Lnc-TRTMFS promotes milk fat synthesis via the miR-132x/RAI14/mTOR pathway in BMECs.

As an important index to evaluate the quality of milk, milk fat content directly determines the nutrition and flavor of milk. Recently, growing evidence has suggested that long noncoding RNAs (lncRNAs) play important roles in bovine lactation, but little is known about the roles of lncRNAs in milk fat synthesis, particularly the underlying molecular processes. Therefore, the purpose of this study was to explore the regulatory mechanism of lncRNAs in milk fat synthesis. Based on our previous lncRNA-seq data and bioinformatics analysis, we found that Lnc-TRTMFS (transcripts related to milk fat synthesis) was upregulated in the lactation period compared to the dry period. In this study, we found that knockdown of Lnc-TRTMFS significantly inhibited milk fat synthesis, resulting in a smaller amount of lipid droplets and lower cellular triacylglycerol levels, and significantly decreased the expression of genes related to adipogenesis. In contrast, overexpression of Lnc-TRTMFS significantly promoted milk fat synthesis in bovine mammary epithelial cells (BMECs). In addition, Bibiserv2 analysis showed that Lnc-TRTMFS could act as a molecular sponge for miR-132x, and retinoic acid induced protein 14 (RAI14) was a potential target of miR-132x, which was further confirmed by dual-luciferase reporter assays, quantitative reverse transcription PCR, and western blots. We also found that miR-132x significantly inhibited milk fat synthesis. Finally, rescue experiments showed that Lnc-TRTMFS could weaken the inhibitory effect of miR-132x on milk fat synthesis and rescue the expression of RAI14. Taken together, these results revealed that Lnc-TRTMFS regulated milk fat synthesis in BMECs via the miR-132x/RAI14/mTOR pathway.

Milk fat is an important index to evaluate the quality of milk. The content of milk fat directly determines the quality and flavor of milk. Studies have shown that milk components can change with the expression of specific genes and noncoding RNA that regulate it in different lactation periods. In this study, after the interference and overexpression of Lnc-TRTMFS on milk fat metabolism in bovine mammany epithelial cells, we found that Lnc-TRTMFS could positively regulate milk fat synthesis in bovine mammary epithelial cells. The ceRNA network of Lnc-TRTMFS-miR-132x-RAI14 was constructed by software prediction and double fluorescein report test, and the salvage effect of Lnc-TRTMFS on milk fat synthesis was confirmed by salvage test. Most importantly, we found that Lnc-TRTMFS and miR-132x can regulate milk fat by regulating the mTOR pathway by regulating RAI14.

MiR-143 Regulates Milk Fat Synthesis by Targeting Smad3 in Bovine Mammary Epithelial Cells.

Milk fat is the main nutritional component of milk and is also an important indicator for evaluating milk quality. Substantial evidence has implicated miRNAs in the synthesis of milk fat. miR-143 is one of the miRNAs closely related to lipid metabolism. Herein, miR-143 upregulation remarkably promoted the production of lipid droplets and increased the level of intracellular triglyceride (TAG). Meanwhile, miR-143 suppression overtly repressed TAG synthesis and lipid droplet accumulation in bovine mammary epithelial cells (BMECs). At the same time, miR-143 significantly upregulated the genes associated with lipid synthesis, including PPARγ, SCD1, CEBPß, and SREBP1. To examine the regulatory mechanism of miR-143 in milk fat synthesis, Smad3 was predicted as a new potential miR-143 target gene by TargetScan. Further studies found that miR-143 expression was inversely related to the levels of Smad3 mRNA and protein. Furthermore, luciferase reporter assays confirmed Smad3 to be a miR-143 direct target. Moreover, Smad3 gene silencing significantly increased intracellular TAG level in BMECs. These findings revealed that miR-143 promotes the TAG synthesis in BMECs via increasing the lipid synthesis related gens expression by targeting Smad3. The results of this study can be exploited in devising novel approaches for improving the nutritional value of milk in dairy cows.