Fig 2: PDIA4 is highly expressed in gliomas and significantly associated with the advanced stage of tumors. (A) Differential expression of LCTL in brain lower grade glioma (LGG) and glioblastoma (GBM) compared to levels in normal brain tissues. *P < 0.05. (B) Relative mRNA expression level of PDIA4 in the normal glial cell line HEB and glioma cell lines. *P < 0.05; **P < 0.01; ***P < 0.001. (C) IHC staining of PDIA4 in WHO grade IV gliomas of and comparison with normal brain tissues resected at Xiangya Hospital. Original magnification, ×100. Scale bar, 50μm. (D) The upper section shows the intensity of IHC staining of PDIA4 in the cytoplasm of glioma tissue arrays. The lower section shows the representative intensity at each level of PDIA4 IHC staining. *P < 0.05; **P < 0.01. (E) PDIA4 expression in gliomas of WHO grade III–V, based on both TCGA and CGGA datasets. *P < 0.05; ***P < 0.001. (F) The expression of PDIA4 in IDH subtypes of gliomas based on both the TCGA and CGGA datasets. ***P < 0.001. (G) PDIA4 expression pattern in different molecular subtypes of glioma (classical, mesenchymal, neural, proneural) in the TCGA dataset. *P < 0.05; ***P < 0.001.

Fig 3: PDIA4 correlates with TME and promotes glioma cell proliferation.(A) PDIA4 expression positively correlated with immune score and stromal score in glioblastoma multiform (GBM) patients. (B) PDIA4 expression positively correlated with immune score and stromal score in lower grade glioma (LGG) patients. (C) PDIA4 expression significantly correlated with 46 cell types, as calculated by xCells in glioma. Data of all grades of glioma were used in this analysis. (D) Protein–protein interaction (PPI) network of PDIA4. Data of all grades of glioma were used in this analysis. (E) Demonstration of PDIA4 knockdown (KD) in U87 and U251 GBM cells by Western blotting analysis. Cells transfected with non-specific shRNA were used as control. (F) A total of 3000 control and PDIA4 KD glioma cells were plated in a 96-well plate in 200 μL medium. Cell viability was assayed using CCK8 assay. Both PDIA4 KD U87 and U251 cells showed significantly reduced cell viability compared to control. ***P < 0.001.

Fig 4: Functional enrichment analysis of PDIA4 in TCGA and CGGA cohorts. Data of all grades of glioma were used in this analysis. (A and B) The top10 biological process terms of GO enrichment analysis based on TCGA and CGGA datasets, respectively. (C and D) The top10 molecular function terms of GO enrichment analysis based on TCGA and CGGA datasets, respectively. (E and F) The top10 cellular component terms of GO enrichment analysis based on TCGA and CGGA datasets, respectively. (G and H) KEGG pathway analysis based on TCGA and CGGA datasets and the top 10 terms were visualized.