Molecular Mechanism Of LncRNA446-Mediated Alix Ubiquitination Regulating Intestinal Tight Junction Proteins Involved In PEDV Infection

Porcine epidemic diarrhea(PED)is a highly contagious disease caused by porcine epidemic diarrhea virus(PEDV),which brings huge economic losses to large-scale pig farms in China.Prevention and control methods unable to achieve efficient immunization because of the genetic variation of PEDV,and it is of great research value to find new prevention and control strategies.Tight junction-associated proteins play an important role during numerous viruses infection,so maintaining the integrity of tight junctions may be a new strategy for the prevention and treatment of PEDV.In recent years,lncRNAs,as regulatory molecules that play an important role in numerous biological functions,have the potential to be used as molecular markers for the diagnosis of human diseases.Screening the key lncRNAs that regulate host tight junctions during PEDV infection can be used as an effective genetic method,and provide a scientific basis and experimental support for further elucidating the pathogenesis of PEDV.In this study,lncRNA sequencing was performed in the jejunal mucosa of PEDV-infected with intestinal barrier damaged and healthy with intestinal barrier intact piglet individuals previously screened by our group,lncRNA446 was used as the research object to reveal the molecular mechanism by which lncRNA446 is involved in the regulation of PEDV replication through tight junctions at the cellular level,further screen the interaction molecules of lncRNA446 at the mRNA and protein levels,identify the interaction proteins that Alix plays an important role in assembling and maintaining the polarity of cellular tight junctions,and deeply validate the function of Alix in PEDV infection by constructing interference and overexpression vectors in order to reveal the important role of lncRNAs in the host process of PEDV infection and lay the foundation for the future development of disease resistance breeding and molecular markers screening of PEDV.1.High-throughput sequencing was used to screen key lncRNAs that regulate tight junctions during PEDV infectionIn order to screen key lncRNAs that regulate tight junctions during PEDV infection,in this study,lncRNA sequencing was performed using extreme phenotypic samples established previously,and a total of 111 differentially expressed lncRNAs were screened,including 39 up-regulated lncRNAs and 72 down-regulated lncRNAs.Five differentially expressed lncRNAs were randomly selected for qRT-RCR to validate the accuracy of high-throughput sequencing,and the results were consistent with lncRNA sequencing.In this study,we further predicted the target genes of differentially expressed lncRNAs based on the cis and trans mechanisms in combination with previous transcriptome sequencing data and performed functional enrichment analysis,and found that the target genes were mainly annotated to metabolic process,cell part and binding.KEGG analysis was mainly enriched into pathways such as ribosome,systemic lupus erythematosus,alcoholism and viral carcinogenesis,and the tight junction signaling pathways closely concerned in this study were also enriched in KEGG analysis results.2.Subcellular localization and functional validation of lncRNA446In this study,we reverse screened differentially expressed lncRNAs with target genes as tight junction signaling pathway genes based on the prediction results of cis and trans mechanisms and preliminarily screened lncRNA446 as a candidate lncRNA for regulating tight junctions after PEDV infection using qRT-PCR.The distribution localization of lncRNA446 in porcine small intestinal epithelial cells was examined using lncLocator,FISH assay and RNA nucleocytoplasmic separation qRT-PCR,and the results indicated that lncRNA446 was mainly expressed in the cytoplasm.In order to further investigate the function of lncRNA446,the expression of lncRNA was interfered using siRNA,and it was found that interference with lncRNA446 could improve the replication and proliferation of PEDV by experiments such as copy number of PEDV M gene,TCID50,indirect immunofluorescence and western blot.Construction of the DSS enteritis model revealed that the expression level of lncRNA446 showed a significant down-regulation after 12 hours of intestinal damage(P<0.05).The transwell monolayer model revealed a highly significant decrease in TEER values after lncRNA446 interference(P<0.01).On this basis,we examined the alkaline phosphatase content in the supernatant of the cells and found that alkaline phosphatase was extremely significantly increased after interfering with lncRNA446(P<0.01),and transmission electron microscopy revealed that the TJ structure of IPEC-J2 was damaged and the gap between cells was expanded after 48 hours of lncRNA interference.Indirect immunofluorescence and western blot detection of the expression distribution of ZO1 revealed that the expression of ZO1 was significantly decreased after 48 h of interference with lncRNA446,and the expression near the cell membrane was disrupted.Through CCK8 assay,it was found that lncRNA446 interfered with the activity of IPEC-J2 cells at different time points showed a highly significant decrease(P<0.01)in a time-dependent manner,and interfering with the expression of lncRNA446 could arrest the cell cycle of IPEC-J2 in G2 phase and significantly increase the apoptosis rate of IPEC-J2 cells(P<0.01).3.lncRNA446 inhibits Alix ubiquitin degradation to regulate tight junctionsThis study screened the expression profile of mRNA after lncRNA446 interference by transcriptome sequencing,and the results showed that 380 genes were differentially expressed after lncRNA446 interference,mainly enriched in GO entries such as defense response to virus,respiratory chain complex and ubiquitin protein ligase binding,as well as KEGG signaling pathways such as influenza A,protein processing in endoplasmic reticulum and antigen processing and presentation.The secondary structure of lncRNA446 was predicted by RNAfold,and lncRNA446 was divided into C-terminal,N-terminal,and intermediate segments for RNApulldown according to the secondary structure of lncRNA446,with 17,12,and 23 interacting proteins screened in each segment,respectively,of which a key protein Alix was present in the C-terminus.The binding of lncRNA446 and Alix in porcine small intestinal epithelial cells was reverse verified using RIP.Indirect immunofluorescence and western blot experiments revealed that the expression distribution of Alix was significantly reduced after lncRNA446 interference,and treatment using MG 132 and leupeptin suggested that lncRNA446-mediated degradation of Alix was located in the proteasome rather than lysosomes.In order to investigate whether lncRNA446 mediates the ubiquitination of Alix,this study analyzed the ability of Alix to bind ubiquitin in IPEC-J2 cells after lncRNA446 interference,and the results showed that the ubiquitination level of Alix increased after lncRNA446 interference,indicating that lncRNA446 may mediate the degradation of Alix by inhibiting the ubiquitination level of Alix.4.Functional mechanism study of Alix during PEDV infectionIn this study,we verified the interaction between Alix and ZOl in IPEC-J2 cell line using CoIP,and western blot results showed that interfering with the expression of Alix could reduce the protein expression of ZO1 in IPEC-J2 cells.In order to further study the role of Alix in the mechanism of resistance to PEDV infection in piglets,this study examined the difference in mRNA expression of Alix between PEDV-infected and healthy piglet individuals,and found that the mRNA expression of Alix in the jejunal mucosa of piglets was extremely significantly up-regulated after PEDV infection(P<0.01),western blot and indirect immunofluorescence were performed for Alix protein expression at different time points of PEDV infection,and the results revealed that Alix showed a significant down-regulation expression after 48 hours of PEDV infection,and the co-localization results showed that Alix distribution and expression trends were highly similar to F-actin.In this study,we successfully constructed the interference and overexpression cell lines of Alix and validated them at mRNA and protein levels,which can be used for the next step of functional analysis experiments.The fluorescence localization of F-actin showed that Alix interference could significantly reduce the expression distribution of F-actin,and then the relationship between Alix and PEDV infection was assessed using PEDV infection of Alix interference and overexpression cell lines in this study.PEDV copy number,indirect immunofluorescence and western blot results showed that interference with Alix could increase the replication and proliferation of PEDV,and overexpression could inhibit the replication and proliferation of PEDV.After bioinformatics analysis of the tertiary structure of Alix and prediction of the ubiquitination sites of Alix,it was found that there were potential ubiquitination sites at amino acid positions 332,344,and 695 of the Alix protein sequence(SVMscore>0.8).In this study,we used co-immunoprecipitation with Alix in porcine intestinal epithelial cells combined with mass spectrometry to screen the interacting proteins of Alix,resulting in a total of 226 interacting proteins of Alix.In order to further screen the E3 ligases of Alix,Alix was predicted by UBibrowser online software in this study,and a total of 37 potential E3 ligases were obtained,and the prediction results were intersected with the IP results to identify CRYAB,an E3 ligase that may play a key role in the ubiquitination and degradation of Alix in porcine intestinal epithelial cells.