[1]陈洁,陈慧,詹轶群,等.RNF31表达下调抑制TNF-α刺激的NF-κB通路的激活[J].南方医科大学学报,2014,(12):1713.
点击复制

RNF31表达下调抑制TNF-α刺激的NF-κB通路的激活()
分享到:

《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2014年12期
页码:
1713
栏目:
出版日期:
2014-12-20

文章信息/Info

Title:
Lentivirus-mediated interference of E3 ubiquitin ligase RNF31 inhibits tumor-necrosis factor-α-induced activation of nuclear factor-κB pathway
作者:
陈洁陈慧詹轶群杨晓明于淼
关键词:
E3泛素连接酶RNF31慢病毒NF-κB转录调节
Keywords:
E3 ubiquitin ligase RNF31 lentivirus nuclear factor-κB transcriptional regulation
摘要:
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pGreenPuro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pGreenPuro-RNF31载
体并获得慢病毒颗粒,病毒滴度可达3×107 pfu/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。
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. Conclusion RNF31 down-regulation inhibits NF-κB pathway activation induced by TNF-α.

相似文献/References:

[1]李川,马纪,张越,等.慢病毒介导的shRNA靶向干扰nestin鼻咽癌稳定细胞株的建立[J].南方医科大学学报,2011,(04):604.
[2]耿海涛,肖乾,徐邓勇,等.HNF6 重组慢病毒载体的构建及对大肠癌SW620细胞侵袭转移能力的影响[J].南方医科大学学报,2012,(01):66.
[3]杨慧,张超,陆滟霞,等.Has-mir-335慢病毒表达载体的构建及其靶基因鉴定[J].南方医科大学学报,2012,(03):306.
[4]荆玉明,罗杰,张艳玲,等.p38丝裂原激活蛋白激酶基因重组慢病毒载体的构建及其在建立人前列腺癌稳定细胞株中的应用[J].南方医科大学学报,2012,(03):317.
[5]刘悦,郝玉娥,詹轶群,等.人甘油激酶慢病毒干涉载体的构建和表达[J].南方医科大学学报,2012,(05):614.
[6]王雪峰,何援利,付霞霏,等.慢病毒介导的Bcl-2基因对磷酰胺氮芥诱导人原代卵巢颗粒细胞凋亡的保护作用[J].南方医科大学学报,2012,(07):932.
[7]彭冬先,何援利,丘立文.慢病毒介导shRNA沉默survivin基因对鸡胚绒毛尿囊膜子宫内膜异位症模型的影响[J].南方医科大学学报,2012,(07):995.
[8]邓海军,孙凯,郭琛,等.人microRNA-338-3p慢病毒表达载体的构建及其靶基因鉴定[J].南方医科大学学报,2012,(08):1098.
[9]陈芳,周畅,陆艳霞,等.Hsa-miR-186在结肠癌细胞中的表达及作用[J].南方医科大学学报,2013,(05):654.
[10]周学亮,刘季春.Notch1胞内结构域慢病毒表达载体及干扰载体的构建[J].南方医科大学学报,2015,(05):692.

更新日期/Last Update: 1900-01-01