Background: PDAC is characterized by high aggressiveness and poor therapeutic response, largely due to the rapid development of gemcitabine resistance. EPS8 regulates cytoskeletal dynamics, cell motility, and pro-survival signaling. Its elevated expression in multiple cancers correlates with enhanced invasiveness and reduced chemotherapy sensitivity. Therefore, EPS8 represents a potential therapeutic target. In this study, CRISPR/Cas9 technology was used to knockout EPS8 in pancreatic cancer cells and assess its impact on gemcitabine resistance and tumor-related cellular behaviors.
Materials and Methods: Two sgRNAs targeting the EPS8 genomic sequence were designed and cloned into a CRISPR/Cas9 expression vector. The construct was introduced into the pancreatic cancer cell line with the highest EPS8 expression. After transfection, puromycin selection was applied to enrich edited cells and isolate single clones. Gene disruption was confirmed by genomic PCR and Sanger sequencing. EPS8 expression levels in edited clones were analyzed using RT-qPCR and western blot. To evaluate the impact of EPS8 loss on chemoresistance, wild-type cells and edited clones were treated with gemcitabine, and cell viability was assessed using an MTS assay.
Results: EPS8 knockout in PDAC cells was confirmed by sequencing, showing loss of target site integrity. Loss of EPS8 expression was further verified in edited clones, which exhibited reduced viability after gemcitabine treatment compared to wild-type controls. Changes in drug-response curves indicated increased sensitivity following EPS8 knockout. No significant morphological abnormalities were observed during routine culture.
Conclusion: Disruption of the EPS8 gene significantly increased the susceptibility of PDAC cells to gemcitabine, indicating that EPS8 contributes to gemcitabine resistance in pancreatic cancer. Targeting EPS8 may help overcome drug resistance and improve the effectiveness of gemcitabine in PDAC chemotherapy. Further studies using additional pancreatic cancer cell lines and in vivo models with clinical samples are needed to clarify the role of EPS8 in gemcitabine resistance.
