The Regulatory Role Of MiR-107 In Coxsackie B3 Virus Replication

Objective:To compare the expression of miR-107 during CVB3 infection in Hela cells,and explore the regulatory role and mechanism of miR-107 on CVB3 replication,hoping to provide new theoretical basis and new biology for the clinical diagnosis of CVB3 infection.landmark.Method:1.Screen miRNAs associated with CVB3 infection.Using uninfected Hela cells as a control,RT-qPCR was used to determine the differential expression of miR-107 over time.2.After overexpression or silencing of miR-107 in Hela cells,the Hela cells was infected with CVB3,the transfection efficiency was detected by RT-qPCR,and the total protein infected up to 6h was collected.Western blot was used to detect the expression of the viral capsid protein VP1,and analysis of miR-107 Effect on CVB3 replication and proliferation.The supernatant was collected for virus plaque experiments,and the effect of miR-107 on the release of CVB3 was analyzed.3.The target genes KLF4 and BACE1 of miR-107 were screened through TargetScan and miRTarBase databases,and target verification was performed using a luciferase reporter vector experiment.4.Hela cells were over-expressed or silenced the target gene and infected with CVB3 for6 h,then we collected total RNA and total protein.RT-qPCR and Western blot were used to analyze the change of target gene expression and the effect of target gene on CVB3 replication and proliferation.Meanwhile,the supernatant was collected for virus plaque experiments.To analyze the effect of target genes on the release of virus CVB3.5.Hela cells were transfected with the upstream lncRNA004787 plasmid of miR-107,RT-qPCR was used to detect the overexpression efficiency,and the total protein was collected after Hela cells were infected with CVB3 for 6h.Capsid protein VP1 was detected by Western blot to analyze the effect of CVB3 replication and proliferation.Viral plaque assay was used to analyze lncRNA004787 on Effects of viral CVB3 release.Result:1.After CVB3 infection of Hela cells,the level of miR-107 gradually increased,the highest at 6 hours.CVB3 capsid protein VP1 can be detected by Western blot in 4 hours.2.Hela cells were infected with CVB3 for 6 hours after miR-107 was overexpressed.Compared with the control group,VP1 expression and the number of viral plaques increased,and cell shrinkage increased under the microscope.When miR-107 was knocked down,both VP1 expression and viral plaques decreased.3.The database predicts that miR-107 targets the 3'UTR of KLF4 and BACE1.The double luciferin experiment found that the expression of fluorescein in the targeted group was lower than that in the control group,and the expression of luciferin in the mutant group was not significantly changed compared to the control group.The results showed that miR-107 has a targeting effect on BACE1.4.Hela cells were infected with CVB3 for 6 hours after KLF4 or BACE1 was overexpressed,the expression of VP1 protein was reduced,and the number of viral plaques was reduced.When hela cells were infected with CVB3 for 6 hours after KLF4 or BACE1 was knockdowned,VP1 protein expression was increased,and the number of viral plaques was increased.5.Hela cells were overexpressed with lncRNA004787 and infected with CVB3 for 6 hours.The VP1 protein expression and the number of viral plaques in the experimental group were lower than those in the control group.After knocking down lncRNA004787,compared with the control group,the expression of VP1 protein and the number of viral plaques in the experimental group decreased.Conclusion: MiR-107 partially promotes the replication and proliferation of CVB3 by negatively regulating the expression of target genes KLF4 and BACE1.