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

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.