Establishment of human pancreatic cancer cell line with stable knockdown of GLI1 gene / 第二军医大学学报

ABSTRACT

Objective To establish a human pancreatic cancer cell line stably transfected with siRNA expression vector targeting GLU gene and examine the interference efficiency. Methods The expression of GLI1 gene in five human pancreatic cancer cell lineswas detected by quantitative real-time PCR (qRT-PCR); the one with the highest expression level of GLI1 was selected as the target cell line and was transfected with three recombinant plasmids pGCti-U6-GLIlsiRNA-1,-2, and -3. The positive cloneswere screened by G418, and the transfection rate was observed by fluorescence microscope. The expression of GLI1 mRNA and protein was analyzed by qRT-PCR and Western blotting analysis, respectively. Results Panc-1 cell line was found to have the highest GLU expression and was selected as the target cell line for transfection. Plasmids pGCti-U6-GLIlsiRNA-1, -2, and-3 were successfully transfected into Panc-1 cells separately. After 4 weeks of G418 screening, three stably transfected cell lines named Panc-1/GIU1siRNA-1, -2, and -3 were obtained, with the transfection rates all higher than 80%. qRT-PCR and Western blotting analysis showed that the expression levels of GLI1 in Panc-1/GIUlsiRNA-1, -2, and -3 cellswere all significantly lower than those in Panc-1/siControl cells and the blank control cells(P<0. 05), with the lowest expression found in Panc-1/GLIlsiRNA-1 cels. Conclusion We have successfully constructed a cell line Panc-1/GLI1siRNA-1 with GLU gene stably silenced, which paving a way for future research.