Study On The Molecular Mechanism Of MiR-122 Inhibiting PCV2 Replication

As a common virus,porcine circovirus produced serious harmness to pig healthy production.Porcine circovirus virus type 2(PCV2)can cause post weaning multisystemic wasting syndrome(Post weaning multisystemic wasting syndrome,PMWS)and congential tremors(Congential Tremors,CT)related diseases,which made damage to the body's immune cells and caused a variety of pathogenic mixed infection,causing severe clinical disease.Porcine circovirus mainly harms fattening pigs and suckling piglets,causing huge economic losses to the world's pig industry.Micro RNAs(miRNAs)are a class of endogenous factors that regulate gene expression at the post-transcriptional level.miRNA play a huge role in biological growth and development,cell differentiation,cell proliferation and apoptosis,disease occurrence and immunity.In recent years,the analysis of the molecular mechanism of miRNA and its regulation of PCV2 replication has gradually become a research hot topic.In this study,based on the previous work of our laboratory that ssc-miR-122 could inhibit the PCV2 replication,we obtained stable cell lines expressing ssc-miR-122 by lentiviral packaging,further verified that the ssc-miR-122 can inhibit PCV2 replication;and through the full-length transcriptome sequencing and bioinformatics analysis on the gene expression differences of PCV2-infected ssc-miR-122 stable cell lines,we revealed the function of differentially expressed genes and expression regulation function.This study laid a foundation for elucidating the molecular mechanism of miR-122 inhibiting PCV2 replication.The main findings are as follows:1.Construction of miR-122 stable cell lines.293 T cells were used to package lentivirus and transfect PK15 cells and 3D4/21 cells.The PK15 control cell line,PK15-miR-122 overexpression,PK15-miR-122 interference stable transgenic cell line and 3D4/21 control cell line,3D4/21 miR-122 overexpression stable transgenic cell line were screened by purinomycin,the expression level of miR-122 was detected by RT-q PCR to verify the constructed stable transgenic strains,and finally the cell lines with stable expression of miR-122 were obtained.2.Analyzed the regulation of miR-122 on PCV2 replication.The copy number of PCV2 infected with PK15 and 3D4/21 stable transgenic strains was detected.The results showed that the DNA copies of PCV2 was significantly reduced(P < 0.01)after overexpression of miR-122 by RT-q PCR.The expression difference of PCV2 virus protein after overexpression of miR-122 was analyzed,and the results showed that the expression level of Rep protein of PCV2 was significantly down-regulated(P < 0.05)after overexpression of miR-122.After knockdown of miR-122 expression in PK15 cells,the DNA copies and viral Rep protein expression were significantly increased(P < 0.05).The expression levels of ORF1?ORF5were significantly down-regulated after PCV2 infected(P < 0.05).All the above results indicated that miR-122 could significantly inhibit the replication of PCV2.3.The effects of miR-122 in PK15 cells on cell proliferation and apoptosis during PCV2 infection were detected.CCK8 and flow cytometry were used to detect the proliferation and apoptosis rate of PK15-miR-122 overexpressed strain cells after PCV2-infected.The results showed that miR-122 could promote the proliferation(P < 0.05)and inhibit the apoptosis(P <0.01)of PK15 cells after PCV2 infection.4.Nanopore full-length transcriptome sequencing.PK15 stable transgenic cells were divided into four groups: group A was PK15-control group,group B was PK15-miR-122 overexpression group,group C was PK15 control PCV2-infected group,and group D was PK15-miR-122 overexpression PCV2-infected group for RNA-seq.Firstly,differences between group A and B were analyzed,that is differences in gene expression caused by miR-122 without PCV2-infected.214 differential transcripts were identified,and the differential transcripts were mainly enriched in signaling pathways such as3'UTR binding pathway and apoptosis.Secondly,differences in group C and D(excluding the same as those in group A and B)were analyzed to eliminate the interference caused by differential expression of miR-122 before PCV2-infected,analysed the expression differences of miR-122 only under the condition of PCV2 infection.A total of 159 differential transcripts were identified,and the differentially expressed transcripts were mainly enriched in the signaling pathways such as immune response and chemokines.The differences between the B and D groups(excluding the same as A and C)were analyzed to eliminate the interference of the expression differences caused by the control group with PCV2-infected,analyzed the expression differences caused by the regulation of miR-122 by PCV2-infected.A total of 103 differential transcripts were screened and enriched into the signaling pathways such as chemokines and inflammatory factors.Finally,through screening analysis,we eliminated the interference caused by miR-122 without PCV2-infected and the differences in gene expression caused by control group with PCV2-infected,identified the differences in gene expression caused by miR-122 and PCV2 infection,and finally screened out 8 differential genes: HMGB2,ERCC3,UBE2 D,CXCL2,CCL2,Novel1241,CEBPZOS and RAB4 B.Protein interaction analysis between the 8 genes and miR-122 target genes revealed that there may be interactions between CCL2,CXCL2,IL1R1 and CANX proteins,which need to be further verified.5.Ten differential genes related to inflammation and immune response were screened for quantitative verification,and the quantitative results showed that the expression trend of candidate genes was consistent with the results of sequencing analysis.In conclusion,cell lines stably expressing miR-122 were successfully constructed in this study and the inhibitory effect of miR-122 on PCV2 was verified.The expression profile changes of miR-122 overexpression and PCV2 infected were obtained by transcriptome sequencing,and the key genes of the interaction between miR-122 and PCV2 were identified,which laid a foundation for elucidating the molecular mechanism of miR-122 inhibiting PCV2 replication,and at the same time provided an important theoretical basis for elucidating the pathogenic mechanism of PCV2.