新疆医科大学学报

目的 探讨基本转录因子3(Basic transcription factor3,BTF3)在食管鳞癌细胞中的下游转录调控网络及其关键靶基因。方法 在食管鳞癌KYSE150细胞中，利用染色质免疫共沉淀测序(Chromatin immunoprecipitation sequencing, ChIP-seq)鉴定BTF3的全基因组结合位点；结合慢病毒转染稳定敲低BTF3后的KYSE150细胞RNA测序(RNA sequencing, RNA-seq)所得差异表达基因，筛选BTF3的直接调控靶点；通过染色质免疫共沉淀-聚合酶链式反应(Chromatin immunoprecipitation-quantitative polymerase chain reaction, ChIP-qPCR)对关键候选靶基因进行验证。结果 ChIP-seq分析揭示了BTF3在基因组上的特异性结合图谱。相较于对照组(Input组),免疫沉淀(Immunoprecipitation, IP)组以C/G富集、特别是包含CCCC或CCC核心的序列作为最显著的基序(Motif),BTF3在基因组中的结合高度富集于基因间区和内含子区域，基因本体论(Gene ontology, GO)-细胞组分(Cellular component, CC)显示靶基因显著富集于胞外区域、细胞外空间和含胶原蛋白的细胞外基质，生物过程(Biological process, GO-BP)显示靶基因主要富集在G蛋白偶联受体信号通路和信号转导生物学过程，分子功能(Molecular function, GO-MF)显示靶基因主要参与G蛋白偶联受体活性、蛋白质结合以及RNA结合。对BTF3敲低的RNA-seq数据中得到的441个差异表达基因与ChIP-seq实验中出现的富集峰(peak)所在的基因进行整合分析，共鉴定出54个重叠基因，即BTF3潜在的直接调控靶基因。这54个重叠基因在胞外区域和细胞外空间有显著富集(GO-CC),重叠基因主要参与正向调节基因表达的生物学过程(GO-BP)以及蛋白质结合(GO-MF)。ChIP-qPCR实验证实BTF3是关键靶基因2′-5′-寡腺苷酸合成酶样蛋白(OASL)、纤维蛋白原样蛋白2(FGL2)、CEBPB反义RNA 1(CEBPB-AS1)和组蛋白H2B簇成员11(H2BC11)的直接转录调控因子。结论 食管癌中BTF3是一个广泛结合在基因组非启动子区域的转录因子，可能通过增强子或内含子调控元件发挥转录调控功能，直接结合并转录调控包括OASL、FGL2、CEBPB-AS1和H2BC11在内的一组下游靶基因，在细胞信号转导和微环境调控中可能扮演重要角色。

Objective To investigate the downstream transcriptional regulatory network and key target genes of basic transcription factor 3(BTF3) in esophageal squamous cell carcinoma(ESCC) cells. Methods In KYSE150 ESCC cells, chromatin immunoprecipitation sequencing(ChIP-seq) was employed to identify the genome-wide BTF3 binding sites. Differentially expressed genes obtained from RNA sequencing(RNA-seq) after stable BTF3 knockdown via lentiviral transfection in KYSE150 cells were combined to screen for direct regulatory targets of BTF3; key candidate target genes were validated via chromatin immunoprecipitation-quantitative polymerase chain reaction(ChIP-qPCR). Results ChIP-seq analysis revealed the specific binding profile of BTF3 across the genome. Compared to the control(Input) group, the immunoprecipitation(IP) group featured C/G-enriched sequences, particularly those containing CCCC or CCC cores, as the most significant motifs. BTF3 binding in the genome was highly enriched in intergenic regions and introns. Gene ontology cellular component(GO-CC) analysis indicated that the target genes were significantly enriched in extracellular regions, extracellular space, and collagen-containing extracellular matrix. GO biological process(GO-BP) analysis showed that target genes were primarily enriched in G protein-coupled receptor signaling pathways and signal transduction biological processes. GO molecular function(GO-MF) analysis revealed that target genes were mainly involved in G protein-coupled receptor activity, protein binding, and RNA binding.Integration analysis of 441 differentially expressed genes identified from RNA-seq data following BTF3-knockdown with genes located at enriched peaks in the ChIP-seq experiment identified a total of 54 overlapping genes, i.e., potential direct regulatory target genes of BTF3. These 54 overlapping genes were significantly enriched in extracellular regions and extracellular spaces(GO-CC), and were primarily involved in the biological process of positive regulation of gene expression(GO-BP) as well as protein binding(GO-MF). ChIP-qPCR experiments confirmed that BTF3 is a direct transcriptional regulator of the key target genes 2′-5′-oligoadenylate synthase-like protein(OASL), fibrinogen-like protein 2(FGL2), CEBPB antisense RNA 1(CEBPB-AS1), and histone H2B cluster member 11(H2BC11). Conclusion In ESCC, BTF3 is a transcription factor that binds extensively to non-promoter regions of the genome and may exert transcriptional regulatory functions through enhancer or intronic regulatory elements. It directly binds to and transcriptionally regulates a set of downstream target genes, including OASL, FGL2, CEBPB-AS1, and H2BC11,potentially playing an important role in cell signal transduction and microenvironment regulation.