Unveiling the role of PYGB in pancreatic cancer: a novel diagnostic biomarker and gene therapy target

Abstract

Purpose: Pancreatic cancer (PC) is a highly malignant tumor that poses a severe threat to human health. Brain glycogen phosphorylase (PYGB) breaks down glycogen and provides an energy source for tumor cells. Although PYGB has been reported in several tumors, its role in PC remains unclear.

Methods: We constructed a risk diagnostic model of PC-related genes by WGCNA and LASSO regression and found PYGB, an essential gene in PC. Then, we explored the pro-carcinogenic role of PYGB in PC by in vivo and in vitro experiments.

Results: We found that PYGB, SCL2A1, and SLC16A3 had a significant effect on the diagnosis and prognosis of PC, but PYGB had the most significant effect on the prognosis. Pan-cancer analysis showed that PYGB was highly expressed in most of the tumors but had the highest correlation with PC. In TCGA and GEO databases, we found that PYGB was highly expressed in PC tissues and correlated with PC's prognostic and pathological features. Through in vivo and in vitro experiments, we found that high expression of PYGB promoted the proliferation, invasion, and metastasis of PC cells. Through enrichment analysis, we found that PYGB is associated with several key cell biological processes and signaling pathways. In experiments, we validated that the MAPK/ERK pathway is involved in the pro-tumorigenic mechanism of PYGB in PC.

Conclusion: Our results suggest that PYGB promotes PC cell proliferation, invasion, and metastasis, leading to poor patient prognosis. PYGB gene may be a novel diagnostic biomarker and gene therapy target for PC.

Keywords: Bioinformatics analysis; Brain glycogen phosphorylase (PYGB); MAPK/ERK; Metastasis; Pancreatic cancer; Proliferation.

Fig. 1

Flowchart of the study design. We identified an oncogenic factor, PYGB, in PC by WGCNA and LASSO regression models. PYGB was then found to be highly expressed in almost all tumors by pan-cancer analysis but had the most significant impact on the prognosis of PC. Then, we verified the effects of PYGB on PC’s proliferation, invasion, and metastasis through a series of cell and animal experiments and found the potential oncogenic mechanism of PYGB in PC through enrichment analysis

Fig. 2

Construction of a hierarchical clustering tree and modeling of risk prognosis. A–C Select appropriate soft thresholds in the GSE16515 dataset and perform average connectivity analysis for 1–30 soft-threshold powers. After creating a hierarchical clustering tree, correlation heatmaps and scatter plots show that the RED module strongly correlates with tumors. D–F Hierarchical clustering trees were created in the GSE16515 dataset in the same way as above. The DARKORANGE2 module had the strongest correlation with tumors. G Taking the intersection of the RED module, the DARKORANGE2 module, and the energy metabolism-related genes module, we found that PYGB, SCL2A1, and SLC16A3 are vital genes in PC. h–J The model consisting of these three genes was verified in the TCGA database to have a strong correlation with both diagnosis and prognosis of PC and is a risk factor for poor prognosis of PC. K Lasso coefficient profiles of the three PC prognostic genes. L Riskscore = (0.152) × PYGB + (0.0767) × SLC2A1 + (0.1482) × SLC16A3 for the three prognostic genes obtained using tenfold cross-validated lasso regression using minimum λ

Fig. 3

Expression levels of PYGB in pan-cancer tissues. A Expression levels of PYGB in pan-cancer tissues in the TIMER 2.0 database. B TCGA and GTEx data show different expressions of PYGB in pan-tumors. C TCGA dataset pairing PYGB expression in cancer tissues and adjacent normal tissues

Fig. 4

PYGB protein expression levels and subcellular localization based on the HPA database. A Immunohistochemical images of protein expression of PYGB in LUAD, LIHC, THCA, HNSC, STAD, and PAAD; B and C mock-ups and immunofluorescence maps of subcellular localization of PYGB

Fig. 5

Prognostic value of PYGB in pan-cancer. A–D The results of OS, DFS, DFS and DSS are presented as forest plots. E Intersection analysis of the effect of PYGB on OS, DFS, DFS and DSS in pan-cancer

Fig. 6

PC’s Prognosis and clinicopathologic features were connected to PYGB expression. A–F TCGA + GTEx, GEO, and UALCAN databases showed that PYGB was highly expressed in PC tissues. G High expression of PYGB indicates poorer OS, PFS, and PFI in patients. H–L High expression of PYGB is associated with multiple clinicopathologic parameters in PC patients. M ROC curves show a high accuracy rate for diagnosing PC. N The expression of PYGB exhibits a favorable correlation with various processes

Fig. 7

Expression of PYGB in PC cells and tissues. A and B PC cell lines express high levels of PYGB mRNA and protein. C–E In PC tissues, PYGB mRNA and protein levels are high

Fig. 8

PYGB promotes the proliferative capacity of PC cells. A Validation of the transfection efficiency of small interfering RNA. B Detection of the transfection efficiency for lentiviruses for the upregulation/downregulation of PYGB. C–F The effect of PYGB gene expression on PC cell proliferation was investigated using EdU, colony formation, and CCK-8 assays

Fig. 9

PYGB promotes the metastatic ability and invasion of PC cells. A and B Transwell and wound healing assays were employed to assess the impact of PYGB gene expression on PC cells' invasive and metastatic potential

Fig. 10

In vivo experiments confirmed that PYGB promotes the proliferation of PC cells. A–C Subcutaneous tumor formation experiments in nude mice revealed the effects of PYGB gene regulation on tumor size and weight. D The effect of the PYGB gene on the formation of liver metastases was observed by constructing a liver metastasis model

Fig. 11

PYGB regulates the MAPK/ERK signaling pathway. A and B GO and KEGG studies revealed that PYGB exhibited associations with many metabolic pathways and signaling pathways implicated in tumorigenesis. C–E GSEA showed that PYGB was associated with multiple critical tumor-related signaling pathways, most notably the MAPK signaling pathway. F PYGB overexpression resulted in enhanced MEK and ERK phosphorylation

Fig. 12

U0126 inhibited the promotion of PYGB in PC. A–C Colony formation, EdU, and CCK-8 assays demonstrated that U0126 blocked the proliferative ability of PYGB in PC cells. D and E The results of the scratch assay and Transwell assay showed that U0126 successfully inhibited the invasive and metastatic properties conferred by PYGB in PC cells