Mechanism Of MiR-92a-1-5p Targeted With SOCS5 In Inhibiting Feline Panleukopenia Virus Replication

Feline panleukopenia is an acute and highly exposed infectious disease caused by feline panleukopenia virus?FPV?.At present,the prevention of this disease is mainly based on vaccination.MicroRNAs?miRNAs?are endogenous small non-coding RNAs with about 18-25 nucleotides in length.A large number of studies have shown that miRNAs can affect many cellular functions and play a major role in regulating interaction between host and pathogen.However,the effects of miRNAs on FPV infection and the mechanism of interaction between FPV and miRNAs have not been reported.To explore the mechanism underlying the involvement of miRNAs in the FPV infection,RNA-seq was used to detect miRNAs expression profiles changes before and after FPV infection in F81 cells and two small RNA databases were obtained.We identified 792 known miRNAs and 271 new miRNAs matures,which includes 57 miRNAs with significantly different expressions.The stem-loop qRT-PCR method was used to identify expression levels of 15 randomly selected miRNAs.The results showed that the differential levels of 14 miRNAs were consistent with the RNA-seq results.9 miRNAs were screened and synthesized for research according to the RNA-seq results and relevant literatures.MiRNA mimic or miRNA control?MC?was transfected into F81 cells and then inoculated with FPV.Subsequently,qRT-PCR and western blot assays were used to detect the effect of screened miRNAs on FPV replication ability.The results showed that miR-92a-1-5p can significantly reduce the FPV genomic DNA and viral protein expression level.Furthermore,the inhibition of miR-92a-1-5p for FPV replication was also verifies using qRT-PCR,western blot,IFA and TCID₅₀.IFN-I can effectively inhibit virus replication.So how FPV infection regulates host miRNA expression?What is the relationship between host miR-92a-1-5p inhibiting FPV replication and IFN-I,and what is the mechanism?Related researches have been conducted on the above issues,and results are as follows:Transcription factor SP1 upregulates miR-92a-1-5p transcriptional level.In order to investigate the transcriptional regulation mechanism of miR-92a-1-5p,the upstream gene sequences of miR-92a-1-5p was found and truncated,and the core sequences region was identified.The important transcription factors of miR-92a-1-5p coresequences region were analyzed using Patch and SIGNAL SCAN.Overexpression and knockdown experiments found that the SP1 is involved in the activation of the core promoter of miR-92a-1-5p.The study found that endogenous SP1 expression levels were upregulated with FPV infection.These results indicate that SP1 upregulates miR-92a-1-5p transcriptional level.miR-92a-1-5p upregulates IFN-I by activating NF-?B pathway to inhibit FPV infection.The expression levels of IFN-?/?and STATs were increased after F81 cells transfecting with miR-92a-1-5p.We found that miR-92a-1-5p could significantly enhance the luciferase activity of NF-?B and ISRE.NF-?B inhibitors can significantly inhibit the promotion of miR-92a-1-5p for IFN-?,IFN-?and ISG15expression.Further research found that miR-92a-1-5p promoted p65 nuclear translocation and phosphorylation,indicating that the NF-?B pathway was activated.Targetscan and miRanda predicted that SOCS5 was the target gene of miR-92a-1-5p and miR-92a-1-5p can downregulate the expression of SOCS5.In summary,this study constructed the miRNAs databases of F81 cells before and after FPV infection.The important transcription factor SP1 that regulates miR-92a-1-5p expression was preliminary discovered,and the upstream regulatory mechanism of miR-92a-1-5p was revealed.Confirmed that host miR-92a-1-5p can activate the NF-?B pathway by regulating SOCS5 gene expression which can inhibit FPV replication.The results of this study elucidate the mechanism of FPV interaction with host cells from the perspective of miRNAs,and provide new evidence for elucidating the pathogenic mechanism of FPV.