ELAVL1-mediated post-transcriptional regulation of EMT transcription factors in pancreatic ductal adenocarcinoma

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths, with a five-year survival rate un-der 10% [1]. Its lethality stems from its aggressive progression and strong chemotherapy resistance, both closely linked to epithelial-mesenchymal transition (EMT). EMT enables epithelial cells to gain migratory mesenchymal traits and is regulated by transcription factors such as SNAIL, SLUG,ZEB1/2, and TWIST [2].Among these, SNAIL plays a major role in metastasis [3], while ZEB1 is as-sociated with poor prognosis [4]. EMT is tightly modulated by networks including ELAVL1, which encodes the RNA-binding protein HuR. HuR stabilizes mRNAs by binding AU-rich elements (AREs) in their 3′ untranslated regions (3′UTRs), promoting their expression [5].Aims & Methods: This study aimed to identify post-transcriptional interactions between ELAVL1 and EMT-related genes and assess their prognostic value in PDAC. mRNA was extracted from cancerous and adjacent normal tissues (n=65), converted to cDNA, and analyzed by RT-PCR.BxPC-3, MiaPaCa-2, and Su.86.86 cell lines were cultured under standard conditions. Immunoprecipitation was performed on 1–2×10⁷ cells using a mouse monoclonal anti-HuR antibody, with IgG as control. qRT-PCR assessed mRNA enrichment. Binding sites were annotated using CISBP-RNA, considering only RNA direct-confirmed 3′UTR motifs. Target 3′UTRsequences were retrieved from GENCODE V47. Statistical analysis was performed using GraphPad Prism, applying the Wilcoxon signed-rank test, Kruskal-Wallis test with Dunn’s correction, and Spearman correlation. Data were presented as median ± interquartile range, with p<0.05considered significant. Results: Expression levels of EMT-related genes and ELAVL1 varied according to survival outcomes. In short-term survivors (1–12 months), SNAIL and SLUG were significantly upregulated (2.19- and 1.92-fold), while ZEB2was downregulated (0.65-fold). Medium-term survivors (13–35 months)showed reduced ZEB1 (0.53), SNAIL (0.30), SLUG (0.35), and TWIST (0.68).Long-term survivors (36–125 months) had the lowest levels of ZEB1 (0.24),ZEB2 (0.50), SLUG (0.24), TWIST (0.35), and ELAVL1 (0.72). ELAVL1 showed strong positive correlations with ZEB1, SNAIL, and SLUG (r = 0.74–0.76),whereas overall survival was weakly negatively correlated with the ex -pression of ZEB1, SNAIL, SLUG, and TWIST (r = -0.25 to -0.32).Although ZEB1, SNAIL, and TWIST did not show significant survival differences individually, low SLUG predicted better early survival (36 vs. 15months, p = 0.0278). ELAVL1 expression was stratified as high and low. High ELAVL1 corresponded to a 71.65-fold increase in ZEB1, whereas highZEB1 reduced ELAVL1 by 96.1%. High ELAVL1 also led to a 312.35-fold in-crease in SNAIL, while low ELAVL1 still supported elevated SNAIL expression (8.45-fold). High SNAIL expression reduced ELAVL1 by 90%, though simultaneous high expression of both resulted in a 3.71-fold ELAVL1 in-crease. High SLUG reduced ELAVL1 by 93%, whereas low SLUG led to a198.11-fold increase. Binding site analysis revealed ELAVL1 targets within3′UTRs: 34 in ZEB1, 1 in SNAIL, and 9 in SLUG. Conclusion: ELAVL1 (HuR) likely plays a central role in regulating EMT in PDAC through stabilization of key transcription factor mRNAs. High levels of ELAVL1, SNAIL, SLUG, and ZEB1 were associated with worse outcomes, particularly in short-term survivors. Conversely, low expression of SLUG was linked to improved survival. The strong correlations and validated binding sites suggest ELAVL1 as a promising prognostic biomarker and potential therapeutic target in PDAC.