Abstract: Objective To study the effect of E3 ubiquitin ligase RNF31 knockdown on nuclear factor-κB (NF-κB) pathway
activation and cell apoptosis. Methods Human RNF31 siRNA sequences were cloned into the lentiviral vector pGreenPuro and
transiently transfected in HEK293T cells to screen the most effective fragments, which were co-transfected along with the
packaging plasmids PMD and SPA in 293T cells. The cell supernatant was collected at 24 h and 48 h after the transfection and
the viral titers were determined with flow cytometry. Real-time PCR and Western blotting were used to evaluate the effect of
RNF31 knockdown on the expression of NF-κB downstream target genes and IκBα activity; the changes of NF-κB pathway
transcriptional activity were assessed with dual luciferase reporter gene. Hochest dying was used to examine the influence of
RNF31 down-regulation on cell apoptosis. Results RNF31 knockdown mediated by the lentiviral vector pGreenPuro-RNF31
suppressed the transcriptional activity of NF-κB and the downstream target genes in HEK293 cells stimulated with TNF-α.
RNF31 knockdown also resulted in suppression of NF-κB-stimulated expression of pIκBα and in increased apoptosis of cells
stimulated with TNF-α for 24 h. Conclusion RNF31 down-regulation inhibits NF-κB pathway activation induced by TNF-α.