CRISPR/Cas9-mediated knockout of c-Met reduces gemcitabine resistance in pancreatic cancer

Background

Pancreatic cancer is one of the most lethal malignancies, and resistance to gemcitabine remains a major obstacle to effective treatment. The receptor tyrosine kinase c-Met has been widely associated with tumor progression and the development of chemoresistance. In this study, we explored whether CRISPR/Cas9-mediated knockout of c-Met could reduce gemcitabine resistance in pancreatic cancer cells.

Methods

c-Met expression was first examined in PANC-1 and AsPC-1 cell lines using RT-qPCR. The sensitivity of these cells to gemcitabine was then evaluated by determining IC₅₀ values using the MTS assay . A CRISPR/Cas9 vector carrying two guide RNAs targeting c-Met was constructed and introduced into the cell line showing higher c-Met expression. After puromycin selection, single-cell clones were isolated, and successful gene knockout was verified by genomic PCR and Sanger sequencing.

Results

AsPC-1 cells exhibited higher c-Met expression and were therefore selected for further experiments. Genomic PCR and Sanger sequencing confirmed efficient disruption of the c-Met gene in selected clones. c-Met knockout led to reduced cell growth and proliferation. Notably, gemcitabine IC₅₀ values were significantly lower in c-Met-deficient clones compared with control cells.

Conclusions

Knockout of the c-Met gene in ASPC-1 cells using the CRISPR/Cas9 system significantly decreased drug resistance to gemcitabine. This research underscores the potential therapeutic advantage of targeting c-Met to overcome chemoresistance in pancreatic cancer, offering a hopeful path for enhancing gemcitabine treatment results.