Changes Of Microrna In Response To Enterovirus 71 Infection And The Related Signaling Pathways In Human Mesenchymal Stem Cells

BackgroundHand,foot and mouth disease(HMFD),a contagious viral disease commonly affecting infants and children under five years old,is characterized by fever,poor appetite,multiple ulcers in mouth,rashes and maculopapule in hand and foot.Therefore,the HFMD outbreaks in the Asia-Pacific region have occurred more and more frequently since 1997,even led to millions of cases and hundreds of children's deaths in Fuyang,China,in 2008,Singapore in 2008,and Guangdong,China,in 2009.It has been a important public health problem throughout the world.Unfortunately,the potential mechanism of HFMD have remained elusive and there are no effective treatments and vaccines by now.EV71 is an RNA virus belonging to the human enterovirus A(HEV-A)species of Picornaviridae,is one of positive single stranded RNA virus.It is one of major causative pathogens of HMFD,and the central nervous system has a high susceptibility to EV71.In 1969,EV71 was first isolated from stool specimens of children with diseases of the central nervous system in California,United States.In the following decades,it has caused a number of epidemic and outbreaks in many countries around the world.Because the poor prognosis and high death rate,it is considered to be the most harmful neurotropic enterovirus after the poliovirus has been controlled.MicroRNAs(miRNAs)are a specie of endogenous and highly conserved non-coding single-stranded RNAs,it exist in a wide range from unicellular to multicellular eukaryotes.Recent studies have demonstrated that miRNAs are involved in the complicated interaction between EV71 virus and host.On one hand,miRNAs encoded by human cells could directly affect the virus replication.On the other hand,virus could change cell normal functions by influencing cellular gene expressions through miRNAs.In this study,we attempted to ues human umbilical cord mesenchymal stem cells(huc-MSCs)as a cell model of EV71 infection in vitro,to invetigate the change of related miRNAs and the underlying mechanism in the infection of EV71 through microarray technology and bioinformatics techniques.ObjectiveTo investigate the possibility of MSC cells as the celluar model of pathogenic mechanism about EV71,to study the related signaling pathway and miRNA in the process of EV71 infection through microarray technology and bioinformatics techniques.Through this study,we want to further clarify the role of miRNA in the underlying pathological process of HFMD derived EV71 infection,and provide valuable reference for prevention and treatment of EV71 infection.Methods1.Culture and Identification of mesenchymal stem cells(1)Recovery and culture of huc-MSC in vitro.(2)The immunophenotype of MSC cells was detected by flow cytometry.2.To detect the virus titer of EV71 and th cytotoxicity text.(1)To calculate the virus titer of EV71 through the method of Reed-Muench.(2)The CCK-8 kit was used to test cell survival rate of MSC infected by EV71 in different MOI(Multiplicity of infection).3.The detection of microarray(1)Using TRNzol reagent to extract the total RNA and purify it through mirVanaTM miRNA Isolation Kit.(2)The integrity of total RNA was tested by formaldehyde denaturing gel electrophoresis.(3)The changes of related mRNA and miRNA in the infection of EV71 were detected by microarray.4.The bioinformatic analysis and qRT-PCR(1)To analyse the fluorescent hybridization image of miRNA microarrays through the Agilent GeneSpring sofeware.(2)To validate the expression changes of miR-6068?miR-575?miR-134-5p?miR-582-5p through qRT-PCR after EV71 infection.(3)To predict the putative target genes of related miRNAs,TargetScan in conjunction with miRanda,PicTa,and miRWalk online bioinformatic softwares were performed.(4)The DAVID gene annotation tools were utilized for GO(Gene Ontology)and KEGG pathway analysis of differential mRNA and related miRNA.Result1.The cellular surface antigenic features were analyzed by flow cytometry,the positive rate of CD34 and CD45 in MSC cells were 0.42%and 0.06%,appeared negative.The CD44 and CD90 were 98.96%and 100%,showed positive.2.To calculate the TCID50(Tissue culture infective dose)of EV71 through the method of Reed-Muench,the TCID50 of the EV71 strain was 10-8.7/0.1ml through calculations,and the virus titer was 3.5x108PFU/ml.3.The CCK-8 test demonstrated that EV71 virus has obvious cytotoxic effect to the MSC cells,the cell survival rate decreased with the increase of MOL4.Totally 76 differential expression of miRNAs and 24 mRNA were found by microarray technology.5.The expression of miR-6068 and miR-575 increased 48 times and 11 times,miR-134-5p and miR-582-5p decreased 16 times and 15 times after EV71 infection through qRT-PCR technique.6.Through GO analysis,we found that EV71 virus may affect the biological processes such as metabolism,signal transduction,regulation of biological process,immune system and so on,and interfere with the function of cells,cell components,organelles and cell membranes,through binding and catalytic molecular function.As to the KEGG pathway analysis,they mainly involves the integrin(31,ECM-receptor,Focal adhesion,PI3K-Akt and other signaling pathway.7.The interaction between integrin ?1 and EV71 could regulate by miR-6068,miR-575,miR-134-5p and miR-582-5p.ConclusionsThe MSC cells were susceptible to EV71 virus;EV71 virus could interfere with the normal function of host cells via regulating integrin ?1,ECM-receptor,Focal adhesion,PI3K-Akt and other signaling pathways;the normal expression of miRNAs were affected after EV71 infection,and totally 76 differentiall expression of miRNAs were found;the interaction between integrin ?1 and EV71 could regulate by miR-6068.miR-575,miR-134-5p and miR-582-5p.