Objective:To establish a human hepatic cancer cell line HepG2 with stable,simultaneous knockdown of three nuclear factor-kappaB inhibitors (IκBα,IκBβ,and IκBε)by RNA interference using pcDNATM6.2-miR-IκBα-IκBβ-IκBε co-targeting IκBα,IκBβ,and IκBε genes,so as to lay a foundation for future study.Methods: We designed and constructed three interfering plasmids pcDNATM6.2-miR-IκBα(1,2,3)targeting human IκBα gene; the three vectors were transfected into HepG2 cells separately and the most effective interfering fragment was identified by RT-PCR and Western blotting analysis. Similarly,we also constructed and selected the most effective interfering vectors targeting IκBβ and IκBε. Then we chained the three most effective interfering fragments (miR-IκBα,miR-IκBβ,and miR-IκBε) into pcDNATM6.2-miR vector to construct pcDNATM6.2-miR-IκBα-IκBβ-IκBε vector,which was used to transfect HepG2 cells and the transfectants were selected by G418.The expression of IκBα,IκBβ,and IκBε in the transfectants was examined by Western blotting analysis.Results: We successfully constructed the pcDNATM6.2-miR-IκBα-IκBβ-IκBε vector. Western blotting analysis showed that,compared with the normal HepG2 cells and those transfected with pcDNATM6.2-miR-Neg vector,the protein expression of IκBα,IκBβ,and IκBε was stably down-regulated in cells transfected with pcDNATM6.2-miR-IκBα-IκBβ-IκBε vector.Conclusion: We have successfully established a HepG2 cell line with simultaneous knockdown of IκBα,IκBβ,and IκBε,paving a way for future study.