miR-2478 inhibits TGFβ1 expression by targeting the transcriptional activation region downstream of the TGFβ1 promoter in dairy goats

In a previous study, miR-2478 was demonstrated to be up-regulated in dairy goat mammary glands during peak lactation compared with the dry period. However, the detailed mechanisms by which miR-2478 regulates physiological lactation and mammary gland development in dairy goats remain unclear. In this study, we used bioinformatics analysis and homologous cloning to predict the target genes of miR-2478 and selected INSR, FBXO11, TGFβ1 and ING4 as candidate target genes of miR-2478. Subsequently, by targeting the 5'UTR of the TGFβ1 gene, we verified that miR-2478 significantly inhibited TGFβ1 transcription and the Pearson's correlation coefficient between miR-2478 expression and TGFβ1 expression was -0.98. Furthermore, we identified the potential promoter and transcription factor binding regions of TGFβ1 and analyzed the potential mechanisms of interaction between miR-2478 and TGFβ1. Dual-luciferase reporter assays revealed that two regions, spanning from -904 to -690 bp and from -79 to +197 bp, were transcription factor binding regions of TGFβ1. Interesting, the miR-2478 binding sequence was determined to span from +123 to +142 bp in the TGFβ1 gene promoter. Thus, our results have demonstrated that miR-2478 binds to the core region of the TGFβ1 promoter and that it affects goat mammary gland development by inhibiting TGFβ1 transcription.