CeRNA Mechanism Research Of Porcine Alveolar Macrophages (3D4/21) Against Swine Influenza Virus (H1N1 And H3N2) Infection

Swine influenza virus(SIV)is an orthomyxovirus,which can infect pigs of different breeds,genders and ages and cause fever,breathing difficulties,coughing,anorexia and weight loss in the pig herd.At present,the main swine influenza virus subtypes are H1N1 and H3N2.In addition,pigs have receptors that both human influenza viruses and avian influenza viruses can bind,which is a huge hidden danger of zoonotic diseases.However,the molecular mechanism and regulatory network of swine influenza virus infection are still unclear.Therefore,revealing the molecular mechanism of host cells against swine influenza virus infection from the genetic level is not only of great significance for screening resistance genes to improve the disease resistance of pigs,but also can provide important reference and basis for the pathological research of pandemic influenza virus.In this study,a cell model of the swine influenza virus(H1N1 and H3N2)infecting the porcine alveolar macrophage cell line(3D4/21)was constructed.It is the first time to reveal the expression profiles of related miRNA,mRNA,lncRNA and circRNA after swine influenza viruses(H1N1 and H3N2)infect 3D4/21 cells at the whole transcriptome level.Combined with bio-information analysis,gene overexpression,gene interference,Real-time Quantitative PCR(qPCR),dual luciferase activity detection and RNA immunoprecipitation(RIP),the two ceRNA networks regulating H1N1 and H3N2 infection in 3D4/21 cells were confirmed in this study.And the important candidate molecules(miR-10391,TCONS?00166432 and novel?circ?0004733)regulating H1N1 and H3N2 infection in 3D4/21 cells were identified using qPCR and enzyme linked immunosorbent assay(ELISA).Finally,gene interference,qPCR,western blot and ELISA were used to identify the regulatory effect and possible molecular mechanism of the target gene MAN2A1 in the process of H1N1 and H3N2 infecting 3D4/21 cells.The main findings are as follows:1.Model establishment of swine influenza virus(H1N1 and H3N2)infecting alveolar macrophage cell line(3D4/21)Swine influenza viruses(H1N1 and H3N2)of different doses(1-fold TCID50,10-fold TCID50 and 100-fold TCID50)were used to infect 3D4/21 cells for different time(24h,48 h and 72 h).The results showed that at the same time point,the expression levels of M and NP genes in 3D4/21 cells infected with 100-fold TCID50 virus were higher than 10-fold and 1-fold TCID50(P<0.05).For the same virus dose,the expression levels of M and NP genes increased with time,and reached a significant level at 48 h(P<0.05 or P<0.01).At the same time point,the expression levels of virus-related genes(RIG-I,TLR7 and NLRP3)in 3D4/21 cells infected by 100-fold TCID50 virus were higher than 10-fold and 1-fold TCID50.In addition,compared with the cells in the virus-free group,the secretion levels of three cytokines(IFN-?,IFN-? and IFN-?)increased significantly after H1N1 and H3N2 infection(P<0.05 or P<0.01).And for 100-fold TCID50 virus,the three cytokines all increased at 48 h,and decreased at 72 h.Indirect immunofluorescence detection results showed that the expression of nucleocapsid protein NP was the strongest when H1N1 and H3N2 infect 3D4/21 cells for 48 h.In summary,infecting 3D4/21 cells with 100-fold TCID50 virus for 48 hours can make swine influenza viruses H1N1 and H3N2 replicate and proliferate better,and cause a strong immune response in host cells.Therefore,100-fold TCID50 virus was selected to infect 3D4/21 cells for 48 h to construct a cell model of swine influenza viruses(H1N1 and H3N2)infecting the porcine alveolar macrophage cell line(3D4/21),which aims to further analyze and explore the molecular mechanism of swine influenza virus infecting 3D4/21 cells.2.Whole transcriptome sequencing analysis of porcine alveolar macrophages(3D4/21)infected with swine influenza viruses(H1N1 and H3N2)In this study,the mRNA,lncRNA,circRNA and miRNA of 3D4/21 cells infected with swine influenza viruses H1N1 and H3N2 were identified.Compared with NC group,there were 119 are common differentially expressed in H1N1 group and H3N2 group.Six common differentially expressed genes(MAN2A1,DMN2,NUBPL,SLC27A1,HSPB8 and TBC1D10C)were identified by qPCR in this study.There were 57 lncRNAs are common differentially expressed in H1N1 group and H3N2 group,in which two common differentially expressed lncRNAs(TCONS?00166432 and TCONS?00087413)were identified by qPCR in this study.There were 22 circRNAs are common differentially expressed in H1N1 group and H3N2 group.Two common differentially expressed circRNAs(novel?circ?0004733 and novel?circ?0006303)were identified by qPCR in this study.There were 5 miRNAs are common differentially expressed in H1N1 group and H3N2 group,in which three common differentially expressed miRNAs(miR-10391,miR-450b-5p and novel?595)were identified by qPCR in this study.The expression profiles of related miRNA,mRNA,lncRNA and circRNA in porcine alveolar macrophages(3D4/21)infected with swine influenza viruses(H1N1 and H3N2)were systematically revealed at the whole transcriptome level for the first time in this study.3.ceRNA mechanism research of influenza A(H1N1 and H3N2)infecting 3D4/21 cellsIn this study,cytoplasmic RNA and nuclear RNA were extracted from 3D4/21 cells,and the expressions of TCONS?00166432 and novel?circ?0004733 in nuclear RNA and cytoplasmic RNA were detected by qPCR.It was found that 49.2%of TCONS?00166432 was expressed in the nucleus and 50.8%of TCONS?00166432 was expressed in the cytoplasm.12.9%of novel?circ?0004733 was expressed in the nucleus,and 87.1%of novel?circ?0004733 was expressed in the cytoplasm.In this study,target binding sites were searched through sequence alignment,and it was found that the seed sequence of miR-10391 completely matched the target gene MAN2A1.miR-10391 has a 5-base complementary pairing with novel?circ?0004733.miR-10391 and lncRNA TCONS?00166432 have a 7-base complementary pairing.The results suggest that they may have strong targeting relationship.The expression levels of miR-10391,MAN2A1,TCONS?00166432 and novel?circ?0004733 in 3D4/21 cells were detected by qPCR after cells were transfected by miR-10391-mimics,miR-10391-inhibitor,lnc-pcDNA3.1,lnc-shRNA,circ-pcDNA3.1 and circ-shRNA.The results showed that miR-10391 could negatively regulate the expression of MAN2A1 gene,TCONS?00166432 and novel?circ?0004733.And the expression trends of MAN2A1 gene and TCONS?00166432 are consistent.The results suggest that there may be targeting relationships between miR-10391 and MAN2A1 gene,TCONS?00166432 and novel?circ?0004733.In addition,direct effect of miR-10391 on the transcriptional activity of MAN2A1 gene,TCONS?00166432 and novel?circ?0004733 targeting binding sites were tested through dual luciferase activity in this study.The results confirmed that miR-10391 could target MAN2A1 gene,novel?circ?0004733 and TCONS?00166432.Finally,the results of the RNA immunoprecipitation experiment further confirmed that miR-10391 can target the MAN2A1 gene,and TCONS?00166432 could also target the MAN2A1 gene.4.The regulatory role of miR-10391,TCONS?00166432 and novel?circ?0004733 in the process of swine influenza virus(H1N1 and H3N2)infecting 3D4/21 cellsAfter the virus infects 3D4/21 cells,the relative expression levels of virus-related genes(RIG-I,TLR7 and NLRP3)in the mimics group cells were up-regulated,in which the expression of NLRP3 was significantly up-regulated(P<0.01).The relative expression levels of the three genes(RIG-I,TLR7 and NLRP3)in inhibitor group cells were down-regulated,in which the expression of TLR7 was significantly down-regulated(P<0.01).In addition,mimics treatment significantly down-regulated the secretion level of IFN-?(P<0.05).The results showed that the expression of miR-10391 does not directly regulate the replication and proliferation of influenza virus,but may participate in the intracellular immune response after swine influenza virus infection.Its high expression inhibits the strength of the immune response to a certain extent.Compared with the H3N2+group,the overexpression of TCONS?00166432 significantly up-regulated the expression level of NP in 3D4/21 cells(P<0.05),and the interference of TCONS?00166432 significantly down-regulated the expression level of NP.gene(P<0.05).In addition,the overexpression of TCONS?00166432 significantly down-regulated the expression level of NLRP3(P<0.01);the interference of TCONS?00166432 significantly up-regulated the expression level of TLR7(P<0.01).Finally,the overexpression of TCONS?00166432 significantly up-regulated the secretion levels of IFN-?and IFN-?(P<0.05),and the interference of TCONS?00166432 significantly down-regulated the secretion of IFN-?(P<0.05).These results suggest that the expression of TCONS?00166432 may directly regulate the replication of swine influenza virus,and the low expression of TCONS?00166432 may be beneficial to inhibit virus replication.In addition,TCONS?00166432 participates in regulating the immune response of host cells to a certain extent,and its low expression may beneficial to 3D4/21 cells to resist swine influenza infection.The overexpression of novel?circ?0004733 significantly up-regulated the expression levels of NP and M genes in virus-infected cells(P<0.05),and the interference of novel?circ?0004733 significantly down-regulated the expression levels of NP gene in H1N1 virus-infected cells(P<0.05).In addition,the overexpression of novel?circ?0004733 significantly down-regulated the expression level of RIG-I in H1N1 virus-infected cells(P<0.05),and the interference of novel?circ?0004733 significantly up-regulated RIG-I and TLR7(P<0.05).Finally,the overexpression of novel?circ?0004733 significantly up-regulated the secretion level of IFN-y(P<0.05),and the interference of novel?circ?0004733 significantly down-regulated the secretion level of IFN-?(P<0.05).The results showed that the expression of novel?circ?0004733 may directly regulate the replication of swine influenza virus,and the high expression of novel?circ?0004733 may be beneficial to virus replication and proliferation.In addition,novel circ?0004733 is involved in regulating the immune response of host cells to a certain extent,and its low expression may beneficial to 3D4/21 cells to resist swine influenza infection.5.The regulatory role of target gene MAN2A1 in the process of swine influenza virus(H1N1 and H3N2)infecting 3D4/21 cellsIn this study,MAN2A1 signaling pathway(N-glycan biosynthesis signaling pathway)genes and interaction protein genes-MAN1A1,MAN1A2,MGAT2 and MGAT3 were predicted combined the results of KEGG pathway analysis and protein interaction prediction websites.It was found that the expression levels of MAN2A1,MAN1A1,MAN1A2,MGAT2 and MGAT3 genes were significantly down-regulated after H1N1 and H3N2 infect 3D4/21 cells(P<0.05).The results suggest that 3D4/21 cells may resist swine influenza virus infection by inhibiting the expression of the entire N-glycan biosynthesis signal pathway.In addition,MAN2A1 interference significantly down-regulated the expression levels of downstream genes MGAT2 and MGAT3 in the N-Glycan biosynthesis signaling pathway(P<0.05).In addition,the interference of MAN2A1 directly significantly down-regulated the expression levels of M and NP(P<0.05).However,it had no significant effect on the expression levels of virus-related genes(RIG-I,TLR7 and NLRP3)in the cells after swine influenza virus infected 3D4/21 cells(P>0.05).The results suggest that the expression of MAN2A1 may mainly regulate the replication and proliferation of swine influenza virus.ELISA results showed that MAN2A1 interference significantly down-regulated the secretion level of IFN-? in cells after virus infection(P<0.05),suggesting that the expression of MAN2A1 affected the cellular immune response to a certain extent.It was found that tunicamycin treatment inhibited the expression of the entire N-Glycan biosynthesis signaling pathway genes(MAN2A1,MAN1A2,MGAT2 and MGAT2)(P<0.01),and significantly down-regulated the expression levels of M and NP.The results suggest that tunicamycin may regulate the replication and proliferation of swine influenza virus by blocking the N-Glycan biosynthesis signaling pathway,which is similar to the effect of MAN2A1 gene interference.It is suggested that the MAN2A1 gene plays an important regulatory role in the process of 3D4/21 resisting the infection of swine influenza viruses(H1N1 and H3N2),and the MAN2A1 gene may directly regulate virus replication and proliferation by mediating the N-Glycan biosynthesis signal pathway.Finally,three core promoter regions of the MAN2A1 gene were identified in this study,and the A/T mutation at the SNP site in the second core promoter region can significantly enhance the transcriptional activity of the MAN2A1 gene(P<0.05).The results suggest that the mutation site may affect the binding of transcription factors,and ultimately affect the expression of the MAN2A1 gene.Based on the above studies,it was the first time to report the ceRNA networks involved in the regulation of the swine influenza virus infecting 3D4/21 cells and determined its possible regulatory mechanism in this study,which provided new insights for revealing the molecular mechanism of 3D4/21 cells resisting swine flu virus infection.