Identification And Regulation Of MiR-371-3 Cluster Promoter In Hepatoma Cells

[Aims]MicroRNAs(miRNAs)are endogenous,noncoding,?22 nt small RNAs that transcripted by RNA polymerase ? and down-regulate genes by targeting 3'UTR of mRNA.MiRNAs are involved in many physiological and pathological progresses.They may have diverse biological functions in developmental and physiological processes,but only a handful of miRNAs have been carefully studied.We have already known that miR-371?-372?-373 express together as a cluster,Here,several methods was performed and identified the mechanism of transcription of miR-371-373 cluster in human hematopoiesis.This study enriches our understanding of the regulation mechanisms of miR-371-373 cluster on hematopoiesis,providing more molecular mechanisms and a new therapy choice for cancer.[Methods]Bioinformatics analysis was performed to pridict the promoter,TFBS,miRNA binding-site and CpG island of miR-371-373 cluster.According to resules of prediction result of transcriptional start site,a set of expression vectors with the different-length promoter was constructed basing on the vector of pGL3-basic and the promoter activity was confirmed by EGFP reporter experiment in HepG2,LM6,QGY-7703 and L-02.Then we used 5'RACE to confirm the transcriptional start site of miR-371-3 73 cluster.The last,we conformed to the results of bio informatics pridictation to regulatory factor and CpG island of miR-371-3 73 cluster and determined the upstream regulatory dement by the technique of real-time PCR and EGFP reporter system.[Results]We cloned the promoter of miR-371-3 73 cluster and found the fragment of p1000,p860 and p148 of promoter could activate EGFP gene transcription in the four cell lines;The p1000 showed highest EGFP activity.5' RACE assay indicated that miR-371-373 cluster transcriptional start sites is located in-591bp.Subsequently,We predicted the miR-371 5'-untranslated region(5'UTR)contains a CpG island and the potential binding site of Spl and GATA1.Overespression of Spl and GATA1 the expression of miR-371-373 were enhanced.The demethylation experiment also confirmed that 5-Aza-CdR can promoter the expression of miR-371-373 cluster.[Conclusions]We cloned the promoter of miR-371-373 cluster and identified the transcriptional start site.It was found that transcriptional factor,Spl and GATA1 and methylation influence the expression of the cluster.The elucidation of regulation mechanisms of miR-371-373 helps us to understand the tumorigenesis,and provides new evidences to cancer diagnosis and therapy.