title: [Lentivirus-mediated interference of E3 ubiquitin ligase RNF31 inhibits tumor-necrosis factor-α-induced activation of nuclear factor-κB pathway].
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.
  
  
  CONCLUSIONS
  RNF31 down-regulation inhibits NF-κB pathway activation induced by TNF-α.
authors.affiliation: Department of Pharmaceutical Engineering, Tianjin University, Tianjin 300072, China.E-mail: yinicj@163.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Yiqun
authors.initials: Y
authors.lastname: Zhan
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Yang
authors.forename: Miao
authors.initials: M
authors.lastname: Yu
mesh_terms: D017209:Apoptosis; D015536:Down-Regulation; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D016086:Lentivirus; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D034741:RNA, Small Interfering; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; C558338:RNF31 protein, human; D044767:Ubiquitin-Protein Ligases