RNF31表达下调抑制TNF-α刺激的NF-κB通路的激活

Lentivirus-mediated interference of E3 ubiquitin ligase RNF31 inhibits tumor-necrosis factor-α-induced activation of nuclear factor-κB pathway

目的利用慢病毒干涉下调内源性RNF31表达,研究NF-κB通路的活化及对细胞凋亡的影响。方法将人RNF31的shRNA片段克隆到慢病毒表达载体p Green Puro中,瞬时转染HEK293T细胞,筛选出有效的干涉片段。将重组表达质粒与包装质粒PMD、SPA共转染293T细胞,在24 h、48 h分2次收集慢病毒上清,用流式细胞术检测病毒滴度。将获得的病毒感染HEK293细胞,提取细胞蛋白,Real-time PCR以及Western Blot检测RNF31干涉效果;报告基因实验检测敲低RNF31对NF-κB转录活性的影响;Real-time PCR检测干涉RNF31对TNF-α诱导的NF-κB下游靶基因的影响;Western Blot检测下调RNF31对IκBα活化的影响;Hochest染色检测下调RNF31对细胞凋亡的影响。结果成功构建RNF31干涉慢病毒p Green Puro-RNF31载体并获得慢病毒颗粒,病毒滴度可达3×107pfu/ml。在HEK293细胞中下调RNF31,抑制TNF-α刺激的NF-κB的转录活性,并抑制NF-κB下游靶基因的表达;下调RNF31抑制TNF-α刺激的IκBα的活化;此外,在TNF-α刺激细胞24 h时,RNF31表达下调使细胞凋亡增多。结论 RNF31表达下调抑制TNF-α刺激的NF-κB通路的激活。

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.