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MicroRNA-199a-3p regulates proliferation and milk fat synthesis of ovine mammary epithelial cells by targeting VLDLR.

ABSTRACT: In our previous study, microRNA (miR)-199a-3p was found to be the most upregulated miRNA in mammary gland tissue during the non-lactation period compared with the peak-lactation period. However, there have been no reports describing the function of miR-199a-3p in ovine mammary epithelial cells (OMECs) and the biological mechanisms by which the miRNA affects cell proliferation and milk fat synthesis in sheep. In this study, the effect of miR-199a-3p on viability, proliferation, and milk fat synthesis of OMECs was investigated, and the target relationship of the miRNA with very low-density lipoprotein receptor (VLDLR) was also verified. Transfection with a miR-199a-3p mimic increased the viability of OMECs and the number of Edu-labeled positive OMECs. In contrast, a miR-199-3p inhibitor had the opposite effect with the miR-199a-3p mimic. The expression levels of three marker genes were also regulated by both the miR-199a-3p mimic and miR-199-3p inhibitor in OMECs. Together, these results suggest that miR-199a-3p promotes the viability and proliferation of OMECs. A dual luciferase assay confirmed that miR-199a-3p can target VLDLR by binding to the 3'-untranslated regions (3'UTR) of the gene. Further studies found a negative correlation in the expression of miR-199a-3p with VLDLR. The miR-199a-3p mimic decreased the content of triglycerides, as well as the expression levels of six milk fat synthesis marker genes in OMECs, namely, lipoprotein lipase gene (LPL), acetyl-CoA carboxylase alpha gene (ACACA), fatty acid binding protein 3 gene (FABP3), CD36, stearoyl-CoA desaturase gene (SCD), and fatty acid synthase gene (FASN). The inhibition of miR-199a-3p increased the level of triglycerides and the expression of LPL, ACACA, FABP3, SCD, and FASN in OMECs. These findings suggest that miR-199a-3p inhibited milk fat synthesis of OMECs. This is the first study to reveal the molecular mechanisms by which miR-199a-3p regulates the proliferation and milk fat synthesis of OMECs in sheep.

SUBMITTER: Wang J

PROVIDER: S-EPMC9391033 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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MicroRNA-199a-3p regulates proliferation and milk fat synthesis of ovine mammary epithelial cells by targeting <i>VLDLR</i>.

Wang Jiqing J Hao Zhiyun Z Hu Liyan L Qiao Lirong L Luo Yuzhu Y Hu Jiang J Liu Xiu X Li Shaobin S Zhao Fangfang F Shen Jiyuan J Li Mingna M Zhao Zhidong Z

Frontiers in veterinary science 20220805

In our previous study, microRNA (miR)-199a-3p was found to be the most upregulated miRNA in mammary gland tissue during the non-lactation period compared with the peak-lactation period. However, there have been no reports describing the function of miR-199a-3p in ovine mammary epithelial cells (OMECs) and the biological mechanisms by which the miRNA affects cell proliferation and milk fat synthesis in sheep. In this study, the effect of miR-199a-3p on viability, proliferation, and milk fat synth ...[more]

PMID: 35990270

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Project description:The mammary gland of the cow is particularly susceptible to infections of a wide range of pathogenic bacteria, including both Gram-positive and Gram-negative bacteria. The endotoxins of these pathogenic bacteria include peptidoglycan (PGN), lipoteichoic acid (LTA) and lipopolysaccharide (LPS), and they are the pathogen-associated molecular patterns (PAMPs) to induce mastitis. LPS can directly inhibit proliferation and milk fat synthesis of bovine mammary epithelial cells (BMECs) while inducing mastitis, but it is unclear whether PGN and LTA also have such effects. Furthermore, since the three PAMPs usually appear simultaneously in the udder of cows with mastitis, their synergistic effects on proliferation and milk fat synthesis of BMECs are worth investigating. The immortalized BMECs (MAC-T cells) were stimulated for 24 h using various concentrations of PGN, LTA and LPS, respectively, to determine the doses that could effectively cause inflammatory responses. Next, the cells were stimulated for 24 h with no endotoxins (CON), PGN, LTA, LPS, PGN + LTA, and PGN + LTA + LPS, respectively, with the predetermined doses to analyze their effects on proliferation and milk fat synthesis of BMECs. PGN, LTA and LPS successfully induced inflammatory responses of BMECs with doses of 30, 30 and 0.1 μg/mL, respectively. Although the proliferation of BMECs was significantly inhibited in the following order: LTA < PGN + LTA < PGN + LTA + LPS, there was no change in cell morphology and cell death. LTA significantly promoted the expression of fatty acid synthesis-related genes but did not change the content of intracellular triglyceride (TG), compared with the CON group. The mRNA expression of fatty acid synthesis-related genes in the LPS group was the lowest among all the groups. Meanwhile, LPS significantly decreased the content of intracellular non-esterified fatty acids (NEFAs) and TG, compared with the CON group. PGN had no effects on milk fat synthesis. Co-stimulation with PGN, LTA and LPS significantly increased the expression of fat acid synthesis-related genes and the intracellular NEFAs, but decreased intracellular TG, compared with sole LPS stimulation. Collectively, PGN, LTA and LPS showed an additive effect on inhibiting proliferation of BMECs. The promoting role of LTA in fatty acid synthesis might offset the negative effects of LPS in this regard, but co-stimulation with PGN, LTA and LPS significantly decreased intracellular TG content.

Dataset Information

MicroRNA-199a-3p regulates proliferation and milk fat synthesis of ovine mammary epithelial cells by targeting VLDLR.

Publications

MicroRNA-199a-3p regulates proliferation and milk fat synthesis of ovine mammary epithelial cells by targeting <i>VLDLR</i>.

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