| Hand,foot and mouth disease（HFMD）,mainly caused by Enterovirus type 71（EV71）,is an acute infectious disease spreading in children under 5 years old.Up to date,the pathogenetic machnism of EV71 is not fully understood.Previous studies showed that non-coding RNAs such as long non-coding RNA（lnc RNA）and micro RNA（mi RNA）play important regulatory roles in the occurrence and development of viral infectious diseases.The host lnc RNA binds to mi RNA and reduces the silencing effect of mi RNA on target gene m RNA,thus forming a lnc RNA/mi RNA/m RNA competitive endogenous RNA（ce RNA）regulatory axis,which further affects virus replication through regulating cellular biological activities.However,whether ce RNA regulatory axis mediates the pathogenesis of EV71 is still unclear.Objective:This study was aimed to predict the key lnc RNAs,mi RNAs and m RNAs probably regulating the EV71 infection process,to verify the influence of EV71 infection on expression of lnc RNA,mi RNA and m RNA,to prove the lnc RNA,mi RNA and m RNA regulatory axis,and to confirm the regulatory role of ce RNA regulatory axis on EV71virus replication.This study will lay a theoretical foundation for elucidating the pathogenetic mechanism of EV71 infection and exploring the targeted drugs against HFMD.Methods:（1）Predicting and screening the key mi RNA molecules induced by EV71infection:Analyze mi RNA transcriptome original data of serum exosomes in mild and severe hand,foot and mouth disease（GSE52780）from Gene expression omnibus（GEO）using R language software.Rhabdomyosarcoma（RD）and SH-SY5Y human neuroblastoma cell were infected with EV71 with the MOI of 0/0.1/0.5/1/2.After 12h and 24 h,mi RNA were detected by quantitive real time–polymerase chain reaction（q RT-PCR）,and the key mi RNA induced by EV71 infection,namely mi R-4443,was confirmed.（2）Predicting and screening the lnc RNA regulating mi R-4443 expression:Predict the lnc RNAs interacting with mi R-4443 using DIANA online tool,and select the lnc RNAs that have been reported to be upregulated by EV71 infection as candidate lnc RNAs.q RT-PCR was used to detect the expression levels of candidate lnc RNAs in RD cells after EV71 infection.Confirm the interaction between ENST00000469812 and mi R-4443 by luciferase reporter assay.Prove the regulatory effect of ENST00000469812on mi R-4443 by overexpression or knockdown of ENST00000469812 followed by q RT-PCR.（3）Predicting and verifying ENST00000469812/mi R-4443/target gene m RNA regulation axis:Predict the target genes of mi R-4443 by using Target Scan,mi RWalk,mi Randa,and DIANA online tools.After RD cells were transfected with mi R-4443mimics or mi R-4443 inhibitor,the expression levels of candidate target genes were detected by q RT-PCR.The transcriptional and translational levels of the target gene nuclear protein 1（NUPR1）induced by EV71 infection were detected by q RT-PCR and Western-blot.The lnc RNA ENST00000469812 influencing the interaction between mi R-4443 and NUPR1 3’-Untranslated region（3’-UTR）was proved by luciferase reporter assay.The regulation of mi R-4443 on translational level of NUPR1 was verified by Western-blot.As well,the reverse effect of mi R-4443 on the upregulation of ENST00000469812 on NUPR1 was confirmed by q RT-PCR and Western-blot.（4）Verifying the regulation effect of ENST00000469812/mi R-4443/NUPR1axis on EV71 virus replication:In RD cells,ENST00000469812,mi R-4443,NUPR1were overexpressed or knockdowned（inhibited）followed by EV71 infection.EV71genomic RNA level,VP1 structural protein expression level,and extracellular mature viral particle levels were detected by q RT-PCR,Western-blot and 50%tissue culture infectious dose(TCID50)method,respectively.mi R-4443 mimics or si NUPR1 treatment on the basis of ENST00000469812 overexpression,EV71 replication levels were also detected.Finally,autophagy in RD cells induced by EV71 infection or ENST00000469812/mi R-4443/NUPR1 axis were assayed by Western-blot.Results:（1）EV71 infection induced downregulation of mi R-4443 expression:It was predicted that 19 mi RNAs were differentially expressed in serum exosomes of mild and severe hand,foot and mouth disease patients.q RT-PCR showed that the expression level of mi R-4443 was significantly reduced in EV71-infected RD and SH-SY5Y cells in MOI dose-dependent manner.Thus,mi R-4443 was selected as one of the key mi RNAs regulating EV71 infection.（2）EV71 infection induced upregulation of lnc RNA ENST00000469812 which downregulated mi R-4443 expression:1272 lnc RNAs were predicted interacting with mi R-4443,and 3 lnc RNAs including ENST00000469812,ENST00000451940 and ENST00000418747 with the highest predicting scores and most upregulating folds reported previously were selected as candidate lnc RNAs.q RT-PCR confirmed that EV71infection induced the upregulation of these 3 lnc RNA,in which ENST00000469812expression was in a MOI dose-and time-dependent manner,suggesting it was the key lnc RNA induced by EV71.The interaction between ENST00000469812 and mi R-4443was confirmed by luciferase reporter assay.q RT-PCR showed ENST00000469812reduced expression of mi R-4443.These suggested that EV71 infection induced upregulation of lnc RNA ENST00000469812 which reduced mi R-4443 expression.（3）Nuclear protein 1（NUPR1）was one of mi R-4443 target genes,and ENST00000469812/mi R-4443/NUPR1 regulatory axis was activated by EV71infection:Nineteen candidate target genes of mi R-4443 were predicted.q RT-PCR and Western-blot confirmed mi R-4443 reduced NUPR1 expression.EV71 infection induced the upregulation of nupr1 gene expression at the transcriptional and translational levels.Luciferase report assay showed interaction between mi R-4443 and NUPR1 m RNA 3’UTR,and ENST00000469812 influencing the above interaction.q RT-PCR and Western-blot confirmed mi R-4443 reversed the upregulation of ENST00000469812 on NUPR1 expression.The above evidence indicated that NUPR1 was the target gene of mi R-4443,and ENST00000469812/mi R-4443/NUPR1 regulatory axis was activated by EV71 infection.（4）The ENST00000469812/mi R-4443/NUPR1 regulatory axis promoted EV71virus replication:After ENST00000469812 overexpression,mi R-4443 inhibition,or NUPR1 overexpression in RD cells,respectively,the levels of the EV71 RNA genome,structural protein VP1,and mature virus particles in the cell supernatant were all upregulated.On the contrary,after ENST00000469812 knockdown,mi R-4443overexpression,and NUPR1 knockdown,the levels of EV71 RNA,protein and virus particles were reduced.Meantime,mi R-4443 overexpression or NUPR1 knockdown could eliminate the promotion effect of ENST00000469812 on EV71 replication,indicating that the ENST00000469812/mi R-4443/NUPR1 regulatory axis promoted EV71 replication.Western-blot confirmed that EV71 induced autophagy in RD cells,and the ENST00000469812/mi R-4443/NUPR1 regulatory axis activated autophagy,suggesting that this regulatory axis might promote EV71 replication through activating autophagy.Conclusion:（1）EV71 infection induced the downregulation of mi R-4443,which might be a key mi RNA involved in regulating EV71 infection.（2）EV71 infection induced upregulation of lnc RNA ENST00000469812 expression.The upregulated ENST00000469812 interacted with mi R-4443 and reduced expression of mi R-4443.（3）EV71 infection induced upregulation of ENST00000469812,which downregulated mi R-4443 expression,thereby eliminated the silencing effect of mi R-4443on the target gene NUPR1.This mechanism led to upregulation of NUPR1 expression,which forms the ENST00000469812/mi R-4443/NUPR1 regulatory axis.（4）EV71 infection induced activation of the ENST00000469812/mi R-4443/NUPR1regulatory axis,which further promoted the EV71 virus replication though enhancing autophagy level.This study laid a foundation for the research on EV71 pathogenic mechanism,and could provide a theoretical basis for exploring antiviral therapy targets. |
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