BLV-miRNAs Regulate The Prediction Of Human Target Genes And Influence On Gene Expression In Human Lymphocytes

Bovine leukemia virus(BLV)is one of the most common infectious viruses worldwide and encodes miRNA(BLV-miRNAs).Studies suggest that exosomal micro RNA(miRNA)transboundarily regulates gene expression through food intake.BLV-miRNAs possibly transboundarily regulates functions of human lymphocytes.We predicte humanized gene can be regulated BLV-miRNAs by Bioinformatics and the universality transboundarily regulation of miRNA as a point of entry.We chose one miRNA,BLV-miR-B4-3p,sharing seed sequences with miR-29 a,as the research objects to identify the impact on cytokine of human lymphocytes and gene expression.We first query mature BLV-miRNA sequences by miRbase and predicte target genes of BLVmiRNAs(BLV-miR-B1-3p,5p;BLV-miR-B2-3p,5p;BLV-miR-B3-3p,5p;BLV-miR-B4-3p,5p;BLV-miR-B5-3p,5p)by miRanda.We selecte the top ten candidate target gene with highest score and analyze functions.We again predicte the candidate target gene what be regulated by multiple miRNAs by RNAhybrid and analyze functions.We again predicte the candidate target gene what be regulated by multiple miRNAs by RNAhybrid and analyze functions.The transfection efficiency of BLV-miR-B4-3p mimics at different time points was determined by qPCR.RNA-Seq was performed on lymphocytes transfected with different concentrations of BLV-miR-B4-3p mimics.The sequencing results were analyzed by differential gene clustering,GO enrichment analysis and KEGG enrichment analysis and the prediction of BLV-miR-B4-3p target genes and the results of transcriptome analysis were verified by qPCR.Finally,the effect of BLV-miR-B4-3p on the transcription level of human lymphocyte immune-related cytokines(Th1 cytokines,Th2 cytokines and proliferation-activated cytokines)was analyzed.A total of 88 candidate target genes ranked in the top 10 were found to have no relevant function reported in 18 of them after functional analysis;thirty-six candidate target genes were correlated with the occurrence and development of neoplastic diseases;two candidate target genes could regulate the cell cycle;sixteen candidate target genes were involved in the regulation of cell signal transduction;fourteen candidate target genes play a role in the formation of cell structure/skeleton proteins;thirteen candidate target genes could regulate cell proliferation and apoptosis;two candidate target genes could regulate cell differentiation and sixteen candidate target genes played a regulatory role in cell migration/invasion function.It is suggested that BLV-miRNA may have a more important association with neoplastic diseases.The candidate target genes that can be jointly targeted by multiple BLV-miRNAs all belong to the MUC family(MUC5B,MUC12 and MUC16)and can be expressed in the colon,influencing the formation of colonic mucosa.The 7 candidate target genes may play an important role in the differentiation,migration and invasion of breast cells,suggesting that the study on the correlation between BLV and human breast cancer can be further explored from the perspective of BLV-miRNA.The candidate target genes COL1A1 and BCR of BLV-miRB4-3p have a regulatory effect on ALL.RNA Seq analysis showed that 556 differentially expressed genes were obtained after BLV-miR-B4-3p entered lymphocytes.GO enrichment and KEGG analysis results show the BLV-miR-B4-3p can separate activation of influenza a virus infection,measles infection pathway,EB virus infection,herpes simplex virus infection pathways,metabolic pathway,toll-like receptors signaling pathways,such as signaling pathways,ultimately to the body's immune system process,stress reaction,response to organic matter and defense reaction,immune response and other biological processes.After BLV-miR-B4-3p enters lymphocytes,it can lead to abnormal lymphocyte immune function,including the significantly increased expression of TNF? mRNA in Th1 cytokines(P < 0.05),the expression of IL-10 mRNA in Th2 cytokines was significantly decreased(P < 0.05),IL-27 mRNA was significantly increased(P <0.001),the mRNA expression level of lymphocyte proliferation and activation related regulatory factors was not affected,tumor suppressor BRCA1 and antimicrobial peptide c AMP were significantly decreased(P < 0.001),the expression of pro-apoptotic factor Caspase9 decreased significantly(P < 0.05).In conclusion,BLV-miRNAs have the risk of transboundary regulation of human cell structure and gene expression related to tumor diseases.BLV-miR-B4-3p can activate multiple viral infection pathways in human lymphocytes,lead to abnormal transcription of cytokines,and affect the immune function of the body.