Plastin-3 is a diagnostic and prognostic marker for pancreatic adenocarcinoma and distinguishes from diffuse large B-cell lymphoma

Abstract

Background: Altered Plastin-3 (PLS3; an actin-binding protein) expression was associated with human carcinogenesis, including pancreatic ductal adenocarcinoma (PDA). This study first assessed differentially expressed genes (DEGs) and then bioinformatically and experimentally confirmed PLS3 to be able to predict PDA prognosis and distinguish PDA from diffuse large B-cell lymphoma.

Methods: This study screened multiple online databases and revealed DEGs among PDA, normal pancreas, diffuse large B-cell lymphoma (DLBCL), and normal lymph node tissues and then focused on PLS3. These DEGs were analyzed for Gene Ontology (GO) terms, Kaplan-Meier curves, and the log-rank test to characterize their association with PDA prognosis. The receiver operating characteristic curve (ROC) was plotted, and Spearman's tests were performed. Differential PLS3 expression in different tissue specimens (n = 30) was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR).

Results: There were a great number of DEGs between PDA and lymph node, between PDA and DLBCL, and between PDA and normal pancreatic tissues. Five DEGs (NET1, KCNK1, MAL2, PLS1, and PLS3) were associated with poor overall survival of PDA patients, but only PLS3 was further verified by the R2 and ICGC datasets. The ROC analysis showed a high PLS3 AUC (area under the curve) value for PDA diagnosis, while PLS3 was able to distinguish PDA from DLBCL. The results of Spearman's analysis showed that PLS3 expression was associated with levels of KRT7, SPP1, and SPARC. Differential PLS3 expression in different tissue specimens was further validated by RT-qPCR.

Conclusions: Altered PLS3 expression was useful in diagnosis and prognosis of PDA as well as to distinguish PDA from DLBCL.

Keywords: Bioinformatic analysis; Biomarker; Differentially expressed genes; Diffuse large B-cell lymphoma; Pancreatic adenocarcinoma.

Fig. 1

Illustration of our search criteria and flow diagram

Fig. 2

The hierarchical cluster heatmaps for DEGs in the MERAV datasets. A DEGs between normal pancreas and lymph node tissue. B DEGs between normal pancreas and DLBCL tissue. The gradual change from red to green represents changes in gene expression from high to low. The black color refers to no difference in gene expression. DEG differentially expressed gene, MERAV the Metabolic Gene Rapid Visualizer, DLBCL diffuse large B-cell lymphoma

Fig. 3

DEGs identified from the batched GEO datasets with the Venn diagrams. A The hierarchical cluster heatmaps of DEGs between PDA and normal pancreas from the four GEO batched datasets. The gradual change from red to green represents changes in gene expression from high to low. The black color indicates no difference in gene expression. B The intersection among “Pancreas > Lymph Nodes”, “Pancreas > DLBCL” and “PDA > Pancreas” groups. C The intersection among “Pancreas < Lymph Nodes”, “Pancreas < DLBCL” and “PDA < Pancreas” groups. DEG differentially expressed gene, GEO Gene Expression Omnibus, PDA pancreatic ductal adenocarcinoma, DLBCL diffuse large B-cell lymphoma

Fig. 4

Gene Ontology terms of the 84 DEGs identified with interaction analysis. Changes in red color saturation represent differences in logFC values

Fig. 5

Validation of DEGs between PDA and normal pancreatic tissue among TCGA, GTEx, and GEO datasets. A The intersection between the 84 DEGs in the GEO datasets and DEGs from the TCGA and GTEx datasets. Forty-six DEGs were identified after comparison of all three databases. B Validation of these 46 DEGs with GSE62165 and GSE62452. Sixteen out of 46 DEGs were confirmed. DEG differentially expressed gene, PDA pancreatic ductal adenocarcinoma, TCGA The Cancer Genome Atlas, GTEx Genotype-Tissue Expression, GEO Gene Expression Omnibus

Fig. 6

Association of NET1, KCNK1, MAL2, PLS1, and PLS3 expression with PDA prognosis, as determined with TCGA, R2, and ICGC data. A, C, E, G, and I Kaplan–Meier curves and log-rank test results for PDA patients from TCGA. B, D, F, H, and J Kaplan–Meier curves and log-rank test results for PDA patients from the R2 and ICGC. Both studies show that patients with high PLS3-expressing PDA had poorer overall survival. PDA pancreatic ductal adenocarcinoma, TCGA The Cancer Genome Atlas, R2 The R2 Genomics Analysis and Visualization Platform, ICGC International Cancer Genome Consortium

Fig. 7

Diagnostic values of PLS3 in PDA vs. normal pancreas. A–D PLS3 expression in PDA. PLS3 levels were higher than those of normal pancreas in all four datasets. E–H ROC curves for PLS3 expression in the four datasets. PDA pancreatic ductal adenocarcinoma, ROC receiver operating characteristic curve. ****P < 0.0001 by Student’s t-test

Fig. 8

Diagnostic value of PLS3 in PDA and pancreas vs. lymph nodes and DLBCL. A, B Level of PLS3. PLS3 was significantly higher in the normal pancreas than in the lymph nodes in the MERAV and GSE71729 datasets. C, D ROC curves of PLS3 in the MERAV and GSE71729 datasets. E, F Level of PLS3. PLS3 was significantly higher in normal pancreas than in DLBCL in the MERAV, TCGA, and GTEx datasets. G, H ROC curves for PLS3 in the MERAV, TCGA, and GTEx datasets. I Level of PLS3. PLS3 expression was notably higher in PDA than in DLBCL in the cell-line data from the MERAV. J ROC curve of PLS3 in the cell line data. K PLS3 level in GSE71729. PLS3 expression was higher in PDA than in lymph nodes. L ROC curve of PLS3 in GSE71729. PDA pancreatic ductal adenocarcinoma, MERAV Metabolic Gene Rapid Visualizer, ROC receiver operating characteristic curve, DLBCL diffuse large B-cell lymphoma, TCGA The Cancer Genome Atlas, GTEx Genotype-Tissue Expression; ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05, analyzed by Student’s t-test

Fig. 9

Correlation of PLS3 with other PDA markers. A Correlation heatmap reporting Spearman correlation values for each comparison. The bar on the left represents the color legend of Spearman correlation values calculated for each paired PDA marker and PLS3 value. B–E The correlation of PLS3 with KRT7, KRT19, SPP1, and SPARC in the batched GEO dataset. F, I The correlation of PLS3 with KRT7, KRT19, SPP1, and SPARC in TCGA. J–M The Kaplan–Meier curves and the log-rank test results of KRT7, KRT19, SPP1 and SPARC for PDA patients from TCGA. N–Q Kaplan–Meier curves and log-rank test results of KRT7, KRT19, SPP1 and SPARC for PDA patients from the R2 and ICGC. PDA pancreatic ductal adenocarcinoma, GEO Gene Expression Omnibus, TCGA The Cancer Genome Atlas, R2 The R2 Genomics Analysis and Visualization Platform, ICGC International Cancer Genome Consortium. ****P < 0.0001 and *** P < 0.001 by Student’s t-test

Fig. 10

RT-qPCR validation of differential PLS3 expression in tissue specimens. Ten pairs of PDA and normal pancreas and five pairs of DLBCL and normal lymph node samples were subjected to total RNA isolation, reverse-transcribed into cDNA, and then subjected to qPCR. Data were quantified using the 2^−∆∆Ct method. ***P < 0.001 and *P < 0.05 by ANOVA