绵羊瘤胃上皮细胞VTN基因启动子转录调控特性研究

doi:10.16431/j.cnki.1671-7236.2025.08.003

摘要/Abstract

摘要： 【目的】探究绵羊瘤胃上皮细胞玻连蛋白(vitronectin，VTN)基因核心启动子的转录活性及转录因子CCCTC-结合因子(CCCTC-binding factor，CTCF)基因过表达对VTN等基因表达的影响。【方法】通过在线生物信息学分析软件预测绵羊VTN基因启动子核心区域并设计6对启动子缺失片段引物，采集湖羊血液提取DNA，克隆VTN基因CDS区上游6个启动子缺失片段区域，构建荧光素酶重组质粒L1-basic、L2-basic、L3-basic、L4-basic、L5-basic及L6-basic，转染绵羊瘤胃上皮细胞，并以荧光素酶报告基因试验验证启动子活性区域位置。通过在线生物信息学分析软件预测转录因子CTCF与VTN基因启动子的结合位点，以绵羊瘤胃上皮细胞为材料，提取RNA后进行反转录，克隆绵羊CTCF基因CDS区，构建CTCF基因的过表达载体，与VTN基因启动子重组质粒L1-basic、L2-basic、L3-basic及L4-basic共转染绵羊瘤胃上皮细胞，以单独转染的重组质粒为对照组，通过荧光素酶报告基因试验验证以确定转录因子CTCF可能结合VTN基因启动子的核心区域；利用实时荧光定量PCR分析CTCF基因过表达转染组CTCF、VTN、C-MYC、GLUT1与SP1基因的表达情况。【结果】试验成功构建绵羊VTN基因启动子区6个重组荧光素酶报告载体。生物信息学网站预测与荧光素酶报告基因试验结果显示，VTN基因核心启动子可能位于―933/―440 bp处，且转录因子CTCF可能与VTN基因启动子核心区域存在互作。CTCF基因过表达后，与对照组相比，VTN、C-MYC与GLUT1基因表达量显著降低(P＜0.05)，而SP1基因表达量则无显著变化(P＞0.05)。【结论】绵羊瘤胃上皮细胞中转录因子CTCF可能主要结合VTN基因启动子的区域(―439/+26 bp)与核心区域(―933/―440 bp)影响VTN基因表达，且CTCF基因过表达影响VTN、C-MYC、GLUT1基因转录，提示CTCF基因潜在影响绵羊瘤胃上皮细胞生长增殖与物质代谢。

关键词: 绵羊; VTN基因; 核心启动子; 瘤胃上皮细胞; CTCF基因

Abstract: 【Objective】 This study was aimed to investigate the transcriptional activity of the core promoter of vitronectin (VTN) gene in sheep rumem epithelial cells and the effects of CCCTC-binding factor (CTCF) gene overexpression on the expression of VTN and related genes. 【Method】 The core region of VTN gene promoter in sheep was predicted using online bioinformatics analysis software,and six pairs of promoter deletion fragment primers were designed.DNA was extracted from blood samples in Hu sheep to clone six promoter deletion fragments upstream of VTN gene CDS region.Luciferase recombinant plasmids L1-basic,L2-basic,L3-basic,L4-basic,L5-basic,and L6-basic were constructed and transfected into sheep rumen epithelial cells.Luciferase reporter gene assays were conducted to verify the location of promoter activity regions.The binding sites between transcription factor CTCF and VTN gene promoter were predicted using online bioinformatics analysis software.RNA was extracted from sheep rumen epithelial cells for reverse transcription,and the CDS region of CTCF gene in sheep was cloned to construct CTCF gene overexpression vector.The vector was co-transfected with VTN gene promoter recombinant plasmids L1-basic,L2-basic,L3-basic,and L4-basic into sheep rumen epithelial cells,with single plasmid transfection as control groups.Luciferase reporter gene assays were performed to determine the core region where transcription factor CTCF binds to VTN gene promoter.Real-time quantitative PCR was used to analyze the relative expression of CTCF,VTN,C-MYC,GLUT1,and SP1 genes in CTCF gene overexpression transfection groups. 【Result】 Six recombinant luciferase reporter vectors in the promoter region of VTN gene in sheep were successfully constructed.The results of bioinformatics prediction and luciferase reporter gene assays indicated that the core promoter of VTN gene might be located at ―933/―440 bp,and transcription factor CTCF potentially interacted with the core region of VTN gene promoter.After CTCF gene overexpression,compared with control group,the relative expression of VTN,C-MYC,and GLUT1 genes were significantly decreased (P＜0.05),while the expression of SP1 gene showed no significant difference (P＞0.05). 【Conclusion】 In sheep rumen epithelial cells,transcription factor CTCF might predominantly bind to the promoter region (―439/+26 bp) and core region (―933/―440 bp) to influence VTN gene expression.CTCF gene overexpression affected the transcription of VTN,C-MYC,and GLUT1 genes,suggesting that CTCF gene potentially influenced the growth,proliferation,and metabolism of sheep rumen epithelial cells.

Key words: sheep; VTN gene; core promoter; rumem epithelial cells; CTCF gene

中图分类号:

S813.3

钟秉潜, 苏比奴尔·艾力, 朱爱文, 孙岩, 王玉涛, 闫伟. 绵羊瘤胃上皮细胞VTN基因启动子转录调控特性研究[J]. 中国畜牧兽医, 2025, 52(8): 3527-3539.

ZHONG Bingqian, SUBINUR Eli, ZHU Aiwen, SUN Yan, WANG Yutao, YAN Wei. Transcriptional Regulation of VTN Gene Promoter in Sheep Rumen Epithelial Cells[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3527-3539.

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