Study On The Mechanism Of CircSIAE Inhibiting CVB3 Replication Through MiR-331-3p/TAOK2

Background:Coxsackievirus B3(CVB3)infection can alter the biological process of cells,causing not only abnormal protein expression but also abnormal circular RNA(circRNA)changes.CircRNA is a kind of non-coding RNA with a closed ring molecular structure and is involved in biological processes such as apoptosis,innate immunity and antivirus.Therefore,it is of great significance to study the effect of circRNA on CVB3 replication.Objective:In order to explore the effect of circRNA on CVB3 replication,circSIAE with abnormal expression after CVB3 infection was selected as the research object to explore the regulatory effect and mechanism of circSIAE/miR-331-3p/ TAOK2 axis on CVB3 replication.Methods:1.The circRNA differentially expressed in Hela cells(a classic cell line infected with CVB3)after infection with CVB3 was screened by qRT-PCR assay,and primers were designed according to the circRNA sequences for ring formation analysis.The expression of circSIAE at different time points of CVB3 Infection was analyzed by qRT-PCR.The multiple of infection(MOI)was 5,and the following MOI values were consistent.Hela cells without CVB3 Infection were used as control.2.The subcellular localization of circSIAE in Hela cells was analyzed by nucleoplasma separation method.Hela cells were treated,and cytoplasmic RNA was extracted from the cell membrane by membrane lysate,and then nuclear RNA was extracted by nuclear lysate.After reverse transcription of nuclear and plasma RNA,qRT-PCR was used to determine the expression level of circSIAE in nuclear and plasma.3.CircSIAE was overexpressed/knocked down in Hela cells and then infected with CVB3.Total protein of cells at 6h and 8h after infection was extracted,and the effect on structural protein VP-1 of CVB3 was verified by western blot assay.The effect of miRNA on virus replication and proliferation was determined by virus plaque assay and CVB3-e GFP plasmid transfection assay.4.Three databases,Circ Bank,Circinteractome and Cancer Specific circRNA Database(CSCD),were used to predict the downstream micro RNA(miRNA)of circSIAE,and their relationship was verified by qRT-PCR assay at m RNA level and their binding sites were verified by dual luciferase assay.The miRNA was overexpressed and inhibited in Hela cells,and its effect on CVB3 structural protein VP-1 was verified by western blot assay.The effect of miRNA on virus replication and proliferation was determined by virus plaque assay and CVB3-e GFP plasmid transfection assay.5.Multiple databases(miRDB,mi Path DB,miRTARbase,Target Scan)were used to predict the downstream target genes of miRNA and analyze the regulatory effect of target genes on CVB3 replication.6.The effects of circSIAE and miRNA on CVB3 virus replication were analyzed,and the effects of NF-?B,p-NF-?B,ERK2/3 and p-ERK2/3 on CVB3 replication were analyzed by western blot.Results:1.Previous studies showed that the expression of circSIAE decreased after CVB3 infection.PCR was performed with circSIAE specific primers,and the target bands were sequenced,and the results showed that the amplified products fully matched the sequence of circSIAE.The circSIAE ring structure was verified by RNase R experiment.QRT-PCR showed that circSIAE decreased with the time gradient of CVB3 infection.The analysis of nucleoplasma separation method showed that circSIAE were mainly distributed in cytoplasm,accounting for 79%(U6 as nuclear reference,18 S as internal plasma reference).2.Western blot showed that overexpression of circSIAE inhibited the replication of CVB3;Virus plaque assay showed that circSIAE inhibited CVB3 proliferation.CircSIAE knockdown promoted CVB3 replication.Virus plaque assay showed that circSIAE promoted the proliferation of CVB3.The fluorescent virus infection of CVB3-e GFP also indicated that circSIAE could inhibit the replication of CVB3.3.Using Circ Bank,Circinteractome and Cancer Specific CircRNA Database(CSCD),it was predicted that miR-331-3p was the downstream target miRNA of circSIAE.The qRT-PCR results showed that the expression level of miR-331-3p was negatively correlated with the expression level of circSIAE,and the double luciferase assay indicated that the two existed binding sites.4.Western blot results showed that miR-331-3p mimics could promote the expression of viral structural protein VP-1,and the number of viral plaques was significantly increased,and the number of fluorescent cells of CVB3-e GFP fluorescence virus was also increased.However,CVB3 was infected after transfection with miR-331-3p inhibitor.Western blot,virus plaque assay and CVB3-e GFP experiments all showed that CVB3 replication was inhibited.Further analysis of multiple databases showed that TAOK2 was one of the target genes of miR-331-3p.In order to analyze the effect of TAOK2 on CVB3,the virus was infected after TAOK2 knockdown in Hela cells,and western blot showed that TAOK2 could promote CVB3 replication.To further analyze the regulatory role of circSIAE/miR-331-3p in CVB3 replication,overexpression of circSIAE in Hela cells promoted the expression of TAOK2 and decreased the expression of p-NF-?B and p-ERK2/3.The expression of TAOK2 was inhibited by circSIAE knockdown,and the expressions of p-NF-?B and p-ERK2/3 were increased.Overexpression of miR-331-3p inhibited the expression of TAOK2 and increased the expression levels of p-NF-?B and p-ERK2/3 proteins.The knockdown of miR-331-3p promoted the expression of TAOK2 and decreased the expression levels of p-NF-?B and p-ERK2/3 proteins.Conclution: CircSIAE negatively regulates TAOK2 by adsorbing miR-331-3p through the inflammation pathway sponge and plays a role in inhibiting CVB3 virus replication.