Background <p>Pancreatic cancer is called “the king of cancer” because of its high degree of malignancy, poor prognosis, and lack of effective treatment. It encompasses multiple subtypes, with pancreatic adenocarcinoma (PAAD) representing its most prevalent form. An increasing number of studies have shown that tripartite motif-containing protein 21 (TRIM21) is associated with the proliferation, migration, and invasion capabilities of various cancers. However, the mechanism of TRIM21 function in pancreatic cancer remains to be further studied.This study explored the mechanisms of TRIM21 in pancreatic cancer through bioinformatics analysis and in vitro experiments and assessed its potential as a prognostic biomarker and therapeutic target.</p> Methods <p>Using the GEPIA 2 platform, which incorporates TCGA, GTEx, and CPTAC datasets along with GEO data, we analyzed the expression levels of TRIM21 mRNA or protein across pan-cancer and specifically in PAAD. The HPA database was utilized to examine differential TRIM21 protein expression between PAAD patient samples and normal tissues, while Western blotting was employed to assess differences in TRIM21 expression between pancreatic cancer cell lines and normal cells. TIMER 2.0 was applied to investigate the correlation between TRIM21 expression and clinical parameters, and the Kaplan-Meier plotter was used to evaluate its prognostic significance. The cBioPortal database was leveraged to analyze the relationship between TRIM21 and immune infiltration in PAAD. The impact of TRIM21 on PAAD cell viability, proliferation, and migration was assessed through colony formation, CCK-8, and wound healing assays, respectively. Flow cytometry was used to measure apoptosis rates and cell cycle distribution. Associations between TRIM21 and pathway genes or proteins in PAAD patients were determined using either TIMER 2.0 or Western blot analysis.</p> Results <p>Analysis of GEPIA 2 and GEO datasets revealed that TRIM21 expression is significantly higher in pancreatic cancer tissues compared to non-tumor tissues. Consistent findings were obtained from immunohistochemical data in the HPA database. Examination of the cBioPortal database indicated that TRIM21 is associated with immune infiltration in PAAD. When TRIM21 was specifically knocked down using siRNA in PANC-1 pancreatic cancer cells, cell viability, proliferation, colony formation, and migration were reduced compared to negative control cells, while apoptosis was significantly increased. TRIM21 knockdown induced noticeable cell cycle arrest at the G1 phase and decreased Cyclin D1 expression. Furthermore, analysis via TIMER 2.0 demonstrated a strong correlation between TRIM21 and the expression of genes related to the Wnt/β-catenin signaling pathway in PAAD patients. Western blot analysis showed that Wnt3a and β-catenin expression decreased in the knockdown group compared to the NC group. Additionally, GSK3β expression was increased, while c-Myc expression was reduced.</p> Conclusions <p>TRIM21 knockdown reduced PANC-1 cell proliferation and migration, induced apoptosis, and affected cell cycle progression. Moreover, TRIM21 knockdown may affect cell growth by influencing the expression of proteins related to the Wnt/β-catenin pathway. Therefore, TRIM21 is expected to be a biomarker for the prognosis evaluation of pancreatic cancer and a potential therapeutic target.</p>