CircRNAs Screening And Functional Validation Of Autophagy Induced By PEDV Infection In Vero-E6 Cells

Objective:the porcine epidemic diarrhea virus（PEDV）is the culprit strain responsible for diarrhea in piglets,which can become vomiting and dehydrated after being infected with PEDV and then cause piglet death,resulting in significant trauma to the global swine breeding industry.The PEDV genome is a single stranded,linear,positive strand RNA that spans approximately 28KB and is a member of the genus coronavirus.The PED epidemic ravaged our country for decades,and currently the most effective means against it remains vaccination.Because the pace of PEDV variation is extremely rapid and variant strain virulence is markedly enhanced,vaccines designed based on the classical strain CV777 do not achieve complete control of the variant strain.PEDV,as a member of coronavirus,can induce the occurrence of cell autophagy,and the relationship between cell autophagy and the replication of the virus is closely related,the two is complicated.Therefore,to find effective molecules against PEDV and improve the resistance of piglets to PEDV from the host perspective,it is only the fundamental approach to control the transmission of PEDV.An increasing number of studies have shown that circ RNAs play important functions when the virus infects host cells and that circ RNAs play very important roles in the processes of cell proliferation, differentiation and autophagy,circ RNAs have great potential as molecular targets for the diagnosis and treatment of diseases,and exploring the regulatory mechanisms of circ RNAs when they infect the host of PEDV can help to find new targets to fight against PEDV.Methods:（1）We established a cell model of PEDV infection by infecting IPEC-J2,Vero,and Vero-E6cells with PEDV in vitro,and the relative expression of PEDV-N was determined by quantitative real-time fluorescence.（2）Total cellular RNA was extracted from Vero-E6 cells infected with PEDV for 24h to prepare samples for transcriptome sequencing.Transcriptome sequencing was used to profile circ RNA expression in Vero-E6 cells infected with PEDV in vitro,and the significantly differentially expressed circ RNAs obtained were subjected to bioinformatics analysis,and the interacting mi RNAs and m RNAs were further analyzed jointly.（3）The differentially expressed circ RNAs obtained from transcriptome sequencing were validated for their expression levels using real-time PCR with primers spanning the circularization sites according to the methods and principles of circ RNA specific primer design; Bioinformatic analysis software analysis such as Miranda,targetscan and KEGG pathway were used to screen autophagy related m RNAs,based on which circ RNAs related to autophagy were reverse selected.（4）Cells were harvested at 24h after viral infection,and the expression levels of the autophagy related circ RNA RBM5 as well as mml-mi R-34c-5p,MDM4,P53 and LC3B were determined by real-time PCR,while P53 and LC3B protein levels were determined by WB.A circ RNA RBM5 overexpression circularization vector was constructed,and the overexpression efficiency of circ RNA RBM5 was detected after transfection into Vero-E6 cells.PEDV were seeded 24h after transfection,and the expression levels of mml-mi R-34c-5p,MDM4,P53 and LC3B were determined from m RNA and protein levels by real-time PCR and WB,respectively.The expression of three genes,PEDV-M,N,and ORF3,was examined and PEDV TCID₅₀ was measured to measure viral replication.Results:（1）PEDV can be stably infected in Vero-E6 cells the expression level of PEDV-N gene in Vero-E6 cells was significantly increased at 24h,48h and 72h（***P<0.01）;The expression level of PEDV-N in IPEC-J2 cells increased significantly（**P<0.05）at 48h,so Vero-E6 cells were chosen as the cellular model for the in vitro replication mechanism study of PEDV.（2）Vero-E6 cells converged at 48h after being infected with PEDV,the cell morphology changed and shrunk together,the cell membrane fused and formed a large number of syncytia,and the infection of Vero-E6 cells with PEDV in vitro successfully established a cytopathic model;The q RT-PCR results showed that the fold changes of viral N,M,or ORF3genes from 12 to 24h after in vitro infection of Vero-E6 cells with PEDV were the largest,so 24h was chosen for all subsequent assays;The differential circ RNA ce RNA network diagram was successfully drawn.（3）Ten circ RNAs with small and more stable numbers of circularized bases were selected and their expression in Vero-E6 cells after PEDV infection was determined by q RT-PCR,which showed the same results as transcriptome sequencing.The autophagy related m RNA was screened by bioinformatics means as MDM4,and on this basis,the circ RNA related to autophagy was obtained as circ RNA RBM5.（4）After PEDV infection of Vero-E6 cells,the expression level of circ RNA RBM5 was dramatically decreased（***P<0.01）.After transfection with circ RNA RBM5 overexpression circularization vector,the expression level of circ RNA RBM5 was dramatically increased（***P<0.01）.After overexpression of circ RNA RBM5,the expression level of mml-mi R-34c-5p was significantly decreased（**P<0.05）,that of MDM4was significantly increased（**P<0.05）,and that of P53 and LC3B was significantly decreased（**P<0.05）.WB results showed that after overexpression of circ RNA RBM5,the expression of P53 and LC3B protein decreased significantly,cell autophagy was inhibited,and circ RNA RBM5-mml-mi R-34c-5p-mdm4-P53-LC3B regulated Vero-E6 cell autophagy.TCID₅₀ and q RT-PCR results showed that inhibition of cell autophagy impeded PEDV replication in Vero-E6 cells.Conclusions:（1）A total of 58 differentially expressed circ RNAs before and after PEDV infection were obtained,and the circ RNA associated with autophagy was circ RNA RBM5.（2）The mi RNAs targeted by circ RNA RBM5 to interact with each other were confirmed as mml-mi R-34c-5p,the downstream target genes of mml-mi R-34c-5p as MDM4,the increased expression level of MDM4 inhibited the expression level of autophagy related proteins P53 and LC3B.（3）Overexpression of circ RNA RBM5 could inhibit cell autophagy,and inhibition of cell autophagy would further hinder PEDV replication in Vero-E6 cells.