To investigate the expression characteristics, prognostic significance, and the association of hephaestin (HEPH) with cell migration, invasion, and epithelial-mesenchymal transition (EMT) in pancreatic cancer.

Expression differences of HEPH in pan-cancer and pancreatic cancer tissues were analyzed through public databases, with validation of HEPH expression levels in clinical samples. Survival analysis and receiver operating characteristic (ROC) curves were used to evaluate the prognostic value of HEPH. Gene set enrichment analysis (GSEA) was performed by grouping samples based on HEPH expression levels and using gene expression fold change as the ranking metric to identify significantly enriched biological pathways. Stable HEPH knockdown pancreatic cancer cell lines, PANC-1 and SW1990, were established. Cells infected with pLKO.1-Puro empty vector lentivirus were designated as the scramble group (negative control), and cells infected with shHEPH lentivirus were designated as shHEPH-1, shHEPH-2, and shHEPH-3 knockdown groups. Knockdown efficiency was verified by RT-qPCR and Western blot. The effects of HEPH on cell migration and invasion were assessed using scratch and Transwell assays. Expression changes of EMT-related markers (E-cadherin, N-cadherin, and Vimentin) were measured. Comparisons between two groups were performed using independent samples t-test. Comparisons among multiple groups were analyzed by one-way ANOVA followed by Dunnett's t-test for pairwise comparisons. Survival analysis was conducted using univariate Cox regression to calculate hazard ratios and 95% confidence intervals, and Kaplan-Meier survival curves were plotted.

HEPH was highly expressed in pancreatic cancer tissues, and high expression was associated with shorter overall survival in patients (HR = 1.55, P = 0.038). ROC analysis showed that the AUCs of HEPH for 3-year and 5-year overall survival of pancreatic cancer patients were 0.638 and 0.678, respectively. Immunohistochemical and paired tissue analyses further confirmed the high expression of HEPH in pancreatic cancer tissues. GSEA revealed that the EMT pathway was significantly enriched in the high HEPH expression group (NES = 3.000, P < 0.001). Compared with the scramble group, knockdown of HEPH reduced the migration (PANC-1: 148.70 ± 19.30 vs 314.30 ± 55.97; SW1990: 175.00 ± 24.58 vs 425.00 ± 26.51) and invasion (PANC-1: 97.67 ± 15.57 vs 227.70 ± 21.73; SW1990: 90.67 ± 11.93 vs 321.30 ± 34.70) abilities of PANC-1 and SW1990 cells (all P < 0.01). Compared to the control group, the HEPH knockdown group showed increased E-cadherin expression (PANC-1: 3.07 ± 0.48 vs 1.00 ± 0.05; SW1990: 2.58 ± 0.41 vs 1.00 ± 0.02; all P < 0.000 1) and decreased N-cadherin (PANC-1: 0.33 ± 0.06 vs 1.01 ± 0.05; SW1990: 0.44 ± 0.08 vs 1.00 ± 0.05; all P < 0.01) and Vimentin (PANC-1: 0.36 ± 0.06 vs 1.00 ± 0.02; SW1990: 0.39 ± 0.06 vs 0.99 ± 0.02; all P < 0.01) expression.

HEPH is highly expressed in pancreatic cancer and is associated with poor prognosis. Knockdown of HEPH suppresses migration and invasion of pancreatic cancer cells, accompanied by altered expression of EMT markers, suggesting that high HEPH expression is correlated with EMT activation and enhanced migratory/invasive capabilities. This suggests that HEPH may serve as a potential biomarker for prognosis and therapeutic intervention in pancreatic cancer.