Cloning Of MiR-101 Promoter And Identification Of Target Genes

[Aims]Herpes simplex virus type-I(human herpes simplex virus type I,HSV-1)is enveloped DNA virus,which can cause diseases such as herpes labialis,and can lie dormant form a latent infection in the trigeminal ganglion.MicroRNAs(miRNAs)are a class of small non-coding RNAs that post-transcriptionally regulate gene expression.Recent evidences indicate that miRNAs not only participate in maintenance of normal cell functions,but also get involved in host-virus interaction and play a key role in the regulation of viral replication.The previous work showed that miR-101 was obviously upregulated in HeLa cells by HSV-1 infection and miR-101 could affect herpes simplex virus-1(HSV-1)replication by suppressing ATP5B.But the mechanism that miRNA impact on viral replication is not clear.So,related miRNA impact on host and virus upon HSV-1 is what this question to illustrate.[Methods]We detected the differencial expression of miR-101 in HeLa cells and its precursor in HeLa cells by real-time PCR.we pridicted the promoter of miR-101 by bioinformation.According to prediction result,the different-length promoter was cloned to pGL3-basic/luciferase reporter vectors and the promoter activity was confirmed in HeLa cell.Then,we used bioinformation and identified the candidate target genes for miR-101.The reliability of the direct target gene was confirmed by fluorescent reporter experiment,real-time PCR and western blot.[Resultsl The RNA levels of miR-101 and its precursor pre-miR-101-1,pre-miR-101-2 were increased in HeLa cells by HSV-1 infection,which miR-101 and pre-miR-101-2 increased particularly evident.Pre-miR-101-2 is located in intron of RCL1 and the expression level of which increased more significant.We cloned the promoter of pre-miR101-2 and verified promoter activity.We identified GRSF1 and COX2 as the candidate target genes for miR-101,which mRNA 3'-untranslated region(3'UTR)contains the potential binding site of miR-101.The fluorescent reporter experiment also confirmed that miR-101 can directly bind to the GRSF1 and COX2 mRNA 3'UTR,positively and negatively regulate the gene expression.The mRNA and protein level of GRSF1 and COX2 was elevated in HeLa cell by HSV-1.[Conclusions]The upregulated miR-101 in HeLa cells upon HSV-1 infection derived from its precursor pre-miR-101-2,meanwhile we cloned the promoter of pre-miR101-2 and verified promoter activity in HeLa cell.,MiR-101 up-regulate and down-regulate the target gene GRSF1 and COX2 in HeLa cells by targeting 3'UTR.The expression level of GRSF1 and COX2 was upregulated upon HSV-1.By clarifying the mechanism between HSV-1 and miR-101,we can further understand the interaction between viral and host,also provides a new basis for the diagnosis and treatment of viral infection.