Abstract:Objective:To study the action of E2F1 protein signal transmission pathway to influence the apoptosis of cholangiocarcinoma QBC939 cells.
Methods :Four siRNAs were designed according to the coding sequence of E2F1 gene, and cloned into the downstream of U6 promoter of pGenesil. The constructed recombinant was analyzed and identified by AseⅠ endonuclease digestion and DNA sequencing. The siRNA constructs were transfected into QBC939 cells via lipofectamine 2000. RT-PCR was used to measure the E2F1 mRNA expression in QBC939, and the rate of apoptosis of QBC939 was detected by the method of flow cytometry after transfection.
Results:The constructed psiRNA plasmid digested with AseⅠwas linearized. The sequencing result confirmed that the sequence of inserted fragment was correct. The expression of E2F1 mRNA was greatly inhibited at 48h after transfection, and the rate of apoptosis of QBC939 increased significantly after transfection.
Conclusions:Eukaryotic expression vector of siRNA targeting E2F1 gene can specifically inhibit E2F1 expression and promote the apoptosis of QBC939 cell. It can be used for later experimental research in cholangiocarcinoma.