Lentivirus-mediated interference of E3 ubiquitin ligase RNF31 inhibits tumor-necrosis fac-tor-α-induced activation of nuclear factor-κB pathway
10.3969/j.issn.1673-4254.2014.12.02
- VernacularTitle:RNF31表达下调抑制TNF-α刺激的NF-κB通路的激活
- Author:
Jie CHEN
1
;
Hui CHEN
;
Yiqun ZHAN
;
Xiaoming YANG
;
Miao YU
Author Information
1. 天津大学制药工程专业
- Keywords:
E3 ubiquitin ligase RNF31;
lentivirus;
nuclear factor-κB;
transcriptional regulation
- From:
Journal of Southern Medical University
2014;(12):1713-1720
- CountryChina
- Language:Chinese
-
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 pIκBαand in increased apoptosis of cells stimulated with TNF-αfor 24 h. Conclusion RNF31 down-regulation inhibits NF-κB pathway activation induced by TNF-α.
