Mechanism Of MiRNA Inhibiting The Replication Of Caprine Parainfluenza Virus Type 3

Caprine parainfluenza virus type 3（CPIV3）,a member of the Respirovirus genus of the Paramyxiviridae family,is an enveloped,single-stranded negative sense RNA virus.CPIV3can be transmitted horizontally through aerosol,so that adjacent healthy goats will be affected by infected goats,presenting these clinical symptoms with cough,high body temperature,runny nose,depression.Goats infected can shed virus and viremia can be detected,which makes it more difficult to prevent and control the infection.Evidence has shown that miRNA in host cells is an indispensable regulator in complex networks of host-pathogen interactions.MiRNA plays an important role in antiviral infection and replication by indirectly acting on a cell component to influence intracellular pathways and regulates innate immune responses.After CPIV3 was inoculated into MDBK cells,249miRNA expressed differentially were obtained by high-throughput sequencing.Among them,bta-miR-98 and bta-miR-222 were down-regulated in the infected group.GO annotation and KEGG enrichment showed that bta-miR-222 plays an important role in antiviral and congenital immunity,and bta-miR-98 affects viral replication by regulating cell apoptosis.In this study,we will carry out the work about molecular mechanism of inhibiting CPIV3replication by transfecting bta-miR-98 and bta-miR-222 mimics in vitro to provide theoretical basis for the prevention and control of the viral disease.1.Molecular mechanism of bta-miR-222 targeting IRF2 inhibiting the replication of CPIV3In order to investigate the effect of bta-miR-222 on CPIV3 replication and its mechanism,the bta-miR-222 mimics were transfected into MDBK cells,inoculated with CPIV3 virus solution,the supernatant and cells were collected,and the virus titer and IFN content were determined.The target genes regulated by bta-miR-222 were predicted by bioinformatics software such as TargetScan and miRBase.The target genes were verified by double luciferase reporting system.The effect of target genes on CPIV3 replication was studied by RNA interference technique.The results showed that bta-miR-222 mimics could inhibit CPIV3 replication,and the expression level of IFN in bta-miR-222 group was higher,and the virus replication titer(10^2.5TCID₅₀/0.1mL)was significantly lower than that in miRNA negative control group(10^3.3TCID₅₀/0.1mL).The results of RT-qPCR and western blot showed that bta-miR-222 could target 3′UTR of IRF2,degrade mRNA of IRF2 and inhibit the expression of IRF2 protein.After CPIV3 infection with MDBK cells knockout IRF2,the viral replication titer(10^2.0TCID₅₀/0.1mL)was lower than that of normal MDBK cells(10^3.2 TCID₅₀/0.1mL).The results showed that bta-miR-222 could inhibit CPIV3 replication by targeting the degradation of IRF2 mRNA to inhibit CPIV3 replication,which provided basic data for the development of potential antiviral drugs based on bta-miR-222.2.Molecular mechanism of bta-miR-98 targeting Caspase 3 inhibiting the replication of CPIV3In order to detect the effect of CPIV3 infection on apoptosis of MDBK cells,CPIV3 was inoculated with MDBK cells for 24 hours,and the mRNA expression levels of Caspase 3,Caspase 8 and Caspase 9 were detected by RT-qPCR.The level of apoptosis activity of Caspase 3,Caspase 8 and Caspase 9 were detected by flow cytometry.MDBK cells were inoculated with CPIV3,for 24 hours after chemical synthesis of bta-miR-98 mimics and bta-miR-98 inhibitor in vitro.CPIV3 mRNA expression levels and titers were detected by RT-qPCR and TCID₅₀,and Caspase 3 enzyme activity was detected by kit.The level of apoptosis was detected by flow cytometry.TargetScan and miRBase bioinformatics software were used to predict the target gene of bta-miR-98.The target gene was verified by double luciferase reporting system.The effect of target gene on CPIV3 replication was studied by RNA interference technique.The results showed that the mRNA expression levels of Caspase 8 and Caspase 9 in Caspase 3 group were about 2.5 times higher than those in normal cells,the activity of Caspase 3 enzyme in exposure group was 3 times higher than that in control group,and the activities of Caspase 8 and Caspase 9 enzyme in exposure group were higher than those in control group.The apoptosis rate of the exposed group（9.81%）was higher than that of the normal cells（2.93%）.The above data suggest that CPIV3 can promote apoptosis.The results showed that bta-miR-98 mimics could inhibit the increment of CPIV3,and the viral replication titer(10^2.7 TCID₅₀/0.1mL)was lower than that of miRNA negative control(10^3.3TCID₅₀/0.1mL).Bta-miR-98 inhibitor could increase the value of CPIV3,and the viral replication titer(10^3.5 TCID₅₀/0.1mL)was higher than that of miRNA negative control(10^3.0TCID₅₀/0.1mL).Bta-miR-98 mimics could effectively inhibit the apoptosis of MDBK cells.The apoptosis rate in the transfer group（24.63%）was lower than that in the miRNA negative control group（28.07%）and the normal cell exposure group（28%）.MiR-98 inhibitor can effectively promote apoptosis of MDBK cells.The apoptosis rate in the transfer group（32.37%）was higher than that in the miRNA negative control group（27.4%）and the normal cell exposure group（28%）.TargetScan predicted that bta-miR-98 could target the 3′UTR,double luciferase reporting system combined with Caspase 3.The results of RT-qPCR and western blot showed that bta-miR-98 could target the 3′UTR of Caspase 3,degrade the mRNA of Caspase 3 and inhibit the expression of Caspase 3 protein.After CPIV3 infection with MDBK cells knockout Caspase 3,the viral replication titer(10^2.5 TCID₅₀/0.1 mL)was lower than that of normal MDBK cells(10^3.2 TCID₅₀/0.1 mL).The results showed that bta-miR-98 could inhibit CPIV3 replication by targeting Caspase 3 to degrade its mRNA expression level,thus inhibiting apoptosis.3.Construction and identification in MDBK cell line of overexpressing stably bta-miR-222 mediated by lentivirusIn order to obtain MDBK cell line stably expressing bta-miR-222,pri-bta-miR-222fragment was amplified from MDBK cell genome by PCR technique,GFP fragment was amplified from pEGFP-N1 plasmid,and the target fragment was recovered b y glue.First cloned into pMD18-T vector,transformed into E.coli,identified by enzyme digestion and sequencing correctly,and then cloned into PCDH-CMV-MCS-EF1-Puro vector to construct miRNA lentivirus recombinant plasmid PCDH-miR-222-GFP.containing bta-miR-222 gene.Recombinant lentivirus V_PCDH-miR-222.containing bta-miR-222 gene was prepared by co-transfection of PCDH-miR-222-GFP and two packaging plasmid PSP,PMD into 293T cells.The prepared lentivirus V_PCDH-miR-222 was inoculated into MDBK cells and screened by puromycin.The infection efficiency was observed by fluorescence microscope.The results showed that the overexpression plasmid of PCDH-miR-222-GFP was constructed successfully and the coincidence rate of sequence alignment was 100%.After co-transfection of PCDH-miR-222-GFP and PSP,PMD into 293T cells,a large amount of fluorescence was observed by fluorescence microscope,which indicated that the recombinant lentivirus V_PCDH-miR-222,was successfully constructed after 10 generations of Puromycin puromycin screening.V_PCDH-miR-222 still has infection efficiency in MDBK cells.The results showed that the MDBK cell line stably expressing bta-miR-222 was successfully obtained,which laid a foundation for the next step to study the function of b ta-miR-222 in MDBK cells infected with virus.