Study on the Role of SMAD4 Gene Deletion in Gemcitabine Resistance in Pancreatic Cancer Cells

OBJECTIVE  To explore the relationship between SMAD4 gene and gemcitabine resistance in pancreatic cancer, and provide new research ideas for individualized treatment of pancreatic cancer.

METHODS  Bioinformatics analysis was used to analysis the SMAD4 gene expression in pancreatic cancer cells, high throughput sequencing was used to detect AsPC-1 and MIA-PaCa2 genes; CCK8 method and Trypan blue staining assay were used to detect the toxicity of gemcitabine at different concentrations on pancreatic cancer cells, and then the optimal concentration and time were selected. Real time fluorescent quantitative PCR and Western blotting were used to detect the mRNA and protein expression of SMAD4 gene. Scratch test and Transwell test were used to detect the effect of silenced or overexpressed SMAD4 gene on cell migration and invasion under the combined action of gemcitabine.

RESULTS  According to cBioPortal database, 33% of pancreatic cancer patients had SMAD4 gene mutations, and most of the mutations were deletion, missense and truncation mutations; High throughput sequencing analysis, real-time fluorescence quantitative PCR, and Western blotting confirmed that the expression levels of SMAD4 gene at mRNA and protein levels in MIA PaCa-2 cells were significantly higher than those in AsPC-1 cells. The optimal concentration of gemcitabine for AsPC-1 cells was 10⁻⁵ mol·L⁻¹, and the optimal treatment time was 48 h. The optimal concentration of gemcitabine for MIA PaCa-2 cells was 10⁻⁷ mol·L ⁻¹, and the optimal treatment time was 48 h. Compared with the control group, silencing the SMAD4 gene significantly increased the survival rate of MIA PaCa-2 cells treated with gemcitabine. Compared with the PBS group, the number of cells migrating and invading in the drug treated group was significantly reduced, but the number of cells migrating and invading in the transfected group was significantly upregulated compared to the non transfected group. After overexpression of the SMAD4 gene, the survival rate of the transfected group cells treated with gemcitabine was significantly lower than that of the control group, and the scratch area of the transfected plasmid drug group decreased the slowest, with a significant reduction in the number of invasive cells passing through the chamber.

CONCLUSION  There are significant differences in the SMAD4 gene between AsPC-1 cells and MIA PaCa-2 cells. SMAD4 gene deletion is associated with gemcitabine resistance in pancreatic cancer cells. SMAD4 gene, as a tumor suppressor gene, can enhance the ability of gemcitabine to inhibit the proliferation, migration and invasion of pancreatic cancer cells, and is expected to become a new target to solve the problem of drug resistance in pancreatic cancer