Regulatory Effect And Preliminary Mechanism Study Of Long Non-coding RNA Lnc-ALPK2 On CVB3 Replication

Objective:Coxsackievirus group B type 3(CVB3)is the main pathogen of acute and chronic viral myocarditis(VCM)in young adolescents,which is seriously harmful to human health.To date,no effective measures are available to treat such disease.Studies have shown that indirect damage caused by host immune response is the main cause of disease progression of VCM,and in-depth analysis the interaction between CVB3 infection and host can provide a new idea for the treatment of viral myocarditis.In recent years,long non-coding RNAs(lnc RNAs)of host cells have been regarded as the new regulator of the interaction between virus and host,and play an important role in the process of virus replication and pathogenesis.Therefore,in this study,we screened the differentially expressed lnc RNAs by high-throughput sequencing in He La cells infected with CVB3,and a highly expressed lnc RNA Lnc-ALPK2 was found.NEDD4L(neural precursor cell expressed developmentally down-regulated 4 like,NEDD4L)was identified as the target gene of lnc-ALPK2 by bioinformatics analysis.Previous studies have shown that NEDD4L can promote the production of type I interferon and enhance the host antiviral immune response.Therefore,this study preliminarily discusses the effect of Lnc-ALPK2-NEDD4L-IFN axis on CVB3 replication.The present study will provide a new perspective for understanding the development and progression of viral myocarditis as well as provides a potential new antiviral target against CVB3 infection.Methods:1.High-throughput sequencing was used to analyze the expression profiles of lnc RNAs and m RNAs in Hela cells infected with CVB3.And we randomly selected five differentially expressed lnc RNAs to verify their expression by q RT-PCR.Then a highly expressed lnc RNA Lnc-ALPK2 was chosen and its expression was verified in different cell lines.2.Hela cells were infected with CVB3 in different MOI(0,0.06,0.6,6,60).The expression of Lnc-ALPK2 was detected by q RT-PCR after CVB3infection.The cells were collected at different time points(0,4,8,24,48 h)after CVB3 infection,and the expression of Lnc-ALPK2 was detected to determine the appropriate virus titer and the time of CVB3 infection.3.The small interference RNAs(si RNAs)of Lnc-ALPK2 was synthesized and the overexpression plasmid pc DNA3.1(+)-Lnc-ALPK2 was constructed to konckdown or overexpress Lnc-ALPK2 in Hela cells,and the cells were then infected with CVB3.The cells were collected to extract the total RNA and total protein at 24h post CVB3 infection.The expression of CVB3 RNA was detected by q RT-PCR and Western blot,and the titer of CVB3 virus in the cell supernatant was detected by TCID₅₀ to determine the effect of Lnc-ALPK2 on CVB3 replication.4.Using bioinformatics databases such as Cis target gene prediction,NCBI,UCSC and Coding Potential Caculator,we predict the target genes of Lnc-ALPK2.According to the predicted results,the possible downstream target of Lnc-ALPK2 is NEDD4L.5.Hela cells were transfected with pc DNA3.1(+)-Lnc-ALPK2 plasmid and si RNA and then infected with CVB3,the cells were collected and total RNA and protein were extracted respectively at 24h after CVB3 infected.The expression of RNA and protein of NEDD4L and the relative expression of downstream genes IFN-?and IFN-?were detected by q RT-PCR and Western blot.Results:1.The expression of Lnc-ALPK2 was significantly higher in CVB3infected different cell lines than that in uninfected cells.The results of q RT-PCR is consistent with that of the chip results.2.The expression of Lnc-ALPK2 was significantly up-regulated in Hela cells after CVB3(MOI=0.6)infected 24 hours(P<0.05).3.The expression of VP1 and 3D RNA,the expression of VP1 protein and the titer of CVB3 virus in the supernatant of the cells with Lnc-ALPK2konckdown were significantly lower than those in the control group(P<0.05).The expression of VP1 and 3D RNA,the expression of VP1 protein and the titer of CVB3 virus in the supernatant of Hela cells with Lnc-ALPK2overexpression were significantly higher than those of the control group.4.Bioinformatics databases such as cis target gene prediction,NCBI,UCSC and Coding Potential Caculator predicted that Lnc-ALPK2 is an antisense chain lnc RNA,and its corresponding sense chain is NEDD4L.Both Lnc-ALPK2 and NEDD4L genes are located on chromosome 18.This confirmed the possibility of Lnc-ALPK2 binding to NEDD4L RNA.5.The expression of NEDD4L,INF-?,IFN-?RNA and NEDD4L protein were significantly decreased in Lnc-ALPK2 overexpressed cells.While NEDD4L,INF-?,IFN-?RNA and NEDD4L protein were significantly up-regulated in Lnc-ALPK2 knock-down cells.Conclusions:1.CVB3 infection can up-regulate the expression of Lnc-ALPK2 in Hela cells.2.As a host factor that promote virus replication,Lnc-ALPK2 can promote CVB3 replication in the process of CVB3 infection.3.NEDD4L is a downstream target gene regulated by Lnc-ALPK2.Lnc-ALPK2 inhibits the expression of NEDD4L by interacting with NEDD4L,resulting in a decrease in the expression of type I interferon and thus promoting CVB3 replication.