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

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STING沉默子区域结合。结论:本实验成功构建了人STING沉默子荧光素酶报告质粒,通过活性比较,推测人STING的核心沉默子区位于+210~+267区域,通过生物信息学分析及点突变实验初步确定人STING沉默子可能含有的潜在转录因子结合位点。并使用免疫印迹试验和染色体免疫共沉淀进一步证实转录因子TEAD1和THAP1与人STING沉默子的结合,为后续研究提供实验资料。

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 region(+210~+267).After transfecting the mutant recombinant plasmid of the transcription factors into HEK293T and HeLa,the relative luciferase activity of THAP1-Mut and TEAD1-Mut were significantly increased,suggesting that STING silencers may contain binding sites of THAP1 and TEAD1.Knockdown of THAP1 and TEAD1 by a siRNA strategy significantly enhanced the transcription activity.Chromosome immunoprecipitation assay showed that the transcription factors TEAD1 and THAP1 combined with hSTING silencer region in the cells.Conclusions The hSTING silencer luciferase reporter plasmid was successfully constructed.By the activity comparison,it is speculated that the core silencer region of human STING is located in the+210~+267 element,which may contain several potential transcription factor binding sequences.The potential binding sites for transcription factors that may be contained in the DNA,and use Western blot and chromosome immunoprecipitation assays to further confirm the combination of transcription factors TEAD1 and THAP1 with hSTING silencer,laying the foundation for subsequent research.