小鼠miR-155基因启动子分析及转录活性鉴定

罗朦莎; 李雪琴; 张莺莺; 吕坤

doi:10.13898/j.cnki.issn.1000-2200.2023.05.002

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

摘要

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2 000 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、1~1 000 nt及1~2 000 nt片段均存在转录激活作用，提示小鼠miR-155基因TSS上游1~2 000 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1~2 000 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1~2 000 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。

Abstract:
ObjectiveTo 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.
MethodsThe 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 2 000 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.
ResultsThe 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, 1-1 000 nt and 1-2 000 nt fragments of the TSS of mouse miRNA-155 gene all had transcriptional activation, suggesting that upstream 1-2 000 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 1-2 000 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.
ConclusionsThe upstream 1-2 000 nt of the TSS of mouse miRNA-155 gene is the promoter region, of which 1-500 nt is the core region of transcription activity. Transcription factors Crp, Pax-6, Elf-1 and Gata-1 all bind to the promoter region of miRNA-155 gene and negatively regulate the transcription of miRNA-155.

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