Changed MicroRNA Expression Profile Induced By HBV Infection And Exploration Of Their Targets In HBV Genes

MicroRNAs(miRNAs) are a set of RNA molecules of 18-23 nt.They were processed from short stem-loop precursors that can be encoded in genomes of plants,animals and viruses.According to the current understanding,microRNA is firstly transcribed as long primary microRNA,which processed into 60-70 nt miRNA precursor(pre-miRNA) by nuclear RNaseⅢDrosha.Then the pre-miRNA is transported from nucleus to cytoplasm by Exportin-5 and further cleaved into～22 nt duplexes.miRNAs were first observed in C. elegans,and now they are known widespread in nature.Thus far,over 3,500 microRNAs have been identified,with greater than 450 found in humans(MiRBase).The sequences of many miRNAs are conserved between distantly related organisms, suggesting that these molecules participate in essential processes,miRNA combines with several proteins,such as Argonaute,to form miRISC(miRNA induced silencing complex). Under the direction of miRNA,miRISC interacts with the target mRNA in an imperfect base pairing manner,which determines that a single miRNA can complement with numerous target mRNAs and regulate gene expression in the post-transcriptional level by pairing with 30-untranslated regions(30-UTRs) of the mRNAs.A computational analysis indicated that more than one-third of protein-encoding genes are regulated by miRNAs.They are involved in the regulation of a variety of biological processes including developmental timing, signal transduction,apoptosis,cell proliferation and tumorigenesis.In one word, miRNAs play an important role in both physiological and pathological process.Since miRNAs have so important functions,do they participate in the pathogenesis of virus infection diseases? At least,miRNAs have several characteristics that make them an attractive option for viruses to utilize in the regulation of gene expression.Due to their small size,miRNAs can be easily encoded in viral genomes,where space is of a premium.In addition,miRNAs are nonimmunogenic and take advantage of a ubiquitous,endogenous host regulatory mechanism.Further,a single miRNA has the potential to alter the expression pattern of a large number of genes.It is conceivable that viruses may seek to alter cellular miRNA expression in ways that benefit viral replication.Extant findings support such a notion since several viruses have been found to encode RNAi suppressors which could function to influence the cell's overall miRNA milieu.So,exploration of changes in microRNA profile of virus infected host cells will contribute to a better understanding of the pathogenesis of virus infection diseases and then provide valuable suggestions about how to diagnose,prevent and control these diseases.Hepatitis B virus(HBV) is a small circular DNA virus,belonging to the hepadnavirus family.The virus attacks liver cells and can cause lifelong infection,scarring of the liver,liver failure,and death.HBV is one of the major threaten to human health and it was estimated that about 350 million people worldwide are chronically infected with HBV.Despite considerable advances in the understanding of the natural history of HBV infection,most of them remain unclear.Seeking for miRNAs with differential expression between HBV infection cells and non-infected cells and further study the functions of these microRNAs may be provide some clues to the pathogenesis of HBV infection.In the present study,we used the technique of microRNA microarray to analyze the miRNAs with differential expression induced by HBV infection,and then tried to find targets in HBV gene of these miRNAs with the aid of a software ViTa.First of all,miRNAs expression profiles of HepG2 and HepG2.2.15 cell were analyzed using Exiqon microRNA microarray.HepG2.2.15 cell line was derived from HepG2 transfected with a plasmid four 5'-3' tandem copies of HBV genome.The cell line could stably express HBV particles and virus proteins,and was a novel in vitro model of HBV infection.Exiqon miRNA microarray is based on LNA technique,and could sensitive, specific and comprehensive profiling of miRNAs in mature forms available in miRBase 8.0 .After analysis with the microarray,in the HepG.2.2.15 cells,there were six microRNAs (hsa-miR-30a-5p,hsa-miR-24,hsa-let-7a,hsa-let-7c,hsa-let-7f and hsa-miR-23b) down-regulated and three microRNAs(hsa-miR-194,hsa-miR-200a and hsa-miR-345) up-regulated.Secondly,the result of microarray analysis was further confirmed by miRNA-specific quantitative real-time reverse transcriptase-polymerase chain reaction(Taqman miR-qRT-PCR),which were purchased from Applied Biosystems(Foster City,America) and was normalized using the 2^{-ΔΔCT} method relative to U6 small nuclear RNA(snRNA),which was nearly equally expressed in all kind of cells.Similar levels of downregulation of hsa-miR-30a-5p,hsa-miR-24,hsa-let-7a,hsa-let-7c,hsa-let-7f and hsa-miR-23b were observed in the assay.The upregulation of hsa-miR-194,hsa-miR-200a and hsa-miR-345 was observed too.These results indicated that the data of microRNA microarray is reliable.We also detected the miRNAs with differential expression,that is hsa-miR-30a-5p, hsa-miR-24,hsa-let-7a,hsa-let-7c,hsa-let-7f,hsa-miR-23b,hsa-miR-194,hsa-miR-200a and hsa-miR-345,in other cell lines stably transfected by feline immunodeficiency virus(FIV), Murine Stem Cell Virus(MSCV),Dengus virus and herpes simplex virus(HSV) to prove that the changed expression profile in HepG2.2.15 cells is HBV specific.The results of qRT-PCR also demonstrate that as the miRNAs above were concerned,there were no significant changes in these cells with non-HBV infection,indicating that the changed expression profile of these miRNAs is HBV-specific.HepG2.2.15 cell line is established by stably transfected with a vector contained the whole HBV genome.Though the cell could stably secrete HBV particles and HBV protein,it is not a cell naturally infected by HBV and could not mimic accurately a process in vivo. PLC/PRF/5 was a hepatoma cell line derived from a patient infected with HBV.So we further detected the miRNAs expression in the cell line using qRT-PCR.Results demonstrated that hsa-miR-194 and hsa-miR-200a were significantly up-regulated,and hsa-let-7a, hsa-let-7c,and hsa-let-7f were significantly down-regulated.The expression of hsa-miR-24, hsa-miR-30a-5p,hsa-miR-23b and hsa-miR-345 was not significantly changed.At last,12 liver needle specimens from chronic HBV infection patients and 3 normal liver specimens from healthy donor were detected using qRT-PCR.Results indicated that only hsa-let-7a and hsa-le-7f were down-regulated in all patients' specimens.The expression of hsa-miR-30a-5p,hsa-miR-345,hsa-miR-23b and hsa-miR-194 was not changed significantly. The expression of hsa-miR-24,hsa-let-7c and hsa-mir-200a were up-regulated in some specimens and down-regulated in other specimens.Considering with the results of miRNA microarray and cell lines,HBV infection induced the down-regulation of hsa-let-7a and hsa-let-7f,which may be play an important role in the pathogenesis of HBV infection. As previous studies have shown that miRNAs could function in viral pathogenesis through several ways,including the regulation of viral gene expression by host miRNAs,the regulation of viral gene expression by virus encoded miRNAs,and the regulation of host genes by virus encoded miRNAs.To explore whether hsa-let-7a and hsa-let-7f play a role in the regulation of HBV gene expression,with the aid of ViTa(available at http://vita.mbc.nctu.edu.tw/),a software using in prediction of host microRNAs targets on viruses,we got one potential target sites of hsa-let-7a in HBV genes and no potential site of hsa-let-7f.Then,we constructed the pMIR-report vector and 293T cell line stably expressed hsa-let-7a to verify whether the site is the location where hsa-let-7a binds with HBV gene.With luciferase assay,hsa-let-7a could efficiently interacts with the potential site and down-regulated the expression of luciferase.The site locates in 3105-3135 of hbv gene, the coding region of HBV polymerase and Presl,one of most important region during HBV life cycle.Then we detected DNA copies and the level of PreS1 in the supernatant of HepG2.2.15 stably transfected with PH1-puro-let-7a and results demonstrated that PreS1 protein and DNA copies in the supernanant were both down-regulated,further indicated that the site is target of let-7a in HBV gene.In summary,in the present study we firstly used the miRNA microarray to seek for miRNAs with differential expression in HepG2.2.15 and HepG2 cells.After verification using qRT-PCR,expression of these microRNA were detected in a series of cell lines and needle specimen of patients with chronic hepatitis B infection,and results indicated that hsa-let-7a and hsa-let-7f may be the miRNAs with significantly changed expression induced by HBV infection.Then,with the aid of ViTa and luciferase assay,the potential target site of hsa-let-7a in HBV gene was identified,that is 3105-3135 of HBV gene,the co-coding region of polymerase and PreS1,which play important role in the life cycle of HBV.Maybe,HBV down-regulates the expression of hsa-let-7a to escape from its inhibition to PreS1 and polymerase through certain mechanism,which contributes to a better understanding about the pathogenesis of HBV infection.In further studies,we will try to find out by which way HBV down-regulate the expression of hsa-let-7a in liver cells.