Lentivirus-mediated interference of E3 ubiquitin ligase RNF31 inhibits tumor-necrosis factor-α-induced activation of nuclear factor-κB pathway / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To study the effect of E3 ubiquitin ligase RNF31 knockdown on nuclear factor-κB (NF-κB) pathway activation and cell apoptosis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>RNF31 down-regulation inhibits NF-κB pathway activation induced by TNF-α.</p>