小鼠miR-155基因启动子分析及转录活性鉴定  

Promoter analysis and transcriptional activity identification of mouse miR-155 gene

作　　者：罗朦莎 李雪琴 张莺莺 吕坤 LUO Meng-sha;LI Xue-qin;ZHANG Ying-ying;L Kun(School of Graduates,Wannan Medical College,Wuhu Anhui 241001;Key Laboratory of Non-coding RNA Transformation Research of Anhui Higher Education Institutes,Wuhu Anhui 241001,China)

机构地区：[1]皖南医学院研究生学院,安徽芜湖241001 [2]重大疾病非编码RNA转化研究安徽普通高校重点实验室,安徽芜湖241001

出　　处：《蚌埠医学院学报》2023年第5期565-569,共5页Journal of Bengbu Medical College

基　　金：国家自然科学基金项目(81772180,81701557,81870017);安徽省高校自然科学研究重点项目(KJ2018A0265)。

摘　　要：目的:鉴定小鼠miR-155基因启动子转录活性区域,预测转录因子结合位点,探讨miR-155在巨噬细胞极化中表达失调的转录调控机制。方法:分别构建小鼠miR-155基因表达质粒和miR-155基因启动子连续截短片段的荧光素酶报告载体。将miR-155表达载体与miR-155启动子荧光素酶报告载体瞬时共转染人胚肾上皮细胞293T,48 h后检测双荧光素酶活性。选取miR-155基因转录起始位点(TSS)上游2000 nt作为启动子区,生物信息学预测该区域可能结合的转录因子,并在体外极化的M1型和M2型巨噬细胞中检测结合的相关转录因子的表达。结果:成功构建小鼠miR-155基因表达质粒和miR-155基因启动子连续截短片段的荧光素酶报告载体。双荧光素酶试验结果显示,小鼠miR-155基因TSS上游1~500 nt、1~1000 nt及1~2000 nt片段均存在转录激活作用,提示小鼠miR-155基因TSS上游1~2000 nt均为miR-155基因的启动子区域,其中1~500 nt为启动子核心区域。转录因子的生物信息学预测结果显示,miR-155基因TSS上游1~2000 nt的序列存在Crp、Pax-6、Elf-1、Gata-1、Hnf-4、BR-C Z4等转录因子结合位点。RT-qPCR结果显示,转录因子中结合分数最高的Crp、Pax-6、Elf-1和Gata-1均在M1型巨噬细胞中低表达,与miR-155的表达方式相反。结论:小鼠miR-155基因TSS上游1~2000 nt均为启动子区域,其中1~500 nt为转录核心区域。转录因子Crp、Pax-6、Elf-1和Gata-1均与miR-155基因启动子区域结合,并负调控miR-155的转录。Objective:To identify the transcriptional active region of mouse miR-155 gene promoter,predict the transcription factor binding site,and explore the transcriptional regulation mechanism of miR-155 expression imbalance in macrophage polarization.Methods:The mouse miR-155 gene expression plasmid and luciferase reporter vector of miR-155 gene promoter continuous truncation fragment were constructed,respectively.The miR-155 expression vector and miR-155 promoter luciferase reporter vector were transiently co-transfected into human embryonic renal epithelial cells 293T,and the double luciferase activity was detected 48 hours later.The upstream 2000 nt of the transcription start site(TSS)of miRNA-155 gene was selected as the promoter region,bioinformatics was used to predict the possible binding transcription factors in this region,and the expression of the related binding-transcription factors was detected in M1 and M2 polarized macrophages.Results:The mouse miR-155 gene expression plasmid and the luciferase reporter vector of miR-155 gene promoter continuous truncation fragment were successfully constructed.The results of dual luciferase analysis showed that upstream 1-500 nt,1-1000 nt and 1-2000 nt fragments of the TSS of mouse miRNA-155 gene all had transcriptional activation,suggesting that upstream 1-2000 nt of the TSS of mouse miRNA-155 gene was the promoter region of the gene,and 1-500 nt was the core region of the promoter.The bioinformatics prediction results of transcription factors showed that there were multiple transcription factor binding sites including Crp,Pax-6,Elf-1,Gata-1,Hnf-4,BR-C Z4 in the upstream 1-2000 nt of the TSS of miR-155 gene.RT-qPCR results showed that the four transcription factors Crp,Pax-6,Elf-1 and Gata-1,which had the highest binding match score among the transcription factors,were all down-regulated in M1 macrophages,which was contrary to the expression of miRNA-155.Conclusions:The upstream 1-2000 nt of the TSS of mouse miRNA-155 gene is the promoter region,of which 1-500 nt is th

关 键 词：MIR-155 启动子 转录因子 巨噬细胞 

分 类 号：Q74[生物学—分子生物学] Q291[医药卫生—免疫学] R392.12[医药卫生—基础医学]