Peripheral Blood MicroRNAs Expression Is Associated With Infant Respiratory Syncytial Virus Infection

Objective Respiratory syncytial virus(RSV)is an enveloped negative strand RNA virus,which belongs to the Paramyxoviridae family.Infants with RSV infection often develop severe bronchiolitis or pneumonia.The lack of understanding of the host-virus interface has hindered prevention and treatment methods,as well as a successful RSV vaccine development.Even though recent antiviral efforts have begun to inhibit virus replication by targeting host pathways and using interfering RNAs,regulation of the host immune responses resulting in bronchiolitis remains a challenge.Mature microRNAs(miRNA)are non-coding transcripts 18 to 25 nucleotides in length,which modulate protein expression at the post-transcriptional level.The miRNA gene family makes up a global regulatory network controlling homeostasis,cell proliferation,differentiation,cell migration,disease progression,and inflammatory responses.Many viruses can make use of the host processing machinery for their biogenesis by encoding miRNAs.Viral infections can also influence host miRNA production.In addition,host miRNAs can regulate the viral life cycle,and target the viral messenger RNA.Recent studies have indicated that miRNAs regulate the inflammatory response associated with RNA virus infection.Let-7c,upregulated in influenza virus-infected epithelium,may inhibit influenza virus replication by directly targeting the viral gene product.RSV non-structural protein-1 modifies mir-24 expression via transforming growth factor beta(TGF-β).The lack of downregulation of miR-125a and miR-429 in severe RSV infection may be associated with RSV manifestations.MicroRNA-221 modulates RSV replication in human bronchial epithelium by targeting nerve growth factor expression.Understanding the changes in miRNA expression profiles and identifying the targets genes and their contribution to viral infection may help elucidate novel mechanisms of host-virus interaction.In this study,we analyzed the miRNAs fingerprint in whole blood of infants with RSV.Methods Five RSV infection children were admitted to Zhongnan Hospital of Wuhan University,Wuhan,China,during the RSV season from December 2014 to March 2015 and December 2016 to February 2017.Healthy controls were five healthy children who received regular development examination.Nasopharyngeal aspirates for virus detection were taken from eligible patients on admission,and the specimens were analyzed using a commercial indirect immunofluorescence(ⅡF)kit(EUROIMMUN,Lubeck,Germany)following the manufacturer’s instructions.Patients with bronchopulmonary dysplasia,chronic lung disease including treated asthma,neurological disease,congenital disorder,hypotonia,failure to thrive,or other specific conditions likely to contribute to a more severe course of disease,were excluded.Whole blood samples were collected within 24 h after a diagnosis of RSV infection.All blood samples were stored at-20℃ within 4 h following collection prior to analysis for miRNAs.RNA extraction and quantitation.Total RNA of peripheral blood(no centrifugation and sedimentation)was extracted by the TRIpure Reagent(BioTeke Corporation,Beijing,China)according to the manufacturer’s instructions.Total RNA purification was performed by mirVanaTM miRNA Isolation Kit(Ambion,Austin,TX).RNA quantity and quality were assessed using the NanoDrop(?)ND-1000(Thermo Fisher Scientific,Waltham,MA).MiRNA microarray hybridization.Three RSV infection samples and one healthy control sample were analyzed using miRNA microarrays.The microarray assays were performed by the Affymetrix GeneChip(?)miRNA 4.0 expression profiling kit at Affymetrix,Inc(Santa Clara,CA).The microarray contained probes for 5214 human,mouse and rat miRNAs probes and generated fluorescent miRNAs with a sample input of 130 ng of total RNA.Labeling was performed using FlashTagTM Biotin RNA Labeling Kit(Genisphere Inc.Hatfield,PA)containing Poly(A)Tailing and FlashTag.Ligation.The array hybridization cocktail was prepared.The mixture was incubated at 99 ℃ for 5 minutes,then 45 ℃ for 5 minutes.Subsequently,the mixtures were transferred to both septa of a microarray slide,and the arrays were placed into hybridization oven and incubated at 48 ℃ and 60 rpm for 16 hours.The slides were then washed with washing buffer(Affymetrix Inc,Santa Clara,CA),and scanned immediately by the GeneChip(?)Scanner 3000(Affymetrix Inc,Santa Clara,CA).Data collection and quality assessment were performed using Affymetrix(?)GeneChip(?)Command Console(?)Software(Affymetrix Inc,Santa Clara,CA).The signal intensity of individual miRNA probes was expressed as a ratio of the internal control.Microarray results are deposited in the NCBI’s Gene Expression Omnibus.MiRNAs for quantitative real-time PCR validation.MiRNAs were selected for further validation and analysis following the criteria of significant differential expression and having predictive target genes in public databases.Five RSV infection and five healthy samples were prepared for selected miRNAs validation by quantitative real-time PCR(qRT-PCR).Total RNA was purified using mirVanaTM miRNA Isolation Kit(AM 1561,Ambion,Austin,TX)and 100 ng per reaction was used for qRT-PCR analysis using the Power SYBR(?)R Green PCR Master Mix and the 7900 HT Fast RealTime PCR system(Applied Biosystems,CA).All primers(miRNA RT primer&PCR primers)were synthesized by Invitrogen(Thermo Fisher Scientific Corp.,CA).Transcript expression was normalized using mammalian’ U6 as the endogenous housekeeping gene.Samples were analyzed in triplicates,using ABI Prism SDS2.4 software(Thermo Fisher Scientific Corporation,CA).Samples were normalized and calibrated using the 2-△△Ct method.Bioinformatics analysis.An extensive analysis of miRNAs target genes was performed by using ten different algorithms DIANAmT,miRanda,miRDB,miRWalk,RNAhybrid,PICTAR4,PICTAR5,PITA,RNA22 and Targetscan.The names of miRNAs were put in these databases,if there were predictive target genes of the miRNA in the database,we marked one score,if they were not predictive,we marked zero.The total score was calculated in the end.The higher the score,the more dependable the target gene was.Higher score target genes were selected for performing enriched pathways and functional analysis by the Kyoto encyclopedia of genes and genomes(KEGG)database and Gene ontology(GO).Statistics.The differences between the RSV and the control groups were analyzed by Mann-Whitney Test.Microarray results were analyzed by the R computing environment.The Affymetrix(?)GeneChip(?)Command Console(?)Software with quintiles normalization was used for pre-processing.Differential expression analysis of microarray data was assessed using fold-change method.Differential expression miRNA was set by fold-change>2 for upper regulation and by fold-change≤0.5 for downregulation.Cluster data of microarray results were analyzed by hierarchical clustering with pairwise average-linkage approach.The 2-△△Ct method was used to calculate the relative expression levels of miRNAs in real-time quantitative PCR experiments.The difference in 2-△△Ct values between the RSV group and the control group was determined using the paired T-test.Adjusted p-values<0.05 were considered statistically significant.Results Clinical characteristics.Five males and five females were included in the study.There was no statistical difference in the Mann-Whitney test for gender,age,weight,and birth weight among children in the RSV and control groups.The diagnosis of RSV infection was based on clinical manifestation,physical signs of lung,laboratory tests,and examination.MiRNA expression profiles.Differential expression analysis of miRNA microarrays between RSV patients and healthy controls indicated that 37 miRNAs were upregulated-fold-change≥2,while 24 miRNAs were downregulated(fold-change≤0.5),and 72 miRNAs(fold-change=0.5-2.0)were not changed.From the differentially expressed miRNAs,the top five upregulated miRNAs(miR-106b-5p,miR-181a-5p,miR-20b-5p,miR-342-3p and miR-652-3p)and six downregulated miRNAs(miR-122-5p,miR-320e,miR-320d,miR-877-5p,miR-92b-5p and let-7c-5p)were selected for further validation.QRT-PCR validation of selected miRNAs.Expression of three of the five upregulated miRNAs was significantly different between the RSV group and the control group.However,expression of miR-181a-5p in RSV2,RSV4,RSV5,and miR-652-3p in RSV4 and RSV5 was not consistent with the microarray data.Expression of four of the six downregulated miRNAs(miR-122-5p,miR-320d,miR-877-5p and miR-92b-5p)showed a similar trend as that of the microarrays.However,there was no significant change of miR-320e in all five RSV infected cases,and let-7c-5p was downregulated in RSV1,RSV2,and RSV3,while there was no difference in RSV4 and RSV5.These results demonstrated that most qRT-PCR results(64%)of miRNA expression were consistent with microarray analyses.Prediction of target miRNAs function.By using ten different algorithms DIANAmT,miRanda,miRDB,miRWalk,RNAhybrid,PICTAR4;PICTAR5,PITA,RNA22,and Targetscan,we obtained a list of genes predicted to be targeted by miR-106b-5p,miR-20b-5p,miR-342-3p,miR-122-5p,miR-320d,miR-877-5p,and miR-92b-5p.Gene ontology(GO)enriched function analysis and KEGG pathway analysis indicated that miR-106b-5p,miR-20b-5p,miR-342-3p,miR-877-5p,miR-122-5p,miR-320d and miR-92b-5p were involved in many common pathways.A large number of pathways were associated with inflammatory and immune processes,such as insulin signaling,TGF-beta signaling,Wnt signaling,T and B cell receptor signaling,and Fc epsilon RI signaling pathway.Natural killer cell mediated cytotoxicity was the common pathway of miR-106b-5p,miR-20b-5p,and miR-342-3p.There were twelve pathways and sixteen predicted genes in the common pathways’ crosstalk networks.MAPK1 was involved in eight pathways,and NFAT5 participated in regulating three pathways.Conclusion In this study,miRNA expression profiles were analyzed in peripheral blood of infants with acute RSV infection,and differential expression of miR-106b-5p,miR-20b-5p,miR-342-3p,miR-877-5p,miR-122-5p,miR-320d,and miR-92b-5p was confirmed by qRT-PCR.The function analysis revealed that the miRNAs participate in signaling pathways involved in inflammation and immune responses.