[刚地弓形虫I型状体蛋白16对肺腺癌细胞程序性细胞死亡配体1表达及其与程序性细胞死亡1结合的影响]。

Q3 Medicine

中国血吸虫病防治杂志 Pub Date : 2024-10-29 DOI:10.16250/j.32.1915.2024162

G Li, Y Zhou, S Ma, M Tian, T Dang, Z Zhao

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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 using 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 (allP 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 IROP16 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 IROP16 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 IROP16 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 IROP16 protein overexpression group than in the blank control group and the negative control group (both P values < 0.05), indicating that T. gondii type IROP16 protein promoted the PD-L1/PD-1 binding in A549 cells in a concentration-dose manner.Conclusions: T. gondii type IROP16 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.","PeriodicalId":38874,"journal":{"name":"中国血吸虫病防治杂志","volume":"37 1","pages":"44-54"},"PeriodicalIF":0.0000,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"[Impact of Toxoplasma gondii type I rhoptry protein 16 on programmed cell death ligand 1 expression and its binding to programmed cell death 1 in lung adenocarcinoma cells].\",\"authors\":\"G Li, Y Zhou, S Ma, M Tian, T Dang, Z Zhao\",\"doi\":\"10.16250/j.32.1915.2024162\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Objective: To investigate the impact of Toxoplasma gondii type I, II and III 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 I ROP16 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. 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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 IROP16 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 IROP16 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 IROP16 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 IROP16 protein overexpression group than in the blank control group and the negative control group (both P values < 0.05), indicating that T. gondii type IROP16 protein promoted the PD-L1/PD-1 binding in A549 cells in a concentration-dose manner.Conclusions: T. gondii type IROP16 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.\",\"PeriodicalId\":38874,\"journal\":{\"name\":\"中国血吸虫病防治杂志\",\"volume\":\"37 1\",\"pages\":\"44-54\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-10-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"中国血吸虫病防治杂志\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.16250/j.32.1915.2024162\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"Medicine\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"中国血吸虫病防治杂志","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.16250/j.32.1915.2024162","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Medicine","Score":null,"Total":0}

引用次数: 0

摘要

目的：探讨刚地弓形虫I、II、III型感染蛋白16 （ROP16）对肺腺癌细胞程序性细胞死亡配体1 （PD-L1）表达的影响，探讨刚地弓形虫I型感染蛋白ROP16对肺腺癌细胞中PD-L1的相对表达、PD-L1在细胞膜表面的相对分布以及程序性细胞死亡1 （PD-1）与PD-L1结合的影响。方法：制备过表达刚地弓形虫ⅰ、ⅱ、ⅲ型ROP16蛋白的慢病毒载体，转染人肺腺癌A549细胞系。A549细胞为空白对照组，转染空慢病毒表达载体的A549细胞为阴性对照组，转染过表达弓形虫I、II、III型ROP16蛋白的慢病毒载体的A549细胞为实验组。用嘌呤霉素选择稳定转染的细胞，并用Western blotting、RT-qPCR和免疫荧光检测进行验证。采用Western blotting和RT-qPCR检测5组A549细胞中PD-L1在翻译和转录水平的表达，采用流式细胞术检测A549细胞膜表面PD-L1的相对分布。此外，采用酶联免疫吸附法（ELISA）检测了弓形虫I型ROP16蛋白对A549细胞PD-1/PD-L1结合的影响。结果:相对ROP16蛋白表达是0,0,1.546±0.091,1.822±0.047,2.334±0.089空白对照组,负对照组,和弓形虫类型I, II和III ROP16蛋白进行靶向治疗组(F = 1 339.00, P < 0.001),和相对ROP16 mRNA表达为2.153±0.949,2.436±1.614,14.343±0.020,12.577±0.285,15.090±0.420空白对照组、阴性对照组和弓形虫类型I, II和III ROP16蛋白进行靶向治疗组,（F = 483.50,P < 0.001）。弓形虫I型、II型和III型ROP16蛋白过表达组在翻译和转录水平上均高于空白对照组（p值均< 0.001）。免疫荧光分析显示，刚地弓形虫I型、II型和III型ROP16蛋白主要定位于A549细胞核。免疫印迹显示相对PD-L1蛋白表达为0.685±0.109,0.589±0.114,1.007±0.117,0.572±0.151,0.426±0.116空白对照组,负对照组,和弓形虫类型I, II和III ROP16蛋白进行靶向治疗组(F = 9.46, P < 0.05), RT-qPCR分析量化相对PD-L1 mRNA表达为1.012±0.190,1.281±0.465,1.950±0.175,0.889±0.251,0.230±0.192空白对照组,负对照组,弓形虫I、II、III型ROP16蛋白过表达组（F = 14.18,P < 0.05）。弓形虫型IROP16蛋白过表达组PD-L1在翻译和转录水平上的表达均高于空白对照组（P值均< 0.05）。流式细胞术检测到PD-L1蛋白在A549细胞膜表面的相对分布分别为（10.83±0.60）%、（11.23±0.20）%和（14.61±0.50）% (F = 28.31, P < 0.05)，且弓形虫型IROP16蛋白过表达组PD-L1蛋白的相对分布高于空白对照组和阴性对照组（P值均< 0.001）。ELISA检测重组PD-1蛋白浓度分别为0.04 （F = 10.45, P < 0.05）、0.08 μg/mL （F = 11.68, P < 0.05）、0.12 μg/mL （F = 52.68, P < 0.05）时，弓形虫型IROP16蛋白过表达组、空白对照组和阴性对照组的吸光度(A)值差异有统计学意义；弓形虫IROP16蛋白过表达组的A值高于空白对照组和阴性对照组（P值均< 0.05），说明弓形虫IROP16蛋白以浓度-剂量方式促进了A549细胞中PD-L1/PD-1的结合。结论：弓形虫型IROP16蛋白过表达可在转录和翻译水平上调A549细胞中PD-L1的表达以及PD-L1在A549细胞膜表面的相对分布，并以浓度依赖的方式影响PD-1/PD-L1的结合。

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[Impact of Toxoplasma gondii type I rhoptry protein 16 on programmed cell death ligand 1 expression and its binding to programmed cell death 1 in lung adenocarcinoma cells].

Objective: To investigate the impact of Toxoplasma gondii type I, II and III 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 I ROP16 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 using 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 (allP 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 IROP16 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 IROP16 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 IROP16 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 IROP16 protein overexpression group than in the blank control group and the negative control group (both P values < 0.05), indicating that T. gondii type IROP16 protein promoted the PD-L1/PD-1 binding in A549 cells in a concentration-dose manner.

Conclusions: T. gondii type IROP16 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.

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来源期刊

中国血吸虫病防治杂志 Medicine-Medicine (all)

CiteScore

1.30

自引率

0.00%

发文量

7021

期刊介绍： Chinese Journal of Schistosomiasis Control (ISSN: 1005-6661, CN: 32-1374/R), founded in 1989, is a technical and scientific journal under the supervision of Jiangsu Provincial Health Commission and organised by Jiangsu Institute of Schistosomiasis Control. It is a scientific and technical journal under the supervision of Jiangsu Provincial Health Commission and sponsored by Jiangsu Institute of Schistosomiasis Prevention and Control. The journal carries out the policy of prevention-oriented, control-oriented, nationwide and grassroots, adheres to the tenet of scientific research service for the prevention and treatment of schistosomiasis and other parasitic diseases, and mainly publishes academic papers reflecting the latest achievements and dynamics of prevention and treatment of schistosomiasis and other parasitic diseases, scientific research and management, etc. The main columns are Guest Contributions, Experts‘ Commentary, Experts’ Perspectives, Experts' Forums, Theses, Prevention and Treatment Research, Experimental Research, The main columns include Guest Contributions, Expert Commentaries, Expert Perspectives, Expert Forums, Treatises, Prevention and Control Studies, Experimental Studies, Clinical Studies, Prevention and Control Experiences, Prevention and Control Management, Reviews, Case Reports, and Information, etc. The journal is a useful reference material for the professional and technical personnel of schistosomiasis and parasitic disease prevention and control research, management workers, and teachers and students of medical schools. 　　 The journal is now included in important domestic databases, such as Chinese Core List (8th edition), China Science Citation Database (Core Edition), China Science and Technology Core Journals (Statistical Source Journals), and is also included in MEDLINE/PubMed, Scopus, EBSCO, Chemical Abstract, Embase, Zoological Record, JSTChina, Ulrichsweb, Western Pacific Region Index Medicus, CABI and other international authoritative databases.