L1 distribution on the A549 cell membrane surface, and affect the PD⁃1/PD⁃L1 binding in a concentration⁃dependent manner.  

Key words: Toxoplasma gondii, Rhoptry protein 16, Lung adenocarcinoma, Programmed cell death ligand 1, Programmed cell death protein 1