Phospholipase A2 enzymes PLA2G2A and PLA2G12B as potential diagnostic and prognostic biomarkers in cholangiocarcinoma

Figure 1 Analyzing phospholipase A2 gene family in cholangiocarcinoma: Expression patterns and correlations. A: The enrichment levels of the 5 phospholipase A2 (PLA2) gene families include cytosolic PLA2, calcium-independent PLA2, lysosomal PLA2, platelet-activating factor acetylhydrolase, and secreted PLA2; B: The expression level of PLA2 gene family in cholangiocarcinoma (T) and normal tissues adjacent (NAT); C: Correlations between the expression of each PLA2 family member in cholangiocarcinoma; D: Differentially expressed genes between the PLA2 gene family in tumor and normal samples were shown in the volcano plot, with blue dots representing significantly down-regulated genes and red dots representing significantly up-regulated genes.; E-J: The expression levels of PLA2G2A and PLA2G12B in public databases (The Cancer Genome Atlas and the Gene Expression Omnibus) were found in the T and NAT groups. . ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. NAT: Adjacent normal tissues

Figure 2 Expression and correlation of PLA2 gene families in cholangiocarcinoma and adjacent normal tissues. A: Genetic alterations of PLA2G2A and PLA2G12B genes in patients from three datasets (each rectangle represents one patient; not all patients were shown (n = 91). Gray represents no mutation, and different colors represent different mutation types; B: Gene Set Enrichment Analysis (GSEA) analyses displayed key pathways associated with PLA2G2A expression; C: GSEA analyses displayed key pathways associated with PLA2G12B expression.

Figure 3 Hub genes linked to PLA2G2A and PLA2G12B in cholangiocarcinoma: expression, correlations, and pathway analysis. A and B: Venn diagram showing the relevant hub genes associated with PLA2G2A, PLA2G12B expression, and differentially expressed in cholangiocarcinoma; C: Correlations between the expression of each relevant hub gene; D: The expression level of the relevant hub genes in cholangiocarcinoma (T) and normal tissues adjacent; E: Pathway enrichment and Genes Ontology (GO) function analysis of down-regulated genes correlated with biological BP and MF; F: Chord plot displaying the top 10 significant GO terms and their genes; G: Chord plot displaying the top 10 significant Kyoto Encyclopedia of Genes and Genomes terms and their genes. ^aP < 0.05. NAT: Adjacent normal tissues.

Figure 4 Analysis of microRNA targets in tumor vs normal samples and their prognostic significance in cancer. A: Using TargetScan and miRDB databases, 29 intersecting microRNA (miRNA) targets for the PLA2G2A gene; B: Using TargetScan and miRDB databases, 19 intersecting miRNA targets for the PLA2G12B gene; C: Differentially expressed genes between miRNA targets in tumor and normal samples were shown in the volcano plot; D: Forest plot of the univariate Cox regression analysis in co-intersection miRNA targets; E-G: High expression of hsa-miR-363-5p, hsa-miR-4267, and hsa-miR-9-5p genes was associated with a worse prognosis; H: Sankey diagram visual analysis of miRNA targets, PLA2G2A, PLA2G12B, and Hub gene network data.

Figure 5 Expression and diagnostic potential of PLA2G2A, PLA2G12B, and microRNAs in cholangiocarcinoma and related conditions. A and B: Expression of PLA2G2A, PLA2G12B in serum and urine. Intrahepatic cholangiocarcinoma (ICC), primary sclerosing cholangitis (PSC), and non-tumor (N); C: The differentially expressed microRNAs (miRNAs) related to PLA2G2A and PLA2G12B in serum were screened; D: The gene expression levels of 6 different miRNA molecules in serum. ICC, cholelithiasis (Ch) and N; E: Receiver operating characteristic (ROC) curves of PLA2G2A and PLA2G12B in serum extracellular vesicles in cholangiocarcinoma (CCA) and N groups; F: ROC curves of PLA2G2A and PLA2G12B in serum extracellular vesicles in CCA and PSC groups; G: ROC curves of PLA2G2A and PLA2G12B in urine extracellular vesicles in CCA and N groups; H: ROC curves of miRNAs were expressed differently in the ICC group and N group; I: ROC curves of miRNAs expressed differently in the ICC group and Ch group. CCA: Cholangiocarcinoma; PSC: Primary sclerosing cholangitis; EVs: Extracellular vesicles; AUC: Area under the curve; ICC: Intrahepatic cholangiocarcinoma.

Figure 6 Least Absolute Shrinkage and Selection Operator model optimization and prognostic analysis of PLA2G2A and PNPLA2 in cholangiocarcinoma survival. A: A coefficient profile plot was generated against the log (λ) sequence. Selection of the optimal parameter (λ) in the Least Absolute Shrinkage and Selection Operator (LASSO) model; B: Partial likelihood deviance for LASSO coefficient profiles. The red dots represent the partial likelihood values, the gray lines represent the standard error. Validation of independent prognostic factors; C: Univariate Cox regression analysis was performed to determine the independence of clinical characteristics as prognostic factors; D: Multivariable Cox regression analysis to identify clinical characteristics as prognostic factors; E-G: The survival curve for patients with, pathologic-stage, PLA2G2A, PNPLA2. The receiver operating characteristic (ROC) curve for 1, 3 and 5 overall survival of cholangiocarcinoma patients; H-J: 1-, 3-, and 5-years ROC curve corresponding to PLA2G2A, PNPLA2, and pathologic-stage. AUC: Area under the curve.