IL-6基因-572C/G多态性调控启动子的核提取物结合活性

doi:10.19871/j.cnki.xfcrbzz.2022.01.004

新发传染病电子杂志 ›› 2022, Vol. 7 ›› Issue (1): 17-20.doi: 10.19871/j.cnki.xfcrbzz.2022.01.004

IL-6基因-572C/G多态性调控启动子的核提取物结合活性

傅佳鹏¹, 刘淑燕¹, 李芳², 欧敏², 梁志航², 吴季霖²

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

收稿日期:2021-07-29 出版日期:2022-02-28 发布日期:2022-07-07
通讯作者: 吴季霖,Email：6296082@qq.com
基金资助:
1.广东省基础与应用基础研究基金（2019B1515120041,2019A1515110055,2020A1515010977）; 2.广东省医学科学技术研究基金（B2021273）; 3.深圳市卫生计生系统科研项目（SZXJ2018047）

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

Fu Jiapeng¹, Liu Shuyan¹, Li Fang², Ou Min², Liang Zhihang², Wu Jilin²

1. Department of the Third Pulmonary Disease, Southern University of Science and Technology, the Third People's Hospital of Shenzhen, Guangdong Shenzhen 518112, China;
2. Department of Liver Research Institute, Southern University of Science and Technology, the Third People's Hospital of Shenzhen, Guangdong Shenzhen 518112, China

Received:2021-07-29 Online:2022-02-28 Published:2022-07-07

摘要/Abstract

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-572C/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启动子位置区域,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。

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

Abstract: 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 -572C/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-572C/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 Resultshowed 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 -572C/G polymorphism can affect the transcriptional activity of the IL-6 promoter and regulate the binding activity of nuclear extracts of IL-6 promoters.

Key words: Interleukin-6, Gene polymorphism, Promoter, Binding activity of nuclear extract

傅佳鹏, 刘淑燕, 李芳, 欧敏, 梁志航, 吴季霖. IL-6基因-572C/G多态性调控启动子的核提取物结合活性[J]. 新发传染病电子杂志, 2022, 7(1): 17-20.

Fu Jiapeng, Liu Shuyan, Li Fang, Ou Min, Liang Zhihang, Wu Jilin. IL-6 gene-572C/G polymorphism regulates the binding activity of nuclear extract with the promoter[J]. Electronic Journal of Emerging Infectious Diseases, 2022, 7(1): 17-20.

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IL-6基因-572C/G多态性调控启动子的核提取物结合活性

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

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