| Transcriptome analyses of mammalian genomes have characterized RNA transcripts with low-protein coding potential,known as long non-coding RNAs(lncRNAs).lncRNAs are longer than 200 nt,with relatively conservative secondary structure,subcellular location and splicing form.The type,quantity and mechanism of lncRNAs have not yet been defined.It has become increasingly apparent that lncRNAs are involved in virus-host interaction.Several investigations have identified thousands of lncRNAs differentially expressed in severe acute respiratory syndrome coronavirus(SARS-CoV)infected mice,IAV-infected human lung cells and enterovirus 71(EV71)-infected rhabdomyosarcoma(RD)cells.Such research indicates that some viruses can modulate host and/or viral gene expression via lncRNAs that maintain the virus latency or replication.Some lnc RNAs assist virus replication,escape from cytosolic surveillance,and decrease antiviral immunity.Such large amount of lncRNAs regulated by the high-risk viruses indicates their importance in host response to viral infection.However,only a small portion of these lncRNAs has been assayed in detail for their function in viral infection.Respiratory syncytial virus(RSV)is an enveloped RNA virus,whose genome is a non-segmented,single-stranded,negative-sense RNA.RSV is one of the most important viral respiratory pathogen in infants and young children.RSV causes upper respiratory tract disease,but in 25~40% of such infections,the lower respiratory tract is involved,resulting in bronchiolitis or pneumonia.Current lines of evidences have supported the functional involvement of lncRNAs in host antiviral response.But the action of lnc RNA in RSV infected respiratory epithelial cells is still unknown.lncRNAdb is a famous database constructed by Johnmattick.We found 4 lncRNAs related with RNA virus or respiratory infection,whose names were NEAT1,PRINS,NEST and NRAV.We found significant differences in NRAV,but not NEAT1,PRINS,or NEST expression in RSV infected cells.Jing Ouyang fond that lnc-NRAV was downregulated in respiratory virus,such as influenza A virus,sendai virus,muscovy duck reovirus,herpes simplex virus,which could promote virus replication by reducing the transcription of ISG genes.Although precise mechanism was underlied,lnc-NRAV is mainly in cytoplasm,which is the main area of mature miRNA.Long non-coding RNAs regulate variety biological responses at DNA,RNA,or protein levels.The competing endogenous RNA(ceRNA)hypothesis showed a post-transcriptional regulatory network mediated by miRNAs.By sharing one or more miRNA response elements(MREs),protein-coding and noncoding RNAs compete for binding to miRNAs and then regulate each other’s expression.Whether lnc-NRAV can act as molecular sponge for some miRNAs in ceRNA network? In this study,we tried to discuss the relationship between lnc-NRAV and RSV in A549 and BEAS-2B cells,which would enrich the understanding of the relationship between lncRNAs and respiratory virus infection.Part one The expression of lnc-NRAV is regulated by RSV infection and the viral multiplication is influenced by lnc-NRAV in respiratory epithelial cells.Objective: To investigate the expression of lnc-NEAT1,PRINS,NEST,and NRAV during RSV infection in A549 cells by RT-qPCR.To identify coding potential and conservation of human NRAV by using bioinformatics analysis.To investigate the expression of lnc-NRAV during RSV infection in A549 or BEAS-2B cells through RT-q PCR.To explore influences on viral multiplication by altering NRAV expression.Methods:1 A549 cells were infected with RSV at moi of 3 for 12,24,36 hours.RT-q PCR was performed to determine NEAT1,PRINS,NEST,and NRAV expression.2 We found significant differences in NRAV,but not NEAT1,PRINS,or NEST expression in RSV infected cells.The protein-coding potential,secondary structure and conservation of NRAV was analyzed by LNCipedia and UCSC data base.3 A549 or BEAS-2B cells were infected with RSV at an moi of 3 for 28 hours.Total RNA was collected every 2 hours since infection.RT-q PCR was performed to determine the NRAV expression.4 The expressions of RSV NS1,N,F,G,M and M2 genes in RSV infected NRAV-overexpressing A549 or BEAS-2B cells were analyzed for 28 hours by qRT-PCR,in which total RNA was collected every 4 hours since infection.The expressions of RSV F protein in RSV infected NRAV-overexpressing were analyzed at 24 h,48h,72 h by Western blot.RSV replication was examined by plaque assay in RSV infected NRAVoverexpressing cells.Virus titers in supernatants were measured at 24-48 hours post infection.The supernatants were collected every 4 hours since infection.5 The expressions of RSV NS1,N,F,G,M and M2 genes in RSV infected NRAV-knockdown A549 or BEAS-2B cells were analyzed at 4-28 h by q RT-PCR.The expressions of RSV F protein in RSV infected NRAV-knockdown A549 or BEAS-2B cells were analyzed at 24 h,48h,72 h by Western blot.RSV replication was examined by plaque assay in RSV infected NRAV-knockdown A549 or BEAS-2B.Virus titers in supernatants were measured at 24-48 hours post infection.The supernatants were collected every 4 hours since infection.Results:1 lnc-NRAV was down regulated in RSV-infected A549 cells(MOI=3)for 24,36 hours post infection(P<0.05).We found no significant differences in NEAT1,PRINS,and NEST expression.(P>0.05)2 Long RNA NRAV,with secondary structure composed of multiple stem-loops,did not have the function of encoding protein.lnc-NRAV shared low homology with other vertebrates such as mice.3 lnc-NRAV was down regulated in RSV-infected A549 cells 20 hours post infection(P<0.05).lnc-NRAV was down regulated in RSV-infected BEAS-2B cells 8 hours post infection(P<0.05).4 The over expression of lnc-NRAV promoted RSV replication in A549 or BEAS-2B cells.The relative expressions of RSV NS1,N,F,G,M,and M2 genes,RSV F protein and virus titers in supernatants were up regulated in NRAV-overexpressing A549 or BEAS-2B cells for indicated hours post infection(P<0.05).5 The knockdown of lnc-NRAV decreased RSV replication in A549 or BEAS-2B cells.The relative expressions of RSV NS1,N,F,G,M,and M2 genes,RSV F protein and virus titers in supernatants were down regulated in NRAV knockdown A549 or BEAS-2B cells for indicated hours post infection(P<0.05).Part Two Mechanisms study of lnc-NRAV binding miR-125a/b-5pObjective: To predict the potential of binding miR-125a/b-5p in lnc-NRAV by using bioinformatics analysis.To validate such potential through dual-luciferase reporter assay and gain or loss function experiments.Methods:1 The potential miRNA targeting sites of lnc-NRAV were predicted by online software miRDB.2 PmirGLO Dual-Luciferase reporter plasmid pmir GLO-NRAV and pmirGLO-NRAV mut(miR-125s)were constructed.Luciferase activity were collected in A549 or BEAS-2B cells cotransfected with miR-125 s and luciferase reporters containing nothing,lnc-NRAV,or mutant transcript.3 lnc-NRAV expression levels were analyzed after transfection of miR-125a/b-5p mimics or inhibitor into A549 or BEAS-2B cells.4 Plasmid pcDNA3.1-NRAVmut containing mutated miR-125a/b-5p binding sites was constructed.Mir-125a/b-5p expression levels after the transfection of pc DNA3.1-NRAV or pc DNA3.1-NRAVmut into A549 or BEAS-2B cells were analyzed.5 The expressions of RSV NS1,N,F,G,M and M2 genes in infected A549 or BEAS-2B cells transfected with pcDNA3.1,pcDNA3.1-NRAV,pc DNA3.1-NRAVmut,or cotransfected with pcDNA3.1-NRAV and miR125a/b mimics were analyzed by qRT-PCR.The expressions of RSV F protein were analyzed by Western blot.RSV replication was examined by plaque assay.6 The expressions of RSV NS1,N,F,G,M and M2 genes in infected A549 or BEAS-2B cells transfected with siRNA-control,si-NRAV,or cotransfected with si-NRAV and miR125a/b inhibitor were analyzed by qRT-PCR.The expressions of RSV F protein were analyzed by Western blot.RSV replication was examined by plaque assay.Results:1 lnc-NRAV could bind to miR-125a/b-5p through incomplete complementary pairing in A549 or BEAS-2B cells.2 We found no significant difference in lnc-NRAV levels after over expression or knockdown of miR-125a/b-5p.3 Ectopically expressed lnc-NRAV WT,but not the mutant reduced the levels of miR-125a/b-5p in A549 or BEAS-2B cells.4 The expression levels of RSV were rescued in infected A549 or BEAS-2B cells cotransfected with pcDNA3.1-NRAV and miR125a/b mimics,or cotransfected with si-NRAV and miR125a/b inhibitor.Part three Target mRNA predictions in ceRNA network containing lnc-NRAVObjective:To predict target mRNA in ceRNA network containing lnc-NRAV and miR-125 in RSV infected respiratory epithelial cells by using bioinformatics analysis.To investigate such potential through dual-luciferase reporter assay and gain or loss function experiments.Methods:1 The potential miRNA targeting mRNA was predicted by online software Targetscan,miRDB and micro RNA.According to the results of prediction,IL-6R,IRF4,IL-31,CD5 L,IL-16 and CXCL13 were the potential targets of miR-125a/b-5p.qRT-PCR showed that only IL-6R was expressed in A549 and BEAS-2B cells.And the follow up experiments were designed around IL-6R.2 Pmir GLO Dual-Luciferase reporter plasmid was constructed.Luciferase activities were measured in A549 or BEAS-2B cells cotransfected with luciferase reporter pmir GLO-IL-6R and pcDNA3.1 containing nothing,lnc-NRAV,lnc-NRAVmut.Luciferase activities in A549 or BEAS-2B cells cotransfected with pmirGLO-IL-6R,pcDNA3.1-NRAV and miR-125 mimics were measured in rescue experiment.Luciferase activities in A549 or BEAS-2B cells cotransfected with pmirGLO-IL-6R and si-NRAV were measured.Luciferase activities in A549 or BEAS-2B cells cotransfected with pmirGLO-IL-6R,pcDNA3.1-NRAV and miR-125 inhibitor were measured in rescue experiment.3 The expressions of IL-6R in A549 or BEAS-2B cells transfected with pc DNA3.1,pcDNA3.1-NRAV,pcDNA3.1-NRAVmut,or cotransfected with pc DNA3.1-NRAV and miR125a/b mimics were analyzed by qRT-PCR or Western blot.4 The expressions of IL-6R in A549 or BEAS-2B cells transfected with si-NC,si-NRAV,or cotransfected with si-NRAV and miR125a/b inhibitor were analyzed by qRT-PCR or Western blot.5 The expressions of IL-6R in A549 or BEAS-2B cells transfected with mimics-NC,or cotransfected miR125a/b mimics with pcDNA3.1-NRAV were analyzed by qRT-PCR or Western blot.6 The expressions of RSV NS1,N,F,G,M and M2 genes in infected A549 or BEAS-2B cells transfected with pcDNA3.1,pcDNA3.1-NRAV,pc DNA3.1-NRAVmut,or cotransfected pcDNA3.1-NRAV or pcDNA3.1-NRAVmut with si-IL-6R were analyzed by qRT-PCR.The expressions of RSV F protein were analyzed by Western blot.RSV replication was examined by plaque assay.7 The expressions of RSV NS1,N,F,G,M and M2 genes in infected A549 or BEAS-2B cells transfected with si-control,si-NRAV,si-IL-6R,or cotransfected si-NRAV with pcDNA3.1-IL-6R were analyzed by q RT-PCR.The expressions of RSV F protein were analyzed by Western blot.RSV replication was examined by plaque assay.Results: 1 IL-6R,IRF4,IL-31,CD5 L,IL-16 and CXCL13 were in the prediction intersection of miR-125a/b-5p targets from Targetscan,miRDB and microRNA.qRT-PCR showed Ct value of IL6-R was about 28-29,but other targets were over 40(undetected).So the follow up experiments were designed around IL-6R.2 Interleukin-6 receptor was found to share the common MRE of miR-125a/b-5p in dual-luciferase reporter assay in A549 and BEAS-2B cells.3 lnc-NRAV was positively correlated with IL-6R,and both of them were able to promote RSV replication in A549 or BEAS-2B cells.3.1 Ectopically expressed lnc-NRAV WT,but not the mutant or cotransfected with miR-125a/b-5p mimics increased the levels of IL-6R in A549 or BEAS-2B cells.3.2 Knockdown of NRAV induced down regulation of IL-6R.We found no significant difference in IL-6R levels after cotransfecting si-NRAV and miR-125a/b-5p inhibitor in A549 or BEAS-2B cells.3.3 Over expression of miR-125a/b-5p induced down regulation of IL-6R.We found no significant difference in IL-6R levels after cotransfecting lnc-NRAV and miR-125a/b-5p mimics in A549 or BEAS-2B cells.3.4 The expression levels of RSV could be increased in infected A549 or BEAS-2B cells transfected with pc DNA3.1-NRAV or pcDNA3.1-IL-6R.We found no significant difference in RSV levels after transfecting with pc DNA3.1-NRAVmut,or cotransfecting pcDNA3.1-NRAV and si-IL-6R in A549 or BEAS-2B cells.3.5 The expression levels of RSV were reduced in infected A549 or BEAS-2B cells transfected with si-NRAV or si-IL-6R.We found no significant difference in RSV levels after cotransfecting si-NRAV and pcDNA3.1-IL-6R in A549 or BEAS-2B cells.Conclusions:1 lnc-NRAV was down regulated in RSV-infected A549 or BEAS-2B cells with the infection prolonged.2 RSV replication was promoted in NRAV-overexpressing A549 or BEAS-2B cells,and reduced in NRAV knockdown cells.3 lnc-NRAV,who acted as a molecular sponge,bound miR-125a/b-5p through complementary pairing.4 lnc-NRAV shared the common MRE of miR-125a/b-5p with IL-6R.The binding of lnc-NRAV and miR-125a/b-5p induced the expression of IL-6R,which promoted RSV replication in A549 or BEAS-2B cells. |
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