NF⁃κB p65调控IRF⁃8基因启动子活性及其启动子结合元件的初步鉴定
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国家自然科学基金(31470853,81603358,31770934,81971468)


The regulatory effects of NF⁃kB p65 on IRF⁃8 gene promoter activity and initial identification of its binding elements
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    摘要:

    目的:探讨大鼠核因子-κB(nuclear factor-κB,NF-κB)p65亚基在细胞内过表达和其活性变化对干扰素调节因子-8(interferon regulatory factor-8,IRF-8)基因启动的影响,并初步筛查IRF-8启动子上可能的p65结合元件。方法:采用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增大鼠p65基因蛋白编码区(complete sequence coding,CDS)序列,将其插入到空载pIRES2-EGFP质粒中,构建大鼠野生型(wild type,WT)p65过表达质粒(pIRES2-p65 WT)。在此基础上,将p65第535位丝氨酸(serine,S)突变为天冬氨酸(aspartic,D)或丙氨酸(alanine,A),分别构建p65持续活化突变型质粒(pIRES2-p65 S535D)和p65显性负性突变型质粒(pIRES2-p65 S535A)。之后应用生物信息学软件预测IRF-8基因启动子区p65的结合元件,并据此构建IRF-8启动子全长(full-length,FL)和3个截短的荧光素酶报告质粒,即pGL3-IRF-8-FL(-1 892 ~ +174 nt)、pGL3-IRF-8-1(-1 360 ~ +174 nt)、pGL3-IRF-8-2(-752 ~ +174 nt)和pGL3-IRF-8-3(-68 ~ +174 nt)。将上述质粒行不同组合共转染人胚肾293T(human embryonic kidney 293T,HEK-293T)细胞,Western blot和荧光素酶实验分别检查p65的表达和IRF-8的启动子活性,并分析IRF-8启动子区可能的p65结合元件。结果:菌液PCR及测序证实上述质粒构建成功。分别将pIRES2-p65 WT、pIRES2-p65 S535D、pIRES2-p65 S535A和pGL3-IRF-8-FL共转染HEK-293T,发现过表达pIRES2-p65 WT或pIRES2-p65 S535D均可明显增加IRF-8启动子活性,且以pIRES2-p65 S535D更为显著;而过表达pIRES2-p65 S535A后,IRF-8启动子活性无明显变化。将pGL3-IRF-8-FL、pGL3-IRF-8-1~3和pIRES2-p65 S535D共转染HEK-293T后发现,pGL3-IRF-8-3的启动子活性显著低于pGL3-IRF-8-FL、pGL3-IRF-8-1和pGL3-IRF-8-2,提示大鼠IRF-8启动子-752~68 nt区域可能存在p65结合元件。结论:在HEK-293T细胞内过表达野生型或持续活化突变型p65可显著促进IRF-8基因的启动,且p65与IRF-8启动子的结合元件可能位于-752~-68 nt部位。

    Abstract:

    Objective:This study aims to investigate the effect of rat nuclear factor-κB(NF-κB)p65 subunit over-expression and its activity changes on the gene promoter activity of interferon regulatory factor-8(IRF-8),and initially screen the possible p65-binding elements within IRF-8 promoter. Methods:To construct the rat wild type p65 over-expression plasmid(pIRES2-p65 WT),complete sequence coding(CDS)of rat p65 was amplified by polymerase chain reaction(PCR) and cloned into pIRES2-EGFP. Then,S535D and S535A mutation was done respectively based on the wild type p65 over-expression plasmid to construct p65 constitutively active mutant(pIRES2-p65 S535D)and p65 dominant negative mutant(pIRES2-p65 S535A). The potential p65-binding elements within IRF-8 promoter were predicted by using bioinformatics software. Based on the predicted results,luciferase reporter plasmids of full-length(FL)and three truncated IRF-8 gene promoter were constructed,namely pGL3-IRF-8-FL(-1 892~+174 nt),pGL3-IRF-8-1(-1 360~+174 nt),pGL3-IRF-8-2(-752~+174 nt),pGL3-IRF-8-3(-68~+174 nt). The above-mentioned plasmids were co-transfected into human embryonic kidney 293T(HEK-293T)cells in different groups. Then,the expression level of p65 was detected by Western blot,and the promoter activity of IRF-8 was detected by luciferase experiment to screen the p65-binding elements. Results:It was verified that above-mentioned plasmid was constructed correctly by PCR analysis and nucleotide sequencing. The plasmids of pIRES2-p65 WT,pIRES2-p65 S535D,pIRES2-p65 S535A were respectively transfected into HEK-293T cells together with pGL3-IRF-8-FL. The luciferase results showed that the activity of IRF-8 promoter was markedly increased in response to pIRES2-p65 WT and pIRES2-p65 S535D,especially the later. However,there was no significant change of IRF-8 promoter activity after over-expression of pIRES2-p65 S535A. The plasmids of pGL3-IRF-8-FL or pGL3-IRF-8-1~3 and pIRES2-p65 were co-transfected into HEK-293T cells,and the result displayed that the activity of pGL3-IRF-8-3 was much lower than that of pGL3-IRF-8-FL,pGL3-IRF-8-1 and pGL3-IRF-8-2,indicating that the region of rat IRF-8 promoter(-752~-68 nt) might contain p65-binding elements. Conclusion:Over-expression of wild-type or continuously activated mutant p65 in HEK-293T cells can significantly promote the activity of IRF-8 promoter,and the p65-binding elements in IRF-8 promoter might be located in the -752~-68 nt region.

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王文博,钱宝梅,罗 灿,彭明玉,张 婧,赵 聃,王迎伟,邱 文,季明德. NF⁃κB p65调控IRF⁃8基因启动子活性及其启动子结合元件的初步鉴定[J].南京医科大学学报(自然科学版),2020,(5):638-644

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  • 收稿日期:2019-11-17
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  • 在线发布日期: 2020-06-10
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