RESUMEN
Objective To investigate the effects of small interfering RNA (siRNA) silencing Nanog gene on the ability of migration and invasion of the human lung adenocarcinoma A549 cells. Methods The human lung adenocarcinoma A549 cells were transfected with siRNA targeting Nanog gene , and three experiment groups were set up. The expression level of Nanog was detected using reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Cell migration was examined by wound healing assay and cell invasion was detected by Transwell assay. Results The Nanog silencing cell group (A549-siNanog) showed much lower level of Nanog mRNA and protein (0.40 ± 0.06, 0.50 ± 0.03) than A549-siNC cell group (0.97 ± 0.03, 0.85 ± 0.02; P < 0.05) under RT-PCR and Western blot analysis. Meanwhile, the wounded area filled rate and the number of invaded cells of A549-siNanog cell group (57% ± 0.04, 69.60 ± 17.14) were decreased significantly compared to A549-siNC cell group (95% ± 0.02, 209.60 ± 15.40; P < 0.05). Conclusion siRNA targeting human Nanog could specially suppress the expression of Nanog gene in lung adenocarcinoma A549 cells. In this way, it couldsignificantly reduce the capability of migration and invasion of A549 cells.
RESUMEN
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.