Identification of key genes involved in the inhibition of inflammation by overexpression of PEDF adenovirus in THP1 cells
10.3760/cma.j.cn115989-20230607-00219
- VernacularTitle:THP1细胞过表达腺病毒介导的 PEDF抑制炎症过程中关键基因的鉴别
- Author:
Yuanyuan ZHANG
1
;
Honglian WU
;
Manhong XU
;
Xiaorong LI
;
Yan SHAO
Author Information
1. 天津医科大学眼科医院 天津医科大学眼视光学院 天津医科大学眼科研究所 天津市视网膜功能与疾病重点实验室 天津市眼科学与视觉科学国际联合研究中心，天津 300384
- Keywords:
Diabetic retinopathy;
Inflammation;
Pigment epithelium-derived factor;
Bioinformatics;
CSF1R gene;
THP1 cell
- From:
Chinese Journal of Experimental Ophthalmology
2024;42(10):887-897
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To identify the key genes in the process inhibiting inflammation by overexpression adenovirus-mediated pigment epithelium-derived factor ( PEDF) gene in human monocytic leukemia cells THP1. Methods:Proteomic analysis of THP1 overexpressing adenovirus-mediated PEDF gene was performed.The THP1 cells were divided into GFP and PEDF groups, transfected with GFP and PEDF adenovirus, respectively.The THP1 cells were divided into mannitol group, high glucose group, high glucose+ GFP group, and high glucose+ PEDF group, which were cultured with mannitol for 4 days, anhydrous glucose for 4 days, GFP adenovirus for 3 days, and PEDF adenovirus for 3 days, respectively.The Pedf-/- mice were divided into Pedf-/- group and Pedf-/- diabetes group according to the random table method, with 12 mice in each group.Another 10 C57BL/6 mice were taken as the control group.Mouse retinas were collected for experiments.The mRNA expression levels of differentially expressed genes (DEGs) in retina and THP1 cells were verified by real-time fluorescence quantitative PCR.The DEGs were intersected with the GSE5504 dataset, and the protein-protein interaction (PPI) network was built using the String database.Modules of the PPI were extracted using the Cytoscape software and the MCODE application.Intersections were taken with the Set1 dataset and key genes were found.The expression levels of key genes in THP1 cells and Pedf-/- mice were verified by Western blot.The feeding and operation of experimental animals were in accordance with the regulations of the State Science and Technology Commission on the management of experimental animals and approved by the Animal Management and Use Committee of Tianjin Medical University (No.TTYY2023120217). Results:Through proteomics and bioinformatics analysis, 105 DEGs in the Set1 dataset were screened.The results of real-time PCR showed that the relative expression levels of ARF5, TCF25 and KCTD9 mRNA were significantly higher and the relative expression levels of RNPS1, CSF1R, OGA, IBA57 and MGST2 mRNA were significantly lower in PEDF group than in GFP group, showing statistically significant differences (all at P<0.001).There were significant overall differences in the relative expression levels of down-regulated TCF25, KCTD9 and ARF5 mRNA and up-regulated CSF1R, RNPS1 and IBA57 mRNA among control group, Pedf-/- group and Pedf-/- diabetes group ( F=64.057, 27.561, 37.179, 65.757, 44.024, 34.248; all at P<0.001).Compared with control group, the relative expression levels of TCF25, KCTD9 and ARF5 mRNA were decreased and the relative expression levels of CSF1R and RNPS1 mRNA were increased in Pedf-/- group, showing statistically significant differences (all at P<0.05).Compared with control group, the relative expression levels of TCF25, KCTD9 and ARF5 mRNA were decreased and the relative expression levels of CSF1R, RNPS1 and IBA57 mRNA were increased in Pedf-/- diabetes group, showing statistically significant differences (all at P<0.05).Compared with Pedf-/- group, the relative expression level of TCF25 mRNA was decreased and the relative expression levels of CSF1R, RNPS1 and IBA57 mRNA were increased in Pedf-/- diabetes group, showing statistically significant differences (all at P<0.05).After intersection with the GSE5504 dataset, 20 differential proteins were obtained, which were mainly enriched in positive regulation of gene expression, positive regulation of ERK1 and ERK2 cascade, positive regulation of insulin secretion involved in cell response to glucose stimulation and antigen processing and presentation pathways.The key gene CSF1R was screened by constructing PPI network and MCODE plugin in Cytoscape software.Western blot results showed that the expression levels of CSF1R in high glucose group and high glucose+ GFP group were 1.961±0.085 and 1.000±0.069, which were higher than 1.000±0.072 in mannitol group and 0.469±0.079 in high glucose+ PEDF group, respectively, and the differences were statistically significant ( t=14.940, 8.765; both at P < 0.01).The expression of CSF1R in the retina of Pedf-/- diabetes group was 1.633±0.192, which was higher than 1.000±0.050 in Pedf-/- group, and the difference was statistically significant ( t=5.537, P<0.01). Conclusions:CSF1R may be a key gene and therapeutic target for the inhibition of inflammation by overexpression of adenovirus-mediated PEDF gene in THP1 cell.
