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

Abstract

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, CEBPβ, 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.

Keywords: Smad3; mammary epithelial cells; miR-143; triglyceride.

Figure 1

Effects of miR-143 mimic on milk fat synthesis. (A) miR-143 expression following miR-143 mimic treatment; (B) triglyceride levels in bovine mammary epithelial cells (BMECs) after treatment with the miR-143 mimic; (C) alteration in the number of lipid droplet in BMECs following miR-143 mimic treatment. ** p < 0.01.

Figure 2

Effects of miR-143 inhibitor on milk fat synthesis. (A) miR-143 expression following miR-143 inhibitor treatment; (B) triglyceride levels in BMECs after treatment with the miR-143 inhibitor; (C) alterations in the number of lipid droplet in BMECs after miR-143 inhibitor treatment * p < 0.05; ** p < 0.01.

Figure 3

Effects of miR-143 on the expression of the genes related to lipid metabolism. (A) BMECs were transfected with miR-143 mimic; (B) BMECs were transfected with miR-143 inhibitor. * p < 0.05, and *** p < 0.001.

Figure 4

Impact of miR-143 on the Smad3 mRNA and protein expression. ** p < 0.01.

Figure 5

(A) Sequence alignment of miR-143 and the 3′-untranslated region (UTR) of Smad3 based on the TargetScan and miRDB algorithms; (B) changes in luciferase activity after the BMECs were co-transfected with miR-143 mimics and a luciferase reporter with a fragment of the Smad3 3′-UTR harboring either the miR-143 binding site (Smad3-3′UTR-WT) or a mutant (Smad3-3′UTR-MUT). * p < 0.05. SV40 = Simian virus 40; poly A = polyadenylic acid; BTA = Bos taurus.

Figure 6

Effects of Smad3 on milk fat synthesis. (A,B) Comparative expression of Smad3 mRNA and protein in BMECs transfected with the si-Smad3 and negative control for 48 h. (C) Triglyceride levels in BMECs after treatment with the si-Smad3. ** p < 0.01.