PSMA5 expression in pan-cancers and Glioma
To explore the involvement of PSMA5 in various types of cancer, RNA-seq data from TCGA and GTEx were acquired from UCSC XENA. PSMA5 exhibited a significant upregulation in 28 cancer types when compared to normal tissue, including low-grade glioma (LGG) and glioblastomas (GBM) as shown in Figure 1A. Subsequently, an immunohistochemical analysis was performed to assess PSMA5 expression in normal brain tissue and 15 glioma tissues, as depicted in Figure 1B. The findings revealed an elevated expression of PSMA5 in glioma tissues relative to normal brain tissues, as illustrated in Figure 1C.
Subtypes of gliomas and PSMA5 expression
In order to ascertain the correlation between PSMA5 expression and glioma subtypes, a total of 310 glioma samples from the Chinese Glioma Genome Atlas (CGGA) RNA-sequencing dataset were acquired for analysis.PSMA5 mRNA was overexpressed in high-grade glioma (Figure 2A) and IDH1 wild-type (Figure 2C). Simailar results were validated in 691 samples from the TCGA datasets (Figure 2 B, D).
PSMA5 serves as an autonomous predictive marker in patients with glioma
A Kaplan-Meier survival curve analysis was conducted to investigate the relationship between PSMA5 mRNA expression and survival duration. The CGGA and TCGA datasets analysis results revealed that higher PSMA5 expression indicated remarkably shorter overall survival within all grade gliomas (Figure 3A and B).The prognostic significance of PSMA5 in glioma patients was assessed through ROC curve analysis, revealing that the area under the curve (AUC) values for PSMA5 expression in predicting survival were 0.706 (1 year), 0.766 (3 years), and 0.781 (5 years) in CGGA datasets (Figure 3C), and 0.750 (1 year), 0.774 (3 years), and 0.757 (5 years) in TCGA datasets (Figure 3D).
Subsequently, independent risk factors were assessed through univariate and multivariate Cox regression analyses. PSMA5 expression (P < 0.001), age (P < 0.001), tumor grade (P < 0.001), and IDH1 status (P < 0.001) were determined as independent risk factors in glioma samples from the CGGA and TCGA datasets (Figure 4A and B). In the multivariate regression analysis, PSMA5 expression remained a significant independent prognostic factor (CGGA: p < 0.01, TCGA: p = 0.032, Figure 4C), along with tumor grade (CGGA: p < 0.01, TCGA: p < 0.01, Figure 4D).
Functional analysis of PSMA5
In order to investigate the impact of PSMA5 on cellular function, a median difference in PSMA5 expression was observed between groups with high and low expression levels. A total of 276 Differentially Expressed Genes (DEGs) from the Chinese Glioma Genome Atlas (CGGA) datasets were identified as statistically significant, with 95 genes upregulated and 181 genes downregulated in the PSMA5 high-expression group compared to the low-expression group (|log fold change (logFC)| > 1.0, P < 0.05) (Figure 5A).We also got 2264 DEGs from TCGA datasets, including 1089 upregulated and 1174 down-regulated(|log fold change (logFC)| > 1.0, P < 0.05) (Figure 5B).The heat map depicted the 20 most up-regulated and 20 most down-regulated Differentially Expressed Genes (DEGs) in the comparison of high- and low-expressed groups of PSMA5 in the Cancer Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) datasets (Figures 5C and 5D).
Hereafter,an analysis of the GO terms and GSEA was performed on PSMA5 in order to determine its biological function. GO annotation revealed that high levels of PSMA5 were positively associated with mitotic sister chromatid segregation, B cell-mediated immunity, adaptive immune response and humoral immune response (Figure 6A and B). GSEA revealed that high levels of PSMA5 were positively associated with G2M checkpoint, epithelial-mesenchymal transition, E2F target and TNF signaling via NFKB in the CGGA and TCGA datasets (Figure 6C and D).
The relationship between immune cell infiltration and PSMA5 expression
The expression levels of PSMA5 in the glioma microenvironment were found to be significantly associated with the degree of immune cell infiltration as determined by single-sample gene set enrichment analysis (SSGSEA), as indicated by Spearman correlation analysis (Figure 7A).The expression of PSMA5 correlated significantly positively with macrophage infiltration and Th2 cell infiltration and adversely with pDC (plasmaacytoid DC) cells(Figure 7B and C).These results indicated that there is a strong correlation between high PSMA5 expression and an immune-activated state of glioma.
PSMA5 promotes proliferation and induces cell cycle arrest of glioma cells in vitro
Furthermore, the correlation of PSMA5 with CDKs expression was investigated. PSMA5 was significantly associated with CDKs (Figure 8A and B). Then, we searched the BioGRID4.3 database and found a physical interaction between PSMA5 and CDK1 and CDK2, proteins well-known for their role in cell cycle regulation, mitotic regulation, and tumorigenesis (Figure 8C) . The results suggested that PSMA5 played a tumor-promoting role in cancers by driving the cell cycle.
To explore the function of PSMA5, we silenced endogenous PSMA5 using siRNA.CCK-8 assays. The depletion of PSMA5 inhibited the proliferation ability of U87 cells and induced G2M cell cycle arrest. Additionally, western blot analyses demonstrated a significant decrease in CDK1 and CDK2 expression in U251 cells following transfection with siRNA (Figure 6B).These findings demonstrate that PSMA5 plays a vital role in cell proliferation and cycle progression.