Chen Jie, Chen Hui, Zhan Yiqun, Yang Xiaoming, Yu Miao
Department of Pharmaceutical Engineering, Tianjin University, Tianjin 300072, China.E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2014 Dec;34(12):1713-20.
To study the effect of E3 ubiquitin ligase RNF31 knockdown on nuclear factor-κB (NF-κB) pathway activation and cell apoptosis.
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.
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.
RNF31 down-regulation inhibits NF-κB pathway activation induced by TNF-α.
研究E3泛素连接酶RNF31敲低对核因子-κB（NF-κB）通路激活及细胞凋亡的影响。
将人RNF31小干扰RNA（siRNA）序列克隆至慢病毒载体pGreenPuro中，并在人胚肾细胞293T（HEK293T）中进行瞬时转染以筛选最有效的片段，然后将其与包装质粒PMD和SPA共转染至293T细胞中。转染后24小时和48小时收集细胞上清液，采用流式细胞术测定病毒滴度。采用实时荧光定量聚合酶链反应（Real-time PCR）和蛋白质免疫印迹法（Western blotting）评估RNF31敲低对NF-κB下游靶基因表达及IκBα活性的影响；采用双荧光素酶报告基因检测法评估NF-κB通路转录活性的变化。采用Hochest染色法检测RNF31下调对细胞凋亡的影响。
慢病毒载体pGreenPuro-RNF31介导的RNF31敲低抑制了肿瘤坏死因子-α（TNF-α）刺激的HEK293细胞中NF-κB的转录活性及其下游靶基因的表达。RNF31敲低还导致NF-κB刺激的磷酸化IκBα（pIκBα）表达受到抑制，并使TNF-α刺激24小时的细胞凋亡增加。
RNF31下调可抑制TNF-α诱导的NF-κB通路激活。
