Abstract
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.
Highlights
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
The present study provides evidence that miR-2478 binds to the region of transcriptional activity downstream of the TGFβ1 promoter and that it might synergistically regulate the inhibition of TGFβ1 via the transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBPJ) to affect goat mammary gland development
We examined changes in TGFβ1 mRNA expression in dairy goat primary mammary gland epithelial cells after transfection of a miR-2478 mimic or inhibitor for 24, 48 or 72 h. quantitative PCR (qPCR) assays showed that the expression of mature miR-2478 was significantly higher at 24 and 72 h after transfection of the miR-2478 mimic than that in control cells (P < 0.01), indicating that the miR-2478 mimic was successfully transfected into the mammary gland epithelial cells (Fig. 2C)
Summary
MiR-2478 was demonstrated to be up-regulated in dairy goat mammary glands during peak lactation compared with the dry period. We identified the potential promoter and transcription factor binding regions of TGFβ1 and analyzed the potential mechanisms of interaction between miR2478 and TGFβ1. We predicted and screened four genes (insulin receptor, INSR; F-box protein 11, FBXO11; transforming growth factor beta 1, TGFβ1; and inhibitor of growth family member 4, ING4) as candidate targets of dairy goat miR-2478. We identified the potential promoter and transcription factor binding regions of TGFβ1 and examined the potential mechanisms of the miR-2478/TGFβ1 interaction. The present study provides evidence that miR-2478 binds to the region of transcriptional activity downstream of the TGFβ1 promoter and that it might synergistically regulate the inhibition of TGFβ1 via the transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBPJ) to affect goat mammary gland development
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