细胞水平筛选鉴定激活核因子κB通路的新基因TMEM9B

Cell-based Screening and Validation of Human Novel Gene TMEM9B Activated Nuclear Factor κB Signaling Pathway

核因子κB(NF-κB)是细胞内重要的转录因子,其介导的细胞信号转导通路在细胞凋亡中的作用是国内外研究的热点.为了筛选NF-κB通路相关新基因,建立了基于细胞水平的报告基因高通量筛选模型.利用双荧光素酶报告系统检测报告基因荧光素酶活性,通过对构建的439个人类未知功能基因的筛选,获得了一批激活NF-κB信号通路的功能基因,其中基因TMEM9B可以明显激活NF-κB通路.进一步实验显示TMEM9B激活NF-κB通路呈明显剂量依赖性,Westernblot及EMSA实验证实,TMEM9B能够促进胞质内NF-κB的抑制分子IκBα的降解,并促使NF-κB由胞质向胞核转移,同时流式细胞术实验发现TMEM9B可引起293T和HeLa细胞的凋亡.总之,所建立的基于细胞水平的NF-κB通路筛选模型稳定高效,筛选并验证TMEM9B可明显激活NF-κB信号转导通路,并从而引起细胞凋亡.

Nuclear factor κB （NF-κB） is an important cellular transcription factor.The important role of NF-κB-mediated cell signal transduction pathway in apoptosis is a hot topic at home and abroad.In order to discover new regulators in NF-κB signaling pathway,a high-throughput cell-based screening model based on dual luciferase reporters system was established,a number of genes that can activate NF-κB signal pathway were obtained by screening of 439 novel function genes.Among them,TMEM9B can obviously activate NF-κB signaling pathway.Further experiments showed that TMEM9B activated NF-κB signaling pathway in a dose-dependent pattern.Western blotting and EMSA experiments confirmed that TMEM9B can promote the degradation of IκBα （a cytoplasm inhibitor of NF-κB）,and cause NF-κB shift from the cytoplasm to nucleus.At the same time,flow cytometry result demonstrated TMEM9B can induce apoptosis in HEK293T and HeLa cells.In short,a stable and effective screening system for NF-κB has been established,through which TMEM9B was identified to be able to significantly activate NF-κB signal transduction pathway and thus cause cells apoptosis.