Abstract: [Objective] To validate the targeting relationship between bta-miR-19b and the B-cell CLL/lymphoma 3 （BCL3） gene and to elucidate the molecular function and regulatory mechanism by which bta-miR-19b mediates immune regulation in bovine colostrum. [Methods] The intersection of TargetScan and miRanda predictions was used to screen the immune-related target genes of bta-miR-19b, and the minimum free energy （MFE） of binding between bta-miR-19b and immune-related candidate genes IL7R, EGFR, TNF, BCL3, NME7, MAPK8 and MAP3K1 was predicted using the RNAhybrid website. The wild-type （WT） and mutant （MT） fragments were synthesized using the predicted binding site of the 3′UTR of the BCL3 gene as template to construct pmirGLO luciferase reporter vectors. The bta-miR-19b mimics （chemically synthesized double-stranded RNA molecules that mimic endogenous miRNA functions） and NC control （NC-mimics） were synthesized and co-transfected with the reporter vector into 293T cells for target validation. The biological processes and key pathways of bta-miR-19b-BCL3 axis in the immune regulation of bovine colostrum were screened by bioinformatics methods. [Results] The binding free energy of bta-miR-19b with BCL3 was the lowest, suggesting that there was a potential binding of BCL3. The results of dual luciferase reporter gene assay confirmed that bta-miR-19b could specifically bind to the 3′UTR region of the BCL3 gene. The bta-miR-19b-BCL3 regulatory axis was enriched in 42 GO terms, of which 24 were immune-related biological processes. The KEGG pathways involving the bta-miR-19b-BCL3 regulatory axis included tumor necrosis factor TNF signaling pathway and C-type lectin receptor signaling pathway. [Conclusion] bta-miR-19b can target the 3′UTR of BCL3. Given that BCL3 is a regulatory factor of the NF-κB signaling pathway, it is speculated that the bta-miR-19b-BCL3-NF-κB-TNF regulatory axis may be one of the core molecular mechanisms for bovine colostrum to exert immune function, and the specific mechanism needs to be validated by functional experiments.

Key words: bta-miR-19b, BCL3, double luciferase reporter gene assay, bovine colostrum, immune regulation