A New Insight Into p53-Inhibiting Genes in Epstein–Barr Virus-Associated Gastric Adenocarcinoma

Abstract

Background: The p53 mutation is uncommon in Epstein–Barr virus-linked gastric carcinoma, but its suppression occurs through mechanisms such as ubiquitin specific peptidase 7 (USP7) inhibitions via Epstein–Barr virus nuclear antigen-1 (EBNA1) activity. This study aimed to evaluate the effect of EBNA1 on p53-inhibiting gene expression and the impact of USP7 inhibition on p53 suppression.

Methods: MKN-45 cells were transfected with the EBNA1 plasmid. A stable EBNA1 expression cell line was developed through selection based on hygromycin B resistance. Murine double minute (MDM)4, MDM2, sirtuin (SIRT)3, histone deacetylase (HDAC)1, proteasome 26S subunit, Non-ATPase (PSMD)10, USP7, and p53 expression were checked using real-time PCR. Also, cells containing EBNA1 or control plasmid were treated with GNE-6776, and the expression of the interested genes and cell survival were assessed.

Results: MDM4, MDM2, and PSMD10 were significantly upregulated in the MKN-45 cell line following EBNA1 transfection. Morphological changes were observed in the cells harboring EBNA1 after 20 days. In the control cells, USP7 inhibition significantly upregulated the HDAC1, PSMD10, MDM4, and MDM2 genes after 24 h, but downregulated these genes after four days. In the EBNA1-harboring cells, MDM2, MDM4, and PSMD10 genes were significantly upregulated after 24 h, and this effect was sustained for all genes except for MDM4, even after four days. Furthermore, USP7 inhibition induced apoptosis in both cell groups.

Conclusion: EBNA1 enhances the expression of p53-inhibiting genes. Two events—p53 protein overexpression and apoptosis activation—followed the suppression of the USP7 protein and provided evidence for its possible function. The significance of the EBNA1-USP7 interaction in p53 suppression warrants additional investigation and possibly reconsideration.

Keywords: Herpesvirus 4; Tumor suppressor protein p53; USP7 protein.

Fig.1

Morphological changes of MKN-45 cell lines transfected with EBNA1 and control plasmid. (A) Cell line transfected with control plasmid after 20 days; (B and C) abnormal morphology (abnormal cells were indicated by arrows) in the culture of the MKN-45 cell line transfected with EBNA1 after 20 days; (D) pathological staining of control plasmid-transfected cell after 20 days; (E and F) pathological staining of EBNA1 plasmid-harboring cell after 20 days

Fig. 2

Expression of p53-inhibiting genes in MKN-45 cells, and immunocytochemistry (ICC) for p53 and PI/Trypan blue exclusion assays. (A) p53-inhibiting genes expression in MKN-45 cell line after transfection with EBNA1 plasmid; (B) Trypan blue exclusion assay and (C) PI spectrofluorometry for cell viability assay, showing that 15 µM of GNE-6776 is nontoxic for the MKN-45 cells. ICC for p53 in MKN-45 cells transfected with (D) the control plasmid and (E) EBNA1 plasmid; (F) ICC for p53 in MKN-45 cells transfected with EBNA1 plasmid and treated with GNE-6776, p53 upregulated in MKN-45 cell-harboring EBNA1 plasmid following GNE6776 treatment

Fig. 3

Evaluation of p53-inhibiting genes expression following treatment of EBNA1-positive and -negative cells with GNE-6776 after 24 hours and after four days by quantitative PCR. (A) The cells harboring EBNAl plasmid were treated with GNE6776 (test) and DMSO (control) to evaluate the effect of USP7 inhibition on p53 and p53-inhibiting genes in the presence of EBNA1; (B) effects of USP7 inhibitor on mRNA expression of p53-inhibiting genes in the control plasmid-transfected cell. The cells harboring control plasmid were treated with GNE6776 (test) and DMSO (control) to evaluate the general effect of USP7 inhibition on p53 and p53-inhibiting genes

Fig. 4

Comparison effect of GNE6776 on the cell harboring EBNA1 plasmid vs. the cell harboring control plasmid and evaluation of GNE-6776 treatment on cell viability by AO/PI. (A) Effects of USP7 inhibitor on the cell harboring EBNA1 plasmid and control plasmid harboring cell after 24 hours and 4 days; (B) AO/PI test for cell viability after GNE-6776 treatment. The cell harboring control plasmid group had higher yellow cells (in early apoptosis) compared to the cell harboring EBNA1 plasmid group following treatment with GNE-6776