PIGU overexpression and regulation of cellular biological functions in bladder cancer

Figure 1 Immunohistochemistry detection of PIGU protein expression in bladder cancer tissues. PIGU protein expression in para-cancerous tissues. A: Negative, × 200 (left), × 400 (right); B: Weakly positive, × 200 (left), × 400 (right); C-E: PIGU protein expression in bladder cancer (BLCA) tissue, all 12 points, × 200 (left), × 400 (right), pathological grade I-III (C: PIGU protein expression in BLCA tissue, positive, × 200-left, × 400-right); F: Scatter plot of immunohistochemical results; G: Receiver operating characteristic curve. IHC: Immunohistochemistry.

Figure 2 Analysis of all included microarray and RNA-seq datasets. A-H: Scatter plots of PIGU expression across datasets; I-P: Receiver operating characteristic curves for PIGU in each dataset. AUC: Area under the curve.

Figure 3 Comprehensive analysis indicates that PIGU mRNA expression is upregulated in bladder cancer. A: Standardized mean difference forest plot for PIGU; B: Begg test; C: Egger’s publication bias test. SMD: Standardized mean difference.

Figure 4 Differential testing of PIGU in bladder cancer and non-bladder cancer tissues. A: Receiver operating characteristic curve summary based on all included datasets; B: Sensitivity and specificity; C: Positive likelihood ratio and negative likelihood ratio; D: Fagan plot; E: Deek’s publication bias test. AUC: Area under the curve; SROC: Summary receiver operating characteristic.

Figure 5 Prognostic value of PIGU mRNA expression levels in bladder cancer. A and B: Kaplan-Meier curves for overall survival and disease-free survival in bladder cancer patients with high- and low-expression PIGU groups, where the bottom 25% represents the low-expression group and the top 25% represents the high-expression group; C: Forest plot of univariate Cox regression analysis; D: Forest plot of multivariate Cox regression analysis.

Figure 6 Relative expression levels of PIGU in EJ-1, 5637, J82, and HT-1376 bladder cancer cells. A: Relative expression levels of the PIGU gene; B: Relative expression levels of PIGU protein. ^aP < 0.001, ^bP < 0.0001.

Figure 7 Relative expression levels of PIGU in bladder cancer cells infected with silencing and empty vector lentiviruses. ^aP < 0.0001 vs group HT-1376, ^bP < 0.0001 vs group HT-1376 + siNC.

Figure 8 Effects of silent PIGU on in vitro proliferation of bladder cancer cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.0001.

Figure 9 Effect of silencing PIGU on apoptosis in bladder cancer cells. ^aP < 0.0001.

Figure 10  Effects of silencing PIGU on the cell cycle of bladder cancer cells. A: Experimental results for the HT-1376 + siNC group; B: Experimental results for the HT-1376 + siPIGU group. ^aP < 0.0001, ^bP < 0.001.

Figure 11  Enrichment analysis of PIGU differentially co-expressed genes and protein-protein interaction network analysis. A: Biological process in Gene Ontology enrichment analysis; B: Kyoto Encyclopedia of Genes and Genomes enrichment analysis; C and D: Cellular component and molecular function analysis of graphene oxide; E and F: Protein-protein interaction network analysis (E: The String database; F: Gene MANIA database). BP: Biological process; KEGG: Kyoto Encyclopedia of Genes and Genomes; CC: Cellular component; MF: Molecular function.

Figure 12  Identification of cell cycle-related hub genes. A: Protein interaction results from the String database visualized in Cytoscape; B: Hub genes with the strongest protein interactions identified by the Cytoscape algorithm using the cytoHubba module; color shading indicates interaction strength; C and D: Protein-protein interaction network analysis (C: The String database; D: GeneMANIA database); E: Heatmap showing PIGU correlations with 30 hub genes; F: Forest plot of COX regression analysis for 30 hub genes; G: Scatterplot showing PIGU correlations with identified genes PCNA, MCM2, RRM1, RRM2, and AURKA.

Figure 13  Relationship between PIGU expression and tumor immune infiltration. A: Gene Ontology enrichment analysis of differentially expressed genes in high- and low-PIGU expression groups; B: Kyoto Encyclopedia of Genes and Genomes enrichment analysis; C: Correlation between PIGU expression and immune cell infiltration levels from the TIMER website; D: Correlation between PIGU expression and stromal score, immune score, ESTIMATE score, and tumor purity; E: Differential expression of 13 immune checkpoint genes in high- and low-PIGU expression groups; F: Correlation scatter plots of PIGU with VTCN1, CD28, CD27, CD70, ICOS, TNFRSF9, and TNFRSF18. ^aP < 0.01, ^bP < 0.001.