Mechanism Of Ebv-miR-BHRF1-1 Regulate EBV Infection Via Inhibits P53 In NPC Cell Line

miRNAs(microRNAs)constitute a large family of approximately 22-nucleotide-long non-coding small RNAs that have been found in recent years,miRNAs have been known as key post-transcriptional regulators of gene expression through translational repression or mRNA degradation in metazoans and plants.However,the mechanistic details of the functions of miRNAs in protein synthesis repression are still poorly understood,miRNAs have been found to located from virus to multicellular organisms.About 100-200 miRNAs are expressed in lower metazoans and plants,but at least 1000 are predicted to operate in humans,miRNA are predicated to control the activity of approximately 30%of all protein-coding genes,which have been shown to participate in the regulation of almost all cellular process investigated so far and the changes in their expression are observed in human pathologies,including cancer.After infecting the host cell,virus regulate its and the host cell's gene expression to maintain its existence and get moderate replication,in witch,latent infectious state maintains the infection and replication results in diffusion.It is very important that the balance between the two states can avoid the host cell death caused by excessive reproduce.Most of viruses have been found to encode miRNAs that widely participate in the host-virus interaction.Epstein-Barr virus,which belongs to the human herpes virus,has high correlation with many human diseases including nasopharyngeal carcinoma.Epstein-Barr virus encodes at least 32 miRNAs within two clusters depending on their gene location in the virus' genome:the BHRF1-cluster and the BART-cluster.The BHRF1-cluster miRNAs located in the BHRF1,one of the lytic early genes,while the BART-cluster miRNAs is included in the transcripts named BART,which is a product of latent infection.It has been proved that the BART-cluster miRNAs can suppress virus reproduction to maintain the low reproduction of virus during latent infection.The high level of BHRF1-cluster miRNAs in cell lines with high level virus reproduction indicates that the BHRF1-cluster miRNAs may correlate to the regulation of lytic cycle.But bioinformatics predict that there are no potential target sites of the BHRF1-cluster miRNAs on the EBV encoded mRNAs.Interestingly,two target sites of miR-BHR1-1 lies in the 3'-UTR (3'-Untranslated regions)of p53 mRNA.P53 is an important tumor suppress gene which mutate in many neoplasm.But p53 mutation is infrequently observed in NPC biopsies. Meanwhile,accumulation of wild-type p53 is observed.During their reproduction many virus need to suppress the function of p53 via multiple mechanisms to provide an s-phase-like host cell environment and to prevent cell apoptosis to get enough time for their reproduction. Present investigation tells us that Zta,the product of EBV lytic immediate early gene BZLF1,can bind to p53 and suppress its transcript active function.It indicates that EBV needs to suppress the p53 function to create an advantageous environment for virus reproduction.So,the wild-type p53 with high level in NPC biopsies may have significance in the maintenance of virus latent infection in NPC.So we assume that the EBV encoded miR-BHRF1-1 may contribute to the induction of lytic cycle of EBV through inhibits the translation of P53 in NPC.First,our study showed that after being induced by TPA into lytic cycle the miR-BHRF1-1 increased while the p53 and its downstream gene production mdm2 decreased.Moreover,the percent of S-phase cell enhanced while apoptosis declined.These phenomena indicate that there may be relations between the up-regulation of miR-BHRF1-1 and the down-regulation of p53,and this relation may be involved in the lytic cycle of EBV in NPC.Second,after being transfected with anti-sense RNA targeted to miR-BHRF1-1,the inductive effect of TPA on EBV to lytic cycle in SUNE1 was retarded.At the same time,the suppression of the p53 and mdm2 after inducing EBV entry lytic cycle was decreased;what's more, the suppression of mdm2 even reversed. Then,we constructed the expression plasmid of miR-BHRF1-1 and transiently transfected SUNE1 for 48 hours.After normalizing the transfective efficiency by observing the GFP expression under fluoroscopy,and we proved the expression of mature miR-BHRF1-1 by RT-PCR.Successively,we detected the Zta and Ea-D and proved that the reproduction of EBV was enhanced.Then we detected the p53 and mdm2 simultaneously and proved that the p53 was suppressed.The above data indicate that EBV encoded miR-BHRF1-1 are involved in the switch between different infective states of EBV to maintain the existence of virus by inhibiting the p53.And this discovery proves a new clue for pathogenesis of EBV-related neoplasm and provides new experimental base for research of using EBV encoded miRNA for the treatment of EBV related neoplasm.