Mechanisms Of MiRNA Targeting Endoplasmic Reticulum Stress Involved In Pseudorabies Virus Infection

Pseudorabies virus(PRV)infection causes great economic losses in the pig industry.Therefore,it is an important means to reduce the occurrence of pseudorabies to carry out research on disease resistance breeding to increase the porcine genetically resistance during PRV infection.Using proteomic analysis,our previous research showed that a group of genes involved in endoplasmic reticulum stress is upregulated in PRV-infected PK15 cells.Based on the previous proteomics research,we explored the pathways to activate the endoplasmic reticulum stress during PRV infection,and bioinformatics methods were used to screen and verify host miRNAs,which targeting key genes of porcine endoplasmic reticulum stress pathway.Finally analyze the effects of miRNAs on PRV replication.The main results are as follows:1.Porcine PRV infection activated endoplasmic reticulum stress pathway.PK15 cells were infected with PRV,then the cells were harvested at indicated time points post-infection.The expression of glucose-regulated protein 78(GRP78)was examined by qRT-PCR and Western blot.The results showed that the expression of the endoplasmic reticulum stress marker GRP78 increased during the early stages of PRV infection,indicating that the endoplasmic reticulum stress was activated.Examination of the three branches of the UPR revealed that the IRE1-XBP1 and e IF2?-ATF4 pathways were activated during PRV infection.In addition,we analyzed the expression of ATF4 and CHOP during PRV infection by qRT-PCR.The results showed that PRV infection significantly upregulated the m RNA expression of ATF4 and CHOP.The expression of Bcl-2,a gene regulated downstream of CHOP,was downregulated in the late stage of infection.These results demonstrated that PRV induced apoptosis in later stages of infection through the CHOP-Bcl-2 axis.2.Effects of endoplasmic reticulum stress pathway on PRV infection.The PcDNA3.1-GRP78 recombinant eukaryotic vector was used to overexpress GRP78 and si RNAs was used to knockdown the expression of GRP78,then infected with PRV.Viral genome replication and viral titers were examined by absolute q PCR and TCID50.The results showed that overexpression of GRP78 could enhance PRV replication and titer.However,knockdown of GRP78 expression did not significantly impact PRV replication.PK15 cells were treated with different concentrations of the endoplasmic reticulum stress inducer Tg and inhibitor TUDCA and then infected with PRV.Western blot,absolute q PCR and TCID50 assays showed that low concentration of Tg treatments significantly increased GRP78 protein levels,PRV replication and virus yield,while PRV replication and titer were decreased in TUDCA-treated cells.At the same time,CCK-8 assays showed that different concentrations of Tg and TUDCA treatment had no effect on cell viability.3.Identification of miRNAs targeting key genes of the porcine endoplasmic reticulum stress pathway.Target Scan was used to predict miRNAs that targeting the key genes ATF6,PERK,GRP78,IRE1 and XBP1 of endoplasmic reticulum stress pathways.The results of Target Scan prediction showed that the intersection of miRNAs targeting ATF6,PERK,GRP78,IRE1 and XBP1 were miR-142-5p,miR-145-5p,miR-150,and miR-199a-5p,and these miRNAs were selected as candidate miRNAs.Dual-luciferase reporter vectors were constructed,including candidate miRNA target sites,and co-transfecting dual-luciferase reporter gene vector with miRNA-mimics into BHK-21 cells.The luciferase assay showed that overexpression of miR-142-5p could inhibite the luciferase activity of ATF6,IRE1 and XBP1 dual-luciferase reporter recombinant vectors,overexpression of miR-199a-5p significantly inhibited the luciferase activity of IRE1 and XBP1dual-luciferase reporter recombinant vectors,and overexpression of miR-145-5p significantly inhibited the luciferase activity of IRE1 and PERK dual-luciferase reporter recombinant vector.These results indicated that miR-142-5p,miR-145-5p and miR-199a-5p might target the key genes of the porcine endoplasmic reticulum stress pathway.Then,the candidate miRNAs were overexpressed in PK15 cells,the m RNA expression levels of miR-142-5p,miR-145-5p,miR-199a-5p and their target genes ATF6,IRE1,XBP1 and PERK were examined by qRT-PCR.The results showed that the expression of miR-142-5p,miR-145-5p and miR-199a-5p were significantly upregulated in miRNA mimics transfected cells compared to the negative control cells.At the same time,overexpression of miR-142-5p could significantly inhibit the expression of ATF6 m RNA.Western blot assays showed that overexpression of miR-142-5p could significantly inhibit the expression of ATF6 protein.The results demonstrated that miR-142-5p might participate in the regulation of ATF6 protein by targeting ATF6 m RNA.4.Effect of miRNAs on PRV infection.PK15 cells were infected with PRV,then total RNA of the cells was extracted for miRNA sequencing.The results showed that 35 differential expression of miRNAs were determined in PRV-infected PK15 cells,indicating that the host miRNAs expression were changed during PRV infection.We speculated that miRNA targeting host genes of endoplasmic reticulum stress involved in PRV infection.The miR-142-5p,miR-145-5p,miR-199a-5p mimics were transfected into PK15 cells for 48 h,then infected with PRV.Viral genome replication and viral titers were examined by absolute q PCR and TCID50.The results showed that overexpression of miR-142-5p,miR-145-5p and miR-199a-5p had no significant effect on PRV infection.In conclusion,our results reveal that PRV infection induces endoplasmic reticulum stress and activates the IRE1-XBP1 and e IF2?-ATF4 pathways,and explored the mechanism of miRNA targeting endoplasmic reticulum stress involved in PRV infection from the perspective of miRNA influencing the expression of key genes of endoplasmic reticulum stress pathways.This study provides new thoughts for the mining of porcine disease resistance candidate genes.