摘要
目的 研究维甲酸诱导基因G（RIG-G）蛋白调控p21基因表达的相关机制.方法 采用Western blot法检测白血病细胞株NB4中过表达RIG-G蛋白对p21蛋白表达的影响,以及U937细胞中过表达RIG-G蛋白对c-Jun和JNK蛋白磷酸化水平的影响.将c-Jun表达质粒与含p21基因启动子的报告基因质粒共同转染至293T细胞,采用荧光素酶报告基因实验研究c-Jun蛋白对p21基因的表达调控作用.结果 Western blot法检测结果显示,在NB4细胞中过表达RIG-G蛋白能明显上调p21蛋白的表达水平;而且在全反式维甲酸（ATRA）诱导NB4细胞分化的过程中,随着内源性RIG-G蛋白被明显地诱导表达,p21蛋白的表达水平也明显升高.在过表达RIG-G蛋白的U937细胞中,JNK和c-Jun蛋白的磷酸化水平均明显下降,而JNK和c-Jun总蛋白的表达水平则无明显变化.当U937细胞中加入JNK抑制剂SP600125后,尽管JNK蛋白的磷酸化被完全抑制,但与对照细胞U937T-pTRE比较,过表达RIG-G蛋白的U937T-RIG-G细胞中,c-Jun蛋白的磷酸化水平依然明显下降.表明RIG-G蛋白不仅能通过JNK信号通路抑制c-Jun蛋白的磷酸化,也可以以不依赖JNK信号途径的方式下调c-Jun蛋白的磷酸化水平.荧光素酶报告基因检测结果则显示,当293T细胞中分别转染0.1、0.5、1.0和2.0 μg c-Jun表达质粒时,荧光素酶报告基因的活性分别为转染空载体组的（83.0±1.7）％、（73.7±0.7）％、（68.9±0.9）％和（64.1±0.9）％,提示c-Jun蛋白能抑制p21基因的转录表达活性.结论 在U937细胞中,过表达RIG-G蛋白能通过多条不同的信号途径共同下调c-Jun蛋白的磷酸化水平,最终使p21基因的表达水平升高,发挥阻断细胞周期进程,抑制细胞生长的功能.
Objective To investigate the mechanisms by which retinoic acid-induced gene G （RIG-G） protein regulates p21 gene expression.Methods Western blot was used to detect the effects of RIG-G protein overexpression on p21 protein expression level in leukemia cell line NB4 cells and the phosphorylation of both c-Jun and JNK in U937 cells.The c-Jun expression plasmid and p21 gene promotercontaining reporter plasmid were co-transfected into 293T cells,to explore the regulatory effect of c-Jun protein on p21 gene expression by luciferase reporter assay.Results Western blot showed that the overexpression of RIG-G protein significantly upregulated p21 protein level in the NB4 cells,and the level of p21 protein largely increased along with the induction of endogenous RIG-G protein during the differentiation of NB4 cells treated by all-trans retinoic acid （ATRA）.Moreover,the phosphorylation of both c-Jun and JNK decreased in RIG-G-overexpressing U937 cells while total c-Jun and JNK proteins remained unchanged.After using the JNK inhibitor SP600125 to block JNK phosphorylation,the level of c-Jun phosphorylation was still dramatically reduced in the RIG-G-overexpressing U937T-RIG-G cells,compared with the control U937T-pTRE cells.These results indicated that the inhibitory effect of Rig-G protein on c-Jun phosphorylation could not only be through the JNK pathway,but also via some JNK-independent pathways.Luciferase reporter assay showed that when 0.1,0.5,1.0 and 2.0 μg c-Jun-expressing plasmids were respectively transfected into 293T cells,compared with the empty vector-transfected group,the relative luciferase activities were （83.0 ± 1.7）%,（73.7 ±0.7）%,（68.9 ±0.9）% and （64.1 ±0.9）%,indicating that the transcriptional activity of p21 gene could be inhibited by c-Jun protein.Conclusions RIG-G protein may suppress the phosphorylation of c-Jun protein through different signal pathways,thereby increasing the expression of p21 gene,arresting the cell cycle and inhibiting the cell growth in U937 cells.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2014年第9期657-661,共5页
Chinese Journal of Oncology
基金
国家自然科学基金（ 81170508,81100365 ）
上海市领军人才“国家队”专项基金
