Identification Of Antiviral MiRNAs In Porcine Airway Epithelial Cells

Swine respiratory disease(SRD)with complicated pathogenesis and seriously threatens the healthy development of pig industry.It could be made by one or more of the virus,bacteria,parasites,and also could improved or worsen as the changes of the environment,nutrition,stress and the health of the pig itself.Virus is one of the main pathogenic bacteria of SRD,which could be identified by innate immune system.The innate immune system detects viral infection through the interaction of pattern recognition receptors(PRRs)and pathogen-associated molecular patterns(PAMPs)and signals through the corresponding signal transduction pathwaysto trigger the synthesis of cytokines such as interferon and interleukins,which then activates the expression of various downstream antiviral molecules.finally suppress the virus proliferation in the body.Epithelial cells,the first barrier against SRD,function not only as a physical barrier to pathogen,but also to initiate innate immune response against the pathogenic invasion.The best way to prevent infectious SRD was to understand the molecular mechanisms underlying antiviral innate immune responses in airway epithelial cells,to identify antiviral related miRNAs and characterizing their function.Mi RNAs are a class of non-coding small RNAs of about 21 to 25 nt in length and participate in various life activities by regulating gene expression at post-transcriptional levels.Studies have shown that miRNAs arre closely related to disease resistance / susceptibility.PAMP of most virus is double-stranded RNA(ds RNA)and Poly(I:C),a synthetic dsRNA,can effectively activate the host antiviral innate immune response.In this study,we thereafter identify antiviral related miRNAs in porcine airway epithelial cells stimulated by Poly(I: C)using high-throughput sequencing technology.At the same time the sequence variation of miRNAs(isomiRs)and novel miRNAs were characterized.On this basis,we selected the miRNAs which are of interest to identify the target genes and their induced expression and tissue expression profile.Additionally,the role of miR-20 a on on the cell growth,migration,autophagy and the expression of inflammatory cytokines was analyzed.The main results are as follows:(1)The porcine airway epithelial cells were successfully obtained by the primary culture method and differential adherence method,and the cells were identified as epithelial type cells with high purity by keratin CK18 monoclonal antibody.(2)13,349,772 and 11,987,962 pure sequences were obtained from the control and the treatment groups,respectively,by high-throughput sequencing.The sequence length was between18 and 26 nt,and the peak was located at 22 nt.There are 23 differentially expressed miRNAs(P<0.01,?log2ratio?>1)between control and treatment group,among which 19 weredown-regulated under Poly(I: C)stimulation and 4 were up-regulated.Using MIRANDA,TARGETSCAN and PICTAR,we predicted a total of 386 potential target genes for differentially expressed miRNAs.These target genes were mainly enriched in cell growth regulation,positive and negative regulation of RNA polymerase ? promoter,embryo development,Cell signaling and other biological processes(p<0.05).(3)Two miRNAs,miR-101 and miR-20 a,were randomly selected to validate the potential target genes predicted by bioinformatics by double luciferase reporter gene analysis and site-directed mutagenesis method.The results showed that miR-101 could effectively restrain the expression of all the potential target gene selected(SOX9,TLK2,RANBP9,BCL9,EZH2,SOCS5,CDH11 and DR1);miR-20 a could inhibit the expression of 5 out 8 potential target genes selected(HLF,ITGB8,ARID4 B,SLC40A1 and MAP3K2).(4)There are a large number of isomiR in miRNAs.78 and 87 miRNAs in the treatment and control groups,respectively,possess isomiRs,accounting for 57.35% and 62.14%,respectively,of the total known miRNAs detected.The isomiR contained all the type of nucleotide substitution,among which A>G substitution was the most isomiR form,in both the control and treatment groups,accounting for 27.89% and 29.38% respectively,followed by G>A(12.2% and 12.59%)and G>T(11.39% and 11.9%)substitution.Poly(I:C)treatment had an effect on isomer: 14 and 5specific isomiRs were present in the control and treatment group,respectively.There are also different in editing at position 5 in the 5' end of mature miRNA between control and treatment group.Using dual-luciferase reporter gene analysis and site-directed mutagenesis method,we have showed that editing at position 5 had important effect on the recognition and binding of miRNA to target gene.When the base at position 5(G / T)was mutated,miR-101 can not regulate the expression of target gene.(5)83 and 82 novelw miRNAs were detected in the treatment and control groups,respectively,47 of which were present in both groups.Homologous analysis showed that a total of 19 novel miRNAs could found counterparts in other species.Six novel miRNAs(NS-1,NS-2,NS-3,NS-6,NS-8 and NS-9)were random selected for verification using cloning and sequencingmethod,and the results demonstrated that these miRNAs were indeed present in pigs.The expression of NS-2,NS-8 and NS-9 was significantly increased by Poly(I: C)stimulation with different concentrations(p<0.05).Tissue expression analysis showed that NS-2,NS-8 and NS-9 were ubiquitously expressed all 13 tissues(heart,liver,spleen,lung,kidney,stomach,tongue,bladder,small intestine,large intestine,fat,Longest muscle and skin)detected,but the expression profiles are different.(6)Over-expression of miR-20 a could shorten the time of pk15 cells staying in the growth retardation zone and advance them into the logarithmic growth phase as revealed by cell growth curve test,which significantly promoted cell proliferation(p<0.05)and inhibition of miR-20 a expression significantly decreased cell proliferation(p<0.05)Cell migration test showed that the relative mobility of pk15 cells at each observation time point was significantly higher than that of the control group(p<0.05)after over-expression of miR-20 a and inhibition of miR-20 a expressionsignificantly decreased cell migration(p<0.05).(7)The effect of miR-20 a on the expression of inflammatory genes including IFN-?,IL-29,IL-1,IL-6 and IL-10 were detected by real-time PCR.The results showed that,in pk15 cells,over-expression of miR-20 a can significantly inhibit the expression of these genes(p<0.05);while inhibition of miR-20 a,can significantly promote the expression of these genes(p<0.05).(8)The effect of miR-20 a on autophagy was studied by inducing autophagy in pk15 cells by serum starvation method.The expression of autophagy marker protein LC3? was significantly decreased(p<0.05)after over-expression of miR-20 a,while significantly increased when inhibiting the expression of miR-20a(p<0.05).