Molecular Regulation Mechanism Of M~6A Demethylase ALKBH5 On Resistance Of Porcine Alveolar Macrophages To PEDV Infection

Porcine Epidemic Diarrhea is an acute,highly contagious disease caused by Porcine Epidemic Diarrhea Virus(PEDV),which causes serious economic losses to pig industry.N6methyladenosine(m6A)as the first to be discovered reversible RNA modification,has become a new direction and hotspot of RNA epigenetics in the field of livestock and poultry genetics.Studies have shown,m6A modification plays an important role in the regulation of virus replication and host immunity.However,the effect of m6A modification on porcine alveolar macrophages(3D4/21)resistance to PEDV infection and its molecular mechanism remains unclear.Firstly,this study detected the differential expression of m6A modification enzymes and confirmed key m6A demethylase-ALKBH5.Secondly,we preliminarily screened out promising targets of ALKBH5 based on transcriptome sequencing and bioinformatics analysis.Finally,using dual luciferase activity,western blot and MeRIP-qPCR,we analyzed the effect of ALKBH5-mediated m6A modification on the expression level of target gene.Main results are as follows:1.Pathogens such as PDCoV,PoRV,TGEV and PEDV were detected in lung tissues of PEDV-diarrhea piglets and normal piglets by RT-PCR.Results showed,PEDV was detected in lung tissues of diarrhea piglets,but PDCoV,PoRV,TGEV are not detected.These results indicated that PEDV could be replicated in the lung tissues of piglets.In this study,we analyzed the differential expression of different m6A enzymes(METTL3,METTL14,WTAP,FTO,ALKBH5)in PEDV-diarrhea piglets and normal piglets.Results showed that the expression of merely ALKBH5 in lung tissue of PEDV diarrhea piglets was significantly higher than that of normal piglets(P<0.01).Tissue expression profile of piglets with 7 days of age showed that ALKBH5 gene was expressed in different tissues,and the highest expression level was found in lung tissues.ALKBH5 high expression may be related to piglet’resistance to PEDV infection.After PEDV strain infection for 36 h,the 3D4/21 cells showed obvious pathological effect.Besides,the mRNA and protein expression levels of ALKBH5 gene were significantly increased in PEDV-infected cells.The stable porcine 3D4/21 cell line with lentivirus interference of ALKBH5 gene was successfully established and the interference efficiency is up to 88%.The copy number of PEDV-M gene after interference was significantly higher than that of control group(P<0.01),which indicated that the up-regulation ofALKBH5 expression level can improve the resistance to PEDV infection in porcine 3D4/21 cells.2.Transcriptome sequencing of porcine alveolar macrophages was performed between shALKBH5 and shNC group by RNA-seq technique.Results showed,a total of 285 Mb clean reads were sequenced and 12 types of alternative splicing were identified,which mainly consist of alternative last exons(TTS),alternative first exons(TSS),skipped exons(SKIP).There were 175 differentially expressed genes(DEGs)between the two groups,thereinto,78 DEGs were up-regulated and 97 DEGs were down-regulated in shALKBH5 group.All DEGs were involved in a total of 802 significant GO function classifications and screened out virusrelated GO terms(virus receptor activity,fusion of virus membrane with host plasma membrane),including DPP4,GAS6 gene.Therefore,GAS6 was initially identified as a candidate gene for possible targeted regulation ofALKBH5 based on transcriptome sequencing.3.qPCR and western blot were used to analyze the expression relationship between ALKBH5 and target gene GAS6.Results showed,the expressions of ALKBH5 and GAS6 were significantly up-regulated in the PEDV infected group(P<0.01).After ALKBH5 knockdown,the expression level of mRNA and protein were significantly decreased.These results indicated that the down-regulation of ALKBH5 could significantly reduce the expression of target gene GAS6.Moreover,SRAMP and RMBase V2.0 software were used to predict the potential m6A modification sites of GAS6.One m6A site(c.1469 bp)was found in GAS6 coding region.The m6A site-mutant and wild-type double luciferase reporter vectors were constructed respectively.Double luciferase activity showed that GAS6-wild type was significantly decreased in siALKBH5 group(P<0.01).There was no significant difference in the activity of the mutant(A1469G)between siALKBH5 and siNC groups(P>0.05).The m6A level between shALKBH5 group and shNC group was detected by MeRIP-qPCR.Results showed,the m6A level was significantly increased in shALKBH5 group(P<0.01).Above results indicated that ALKBH5 probably reduced the m6A level of target GAS6 and improved its expression.