人干扰素基因刺激因子(STING)基因沉默子的克隆鉴定及功能初探  

Identification and characterization of stimulator of interferon gene silencer

作　　者：庞晓雨 陈海燕 陈林媛 徐华国 Pang Xiaoyu;Chen Haiyan;Chen Linyuan;Xu Huaguo(Department of Laboratory,The first Affiliated Hospital to Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南京医科大学第一附属医院检验学部,210029

出　　处：《中华微生物学和免疫学杂志》2021年第5期353-360,共8页Chinese Journal of Microbiology and Immunology

基　　金：江苏省自然科学基金(BK20181492);江苏省高层次卫生人才"六个一工程"拔尖人才科研项目(LGY2018058);江苏省研究生科研创新项目(KYCX19_1169)。

摘　　要：目的:构建人干扰素基因刺激因子(stimulator of interferon genes,STING)沉默子的荧光素酶报告质粒,在人胚肾细胞(HEK293T)和人类宫颈癌细胞(HeLa)中检测荧光素酶活性,生物信息学预测其可能结合的转录因子并通过实验验证。方法:通过PCR扩增人STING-5-1a(-124~+267,相对于转录起始位点,TSS:0)和STING-5-2a(-124~+168)区域,亚克隆至pGL3-Basic质粒;将人STING沉默子区域STING-5-1b(+169~+267)正向反向亚克隆至pGL3-Control质粒(pGL3-C-5-1b-positive/negative),并把STING-5-1b分为两段互补片段,亚克隆至pGL3-Control载体上,命名为pGL3-C-STING-5-1b-α(+169~+209)/pGL3-C-STING-5-1b-β(+210~+267),双荧光素酶报告活性分析检测上述重组质粒在HEK293T和HeLa中的活性。应用生物信息学方法预测人STING沉默子的转录因子结合位点,将预测结合位点进行多点突变,检测荧光素酶活性。敲低转录因子,免疫印迹试验检测STING的表达水平。染色体免疫共沉淀反应进一步验证转录因子与人STING沉默子的结合。结果:核酸测序证实,上述重组质粒构建成功。截短STING-5-1b(+169~+267)片段后相对荧光素酶活性上升。同时,与pGL3-Control质粒相比,pGL3-C-5-1b-positive重组质粒的相对荧光素酶活性下降(P<0.05),其中,STING-5-1b-β(+210~+267)序列起主要抑制作用。应用生物信息学软件预测发现转录因子PR结构域锌指蛋白4(PRDM4)、Thanatos相关蛋白1(THAP1)、TEA域转录因子1(TEAD1)、核受体亚家族4 A组成员1(NR4A1)、类Krueppel因子4(KLF4)和叉头转录蛋白O3(FOXO3)可能与人STING沉默子区域(+210~+267)结合。对上述转录因子的结合位点进行多点突变构建突变体并转染至HEK293T和HeLa,发现THAP1-Mut和TEAD1-Mut的相对荧光素酶活性显著上升,提示STING沉默子可能含有THAP1和TEAD1的结合位点。将转录因子THAP1和TEAD1敲低后,STING的表达水平有显著上升。染色体免疫共沉淀试验表明,转录因子TEAD1和THAP1在细胞内与人Objective To Clone the silencer sequences of human stimulator of interferon genes(STING)and evaluate its activity in HEK293T and HeLa,and to preliminarily investigate the transcriptional regulatory mechanisms,screening and verifying the possible binding elements for the silencer sequences of STING.Methods The human STING-5-1a(-124~+267,transcription start site,TSS:0)and STING-5-2a(-124~+168)regions were amplified by PCR,subcloned into pGL3-Basic plasmid,and the luciferase activity was detected in HEK293T and HeLa;the human STING silencer region STING-5-1b(+169~+267)was subcloned into the pGL3-Control plasmid(pGL3-C-5-1b-positive/negative)and STING-5-1b is divided into two complementary elements,subcloned into pGL3-Control vector,named pGL3-C-STING 5-1b-α(+169~+209)and pGL3-C-STING-5-1b-β(+210~+267),detecting the relative luciferase activity of the above plasmid in HEK293T and HeLa.Using bioinformatics methods to predict the transcription factor binding site of human STING silencer,make site-directed mutagenesis at the predicted binding site,and detect luciferase activity.Knockdown of THAP1 and TEAD1 by a siRNA strategy and detect the expression level of STING by western blot analysis.Chromosome immunoprecipitation assay further verified the binding of transcription factor to hSTING silencer.Results It was verified that plasmids mentioned above were constructed correctly by nucleotide sequencing.The relative luciferase increased after truncating the STING-5-1b(+169~+267)fragment.The relative luciferase activity of pGL3-C-5-1b-positive recombinant plasmid decreased(P<0.05),compared with pGL3-Control plasmid.Among them,STING-5-1b-β(+210~+267)fragment play a major inhibitory role.Using bioinformatics software to predict that the transcription factors PR domain zinc finger protein 4(PRDM4),Thanatos-associated protein 1(THAP1),TEA Domain Transcription Factor 1(TEAD1),Nuclear receptor subfamily 4 group A member 1(NR4A1),Krueppel-like factor 4(KLF4)and Forkhead box protein O3(FOXO3)may bind to the human STING silencer

关 键 词：干扰素基因刺激因子 沉默子 转录调控 

分 类 号：R392[医药卫生—免疫学]