Establishment of a stable HepG2 cell line with simultaneous knockdown of nuclear factor-kappaB inhibitors IκBα, IκBβ and IκBε genes / 第二军医大学学报

ABSTRACT

Objective: To establish a human hepatic cancer cell line- HepG2 with stable, simultaneous knockdown of three nuclear factor-kappaB inhibitors (IKappa;Bα, IκBβ, and IκBε) by RNA interference using pcDNA™ 6.2-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 pcDNA™ 6.2-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 pcDNA™ 6.2-miR vector to construct pcDNA™ 6.2-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 pcDNA™ 6.2-miR-IκBα- IκBε-IκBε vector. Western blotting analysis showed that, compared with the normal HepG2 cells and those transfected with pcDNA™ 6.2-miR-Neg vector, the protein expression of IκBα, IκBβ, and IκBε was stably down-regulated in cells transfected with pcDNA™ 6.2-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.