IL-6基因-572 C/G多态性调控启动子的核提取物结合活性  

IL-6 gene-572 C/G polymorphism regulates the binding activity of nuclear extract with the promoter

作　　者：傅佳鹏 刘淑燕 李芳 欧敏 梁志航 吴季霖 Fu Jiapeng;Liu Shuyan;Li Fang;Ou Min;Liang ZhiHang;Wu Jilin(Department of the Third Pulmonary Disease,Southern University of Science and Technology,the Third People's Hospital of Shenzhen,Guangdong Shenzhen 518112,China;Department of Liver Research Institute,Southern University of Science and Technology,the Third People's Hospital of Shenzhen,Guangdong Shenzhen 518112,China)

机构地区：[1]南方科技大学第二附属医院,深圳市第三人民医院肺病三科,广东深圳518112 [2]南方科技大学第二附属医院,深圳市第三人民医院肝病研究所,广东深圳518112

出　　处：《新发传染病电子杂志》2022年第1期17-20,共4页Electronic Journal of Emerging Infectious Diseases

基　　金：广东省基础与应用基础研究基金(2019B1515120041,2019A1515110055,2020A1515010977);广东省医学科学技术研究基金(B2021273);深圳市卫生计生系统科研项目(SZXJ2018047)。

摘　　要：目的探讨-572C/G多态性位点对IL-6基因启动子与核提取物结合活性的影响,为预测疾病的发生发展提供参考。方法将携带-572位点C等位基因型的IL-6基因启动子区(-1029~52bp)克隆到pGL3-Basic载体中,构建pGL3/-572C载体,采用定点突变试剂盒获得pGL3/-572G载体。将构建的IL-6启动子荧光素载体转染至HeLa细胞,利用双荧光素酶系统分析-572C/G基因多态性对启动子活性的影响。设计合成了,涵盖IL-6启动子区-572位点C/G基因型的单链互补寡核苷酸,并在3'端进行生物素标记。然后退火形成双链寡核苷酸,之后与HeLa细胞核提取物孵育,用电泳迁移率实验(EMSA)分析IL-6基因-572 C/G多态性对核提取物结合活性的影响。结果pGL3/-572C在HeLa细胞中的转录活性显著高于pGL3/-572G,差异有统计学意义(P<0.01);EMSA结果显示当核提取物与用生物素标记的C探针孵育后出现一条强迁移条带(复合物1)及一条超级迁移条带(复合物2),而G探针与核提取物孵育后仅出现一条弱迁移条带(复合物1),且用未标记生物素的C或G竞争探针进行竞争性结合,可以特异性抑制复合物1的形成,表明-572C等位基因突变能增加IL-6启动子与核蛋白的结合活性,使更多的核蛋白结合在IL-6启位置区域,形成起始转录复合物。结论-572C/G多态性能影响IL-6启动子的转录活性,同时对IL-6启动子的核提取物结合活性具有调控作用。Objective To investigate the effect of-572C/G polymorphism on the binding activity of IL-6 gene promoter and nuclear extract,it provides a reference for predicting the occurrence and development of diseases.Method A sequence of the IL-6 promoter region,from-1029 to 52 obtained from a subject with C allelotype at position-572 was cloned into the pGL3-Basic vector.The pGL3/-572C vector was mutated to pGL3/-572G,using Site-DirectedMutagenesis Kit.The IL-6 promoter luciferase vectors were transfected into HeLa cells,and the effects of-572 C/G gene polymorphism on promoter activity was determined using Dual-Glo luciferase assay system.The single-stranded complementary oligonucleotides covering the IL-6 promoter region-572 site were designed and synthesized,and biotin-labeled at the 3'end.Then annealed to form a double-stranded oligonucleotide,which is then incubated with HeLa cell nuclear extracts,and the effects of IL-6 allele-572 C/G on the binding activity of nuclear extract was analyzed by electrophoretic mobility-shift assay(EMSA).Result The transcription activity of pGL3/-572C in HeLa cells was significantly higher than that of pGL3/-572G,and the difference was statistically significant(P<0.01);EMSA Result showed that one strong band (complex 1) and one super shift band was shifted when the nuclear extracts were incubated with biotin-labeled C probe, In contrast, there was much less complex 1 formation on the G probe. Furthermore, complex 1 formation was specifically blocked by unlabeled C or G competitor. These result indicated that -572 C alleles variant increased the binding activity of more nuclear protein to an IL-6 promoter region, to form the transcription initiation complex. Conclusion The -572 C/G polymorphism can affect the transcriptional activity of the IL-6 promoter and regulate the binding activity of nuclear extracts of IL-6 promoters.

关 键 词：白细胞介素-6 基因多态性 启动子 核提取物结合活性 

分 类 号：R392[医药卫生—免疫学]