Sterol regulatory element-binding proteins (SREBPs) belong to a family of nuclear transcription factors. The question of which is the most important positive regulator in milk fat synthesis in dairy cow mammary epithelial cells (DCMECs) between SREBPs or other nuclear transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ), remains a controversial one. Recent studies have found that mTORC1 (the mammalian target of rapamycin C1) regulates SREBP1 to promote fat synthesis. Thus far, however, the interaction between the SREBP1 and mTOR (the mammalian target of rapamycin) pathways in the regulation of milk fat synthesis remains poorly understood. This study aimed to identify the function of SREBP1 in milk fat synthesis and to characterize the relationship between SREBP1 and mTOR in DCMECs. The effects of SREBP1 overexpression and gene silencing on milk fat synthesis and the effects of stearic acid and serum on SREBP1 expression in the upregulation of milk fat synthesis were investigated in DCMECs using immunostaining, Western blotting, real-time quantitative PCR, lipid droplet staining, and detection kits for triglyceride content. SREBP1 was found to be a positive regulator of milk fat synthesis and was shown to be regulated by stearic acid and serum. These findings indicate that SREBP1 is the key positive regulator in milk fat synthesis.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate the expression of target genes at the post-transcriptional level by transcript degradation or translational inhibition. The role of bta-miR-15a in bovine mammary gland hasn’t been reported. Using miRNAs prediction software, GHR gene was predicted to be a potential target of bta-miR-15a. In this study, bovine mammary epithelial cell line was used as an in vitro cell model to address the function of bta-miR-15a on bovine mammary epithelial cells. The expression changes of bta-miR-15a and Ghr after bta-miR-15a transfection were detected by qRT-PCR; the expression of GHR protein and casein was detected by western blotting. To determine whether bta-miR-15a can affect cell viability, cells were examined using an electronic Coulter counter (CASY-TT). In conclusion, bta-miR-15a inhibited the expression of casein of bovine mammary epithelial cells, and cell number and viability were reduced by bta-miR-15a expression. Bta-miR-15a inhibited the viability of mammary epithelial cells as well as the expression of GHR mRNA and protein level, therefore suggesting that bta-miR-15a may play an important role in mammary gland physiology.

The mammary gland undergoes intensive remodeling during the lactation cycle, and the involution process of mammary gland contains extensive epithelial cells involved in the process of autophagy. Our studies of mice mammary glands suggest that miR-30a-3p expression was low during involution compared with its high expression in the mammary glands of lactating mice. Then, we revealed that miR-30a-3p negatively regulated autophagy by autophagy related 12 (Atg12) in mouse mammary gland epithelial cells (MMECs). Restoring ATG12, knocking down autophagy related 5 (Atg5), starvation, and Rapamycin were used to further confirm this conclusion. Overexpression of miR-30a-3p inhibited autophagy and altered mammary structure in the involution of the mammary glands of mice, which was indicative of alteration in mammary remodeling. Taken together, these results elucidated the molecular mechanisms of miR-30a-3p as a key induction mediator of autophagy by targeting Atg12 within the transition period between lactation and involution in mammary glands.