Establishment of mouse hepatic cancer cell line Hca-F with knockdown of annexin A7 gene

Abstract

Objective:To establish a mouse hepatic cancer cell line Hca-F transfected with shRNA (small hairpin RNA) targeting annexin A7,so as to provide a basis for future study. Methods: Three shRNAs(shRNA1,2 and 3) were designed and inserted into the pSilencer vector to silence annexin A7 gene. The three pSilencer-shRNA vectors were transfected into Hca-F cells separately,and the most effective pSilencer-shRNA vector was selected based on the results of RT-PCR and Western blotting. The Hca-F cells were transfected with the most effective pSilencer-shRNA vector and the transfectants were selected by 400 μg/ml G418. The cells with annexin A7 stably knockdown were passaged and the expression of annexin A7 was confirmed by Western blotting,and the result was compared with those transfected with empty vector and normal controls.Results: The sequencing results confirmed that the sequences of the 3 shRNAs were correct,and shRNA1 was found to have the best inhibitory effect against annexin A7. Compared with normal Hca-F cells and those transfected with empty vectors,the annexin A7 protein expression was significantly down-regulated in cells transfected with pSilencer-shRNA(0.318 6 vs 0.824 3,0.798 7,P0.05),with no significant difference found between the former 2 groups. Conclusion: We have successfully established a Hca-F cell line with annexin A7 stably down-regulated using shRNA technique,paving a way for future study.