Objectives

Understanding the molecular mechanisms of lipid synthesis in the mammary gland is crucial for regulating the level and composition of lipids in milk. This study aimed to investigate the functional and molecular mechanisms of miR-204-5p in mammary epithelial cells to provide a theoretical basis for milk lipid synthesis.

Methods

Real-time quantitative PCR was performed to detect the transcriptional levels of miR-204-5p and related mRNA abundance in mammary epithelial cells. Western blotting was conducted to determine protein expression. Cell proliferation was assessed by Cell Counting Kit-8. A dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-204-5p and SIRT1. siRNA and overexpression plasmids were transfected into mouse HC11 mammary epithelial cells.

Results

The abundance of miR-204-5p was much higher in lactating mouse mammary glands than in other tissues, which indicated that miR-204-5p may be involved in regulating milk production. MiR-204-5p affected the expression of β-casein and milk lipid synthesis in HC11 mouse mammary epithelial cells but did not influence the proliferation of HC11 cells. Overexpression of miR-204-5p significantly increased the number of Oil Red O⁺ cells, triglyceride accumulation and the expression of markers associated with lipid synthesis, including FASN and PPARγ, whereas inhibition of miR-204-5p had the opposite effect. miR-204-5p promotes lipid synthesis by negatively regulating SIRT1. Overexpression of SIRT1 can repress the promotion of miR-204-5p on lipid synthesis.

Conclusion

Our findings showed that miR-204-5p can promote the synthesis of milk lipids in mammary epithelial cells by targeting SIRT1.

中文翻译：

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MiR-204-5p通过靶向SIRT1促进乳腺上皮细胞中的脂质合成

目标

了解乳腺脂质合成的分子机制对于调节牛奶中脂质的水平和组成至关重要。本研究旨在研究miR-204-5p在乳腺上皮细胞中的功能和分子机制，为合成乳脂提供理论依据。

方法

进行实时定量PCR，以检测乳腺上皮细胞中miR-204-5p的转录水平和相关的mRNA丰度。进行蛋白质印迹以确定蛋白表达。通过细胞计数试剂盒8评估细胞增殖。进行了双重荧光素酶报告基因测定，以验证miR-204-5p和SIRT1之间的靶向关系。siRNA和过表达质粒被转染到小鼠HC11乳腺上皮细胞中。

结果

泌乳小鼠乳腺中miR-204-5p的丰度比其他组织高得多，这表明miR-204-5p可能参与调节乳汁的产生。MiR-204-5p影响HC11小鼠乳腺上皮细胞中β-酪蛋白的表达和乳脂合成，但不影响HC11细胞的增殖。miR-204-5p的过表达显着增加了油红O ⁺细胞的数量，甘油三酸酯的积累以及与脂质合成相关的标志物（包括FASN和PPARγ）的表达，而抑制miR-204-5p具有相反的作用。miR-204-5p通过负调节SIRT1促进脂质合成。SIRT1的过表达可以抑制miR-204-5p对脂质合成的促进作用。

结论

我们的研究结果表明，miR-204-5p可以通过靶向SIRT1来促进乳腺上皮细胞中乳脂的合成。