Role of PA2G4P4 pseudogene in bladder cancer tumorigenesis

Abstract

Background: Many pseudogenes possess biological activities and play important roles in the pathogenesis of various types of cancer including bladder cancer (BlCa), which still lacks suitable molecular biomarkers. Recently, pseudogenes were found to be significantly enriched in a pan-cancer classification based on the Cancer Genome Atlas gene expression data. Among them, the top-ranking pseudogene was the proliferation-associated 2G4 pseudogene 4 (PA2G4P4).

Methods: Genomic and transcript features of PA2G4P4 were determined by GeneBank database analysis followed by 5' RACE experiments. Therefore, we conducted a retrospective molecular study on a cohort of 45 patients of BlCa. PA2G4P4 expression was measured by RT-qPCR, whereas PA2G4P4 transcript distribution was analyzed by in situ hybridization on both normal and cancerous histological sections and compared to the immunolocalization of its parental PA2G4/EBP1 protein. Finally, we tested the effects of PA2G4P4 depletion on proliferation, migration, and death of BlCa cells.

Results: We showed for the first time PA2G4P4 overexpression in BlCa tissues and in cell lines. PA2G4P4 distribution strictly overlaps PA2G4/EBP1 protein localization. Moreover, we showed that PA2G4P4 knockdown affects both proliferation and migration of BlCa cells, highlighting its potential oncogenic role.

Conclusions: PA2G4P4 may play a functional role as an oncogene in BlCa development, suggesting it as a good candidate for future investigation and new clinical applications.

Keywords: EBP1; bladder cancer; expression; noncoding RNA; oncogene.

Figure 1

PA2G4P4 genomic location and structure. In the upper part of the figure, the relative genomic position and organization of PA2G4P4 and LINC00886 are schematically represented. In the lower part, the PA2G4P4 region is expanded. The gray line schematically represents the annotated PA2G4P4 transcript. Segment A corresponds to the 5′ RACE product, extending the PA2G4P4 region with 104 nucleotides; segment B corresponds to the in situ hybridization probe sequence; segment C spans the PA2G4 homolog region (modified from UCSC BLAT). PA2G4P4, proliferation-associated 2G4 pseudogene 4.

Figure 2

PA2G4P4 expression in BlCa cell lines and tissues. (A) PA2G4P4 expression level measured by RT-qPCR in J82 and RT112 cells (*** p < 0.001). (B) PA2G4P4 expression in BlCa patients (n = 45) compared to NBE samples (n = 34) (* p < 0.05). Data are representative of three indipendent experiments. P-values were obtained in panel (A) using the Student’s t test for independent samples and in panel (B) using the non-parametric Mann–Whitney U test.

Figure 3

Histological analysis of the PA2G4P4 transcript and EBP1 protein. Analysis of PA2G4P4 transcript distribution and comparison with EBP1 protein localization in both urothelial carcinomas and adjacent non-tumoral bladder tissues. Panel (A) refers to non-tumoral bladder tissues, while panel (B) shows the results on urothelial carcinomas. In both cases, serial sections were analyzed by digoxigenin in situ hybridization for PA2G4P4 transcript detection (upper part of the panels) or EBP1 immunohistochemistry (lower part of the panels). Squares indicate the magnified areas. Bars represent 200 μm. PA2G4P4, proliferation-associated 2G4 pseudogene 4; EBP1, ErbB3-binding protein 1; LG, low-grade; HG, high-grade.

Figure 4

Proliferation phenotype of J82 bladder cells following PA2G4P4 silencing. (A) Growth rates evaluated at different time points counted using a Bürker chamber and trypan blue staining. (B) Analysis of cell cycle distribution in the G0/G1, S, and G2/M phases. The upper panel represents the mean of three independent experiments; the lower panel shows flow cytometry histograms of a single representative experiment (*** p < 0.001,* p < 0.05). (C) Analysis of apoptosis following Annexin V and PI staining. The dot plots show the percentage of cell distribution in early apoptosis (positive for Annexin V staining), late apoptosis (double positive for both Annexin V and PI staining), and necrotic cells (single positive for PI staining). PA2G4P4, proliferation-associated 2G4 pseudogene 4. (D) Summary graph of three independent experiments of apoptosis analysis (** p < 0.005).

Figure 5

PA2G4P4 silencing effects on J82 bladder cancer migration. (A) Representative views of the wound healing assay were captured at 0 and 24 h, demonstrating a reduced migration of J82 cells following pseudogene silencing, as compared to the control. The scale bar in the image is 100 µm. Magnification 10X. (B) Quantification of cell migration by measuring the distance between the invading front of the cells in three randomly selected microscopic fields (magnification, x20) for each condition and time point. The degree of motility is expressed as the percentage of wound closure, as compared with the 0-time point. PA2G4P4, proliferation-associated 2G4 pseudogene 4. (*** p < 0.001,* p < 0.05).