探讨膜铁转运辅助蛋白（HEPH）在胰腺癌中的表达特征、预后意义及其与细胞迁移、侵袭和上皮间质转化（EMT）表型的相关性。

通过公开数据库分析HEPH在泛癌及胰腺癌组织中的表达差异，结合临床样本检测HEPH在胰腺癌组织中的表达水平。采用生存分析和受试者工作特征曲线（ROC）评估HEPH的预后价值。根据HEPH表达水平对样本进行分组，以基因表达倍数变化为排序指标进行基因集富集分析（GSEA），筛选其显著富集的生物学通路。构建HEPH稳定敲低的胰腺癌细胞株PANC-1和SW1990，将感染pLKO.1-Puro空载体慢病毒设为scramble （阴性对照）组，感染shHEPH慢病毒设为shHEPH-1、shHEPH-2和shHEPH-3敲低组；采用RT-qPCR和Western blot验证敲低效率；通过划痕实验和Transwell实验检测HEPH对胰腺癌细胞迁移和侵袭能力的影响；同时检测EMT相关标志蛋白E-cadherin、N-cadherin和Vimentin的表达变化。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，组间两两比较采用Dunnett-t检验。采用单因素Cox回归进行生存分析，计算风险比（HR）及95%置信区间，并绘制Kaplan-Meier生存曲线。

HEPH在胰腺癌组织中高表达，且高表达与患者较短的总体生存期相关（HR = 1.55，P = 0.038）。ROC曲线分析显示，HEPH评估胰腺癌患者3年及5年总体生存状态的AUC分别为0.638和0.678。免疫组织化学及配对组织检测结果进一步证实，HEPH在胰腺癌组织中呈高表达。富集分析显示，HEPH高表达分组的基因富集分析在EMT通路富集（NES = 3.000，P < 0.001）。与scramble组相比，敲低HEPH后，PANC-1和SW1990细胞迁移[PANC-1：（148.70 ± 19.30）比（314.30 ± 55.97）个；SW1990：（175.00 ± 24.58）比（425.00 ± 26.51）个]和侵袭细胞数[PANC-1：（97.67 ± 15.57）比（227.70 ± 21.73）个；SW1990：（90.67 ± 11.93）比（321.30 ± 34.70）个]下降（P 均 < 0.01）。与对照组相比，HEPH敲低组细胞中E-cadherin （PANC-1：3.07 ± 0.48比1.00 ± 0.05；SW1990：2.58 ± 0.41比1.00 ± 0.02）升高（P 均 < 0.000 1），而N-cadherin （PANC-1：0.33 ± 0.06比1.01 ± 0.05；SW1990：0.44 ± 0.08比1.00 ± 0.05）和Vimentin （PANC-1：0.36 ± 0.06比1.00 ± 0.02；SW1990：0.39 ± 0.06比0.99 ± 0.02）降低（P 均 < 0.01）。

HEPH在胰腺癌组织中高表达，与患者不良预后相关。敲低HEPH可抑制胰腺癌细胞迁移与侵袭能力，伴随EMT标志蛋白表达改变，提示HEPH高表达与EMT激活及细胞迁移侵袭能力增强相关，HEPH可能成为胰腺癌预后评估及潜在治疗干预的候选分子。

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.