Wang Chuanchuan, Zhao Jinyan, Feng Xiaofang, Zhao Wei, Ma Ruoshuang, Yu Baojun, Xue Lin, Wang Hua, Chen Yafei, Zhang Juan, Gu Yaling
College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China; Key Laboratory of Ruminant Molecular Cell Breeding in Ningxia, Ningxia University, Yinchuan 750021, China.
Yinchuan Animal Husbandry Technical Extension and Service Centre, Yinchuan 750021, China.
Genomics. 2024 Nov;116(6):110955. doi: 10.1016/j.ygeno.2024.110955. Epub 2024 Oct 29.
Milk fat is produced and secreted by the mammary gland, which is mainly regulated by diet and gene-molecule network. Therefore, understanding the molecular mechanism of milk fat synthesis is of practical significance for improving milk quality. Fatty acid-binding protein 4 (FABP4) is a candidate messenger RNA (mRNA) closely linked to milk fat metabolism obtained from transcriptomic analysis of mammary epithelial cells of cows in the pre-existing high- and low-milk-fat groups, and its expression pattern and function are still unclear. The qRT-PCR results depicted that FABP4 was highly expressed in bovine mammary epithelial cells (BMECs) in breast tissues and the high milk fat group. Subsequently, the regulatory effects of FABP4 on BMECs were analyzed by CCK8, EdU, and flow cytometry, and the results demonstrated that FABP4 inhibited the proliferation and viability of BMECs and promoted their apoptosis. Next, the effect of FABP4 on milk lipid metabolism was explored. pEGFP-N1-FABP4 was transfected into BMECs, and FABP4 upregulated the expression levels of the milk lipid marker genes XDH, PPARG, and ACSS2, and promoted the formation of triglycerides (TGs), cholesterol, lipid droplets, and β-casein. Strong interactions between FABP4 and PPARG were identified using STRING prediction. Western blotting revealed that FABP4 interacted with PPARG to promote PPARG expression, while the opposite result was observed after interfering with FABP4. The gene regulation of microRNA (miRNA) is essential for fatty acid metabolism and synthesis. Predicted by website and combined with pre-miRNA transcriptome sequencing results, we hypothesized that FABP4 might be the target gene of bta-miR-224. The results of the dual-luciferase reporter gene and qRT-PCR revealed that bta-miR-224 negatively regulated FABP4 expression by targeting the 3'-UTR of FABP4. By exploring the function of bta-miR-224, we observed that bta-miR-224 mimics downregulated the expression of the milk fat marker genes AGPAT6, ACSS2, and XDH and inhibited TG synthesis and lipid droplet secretion. However, the bta-miR-224 inhibitor depicted the opposite results. In conclusion, FABP4 plays a crucial role in regulating BMEC proliferation and differentiation. Bta-miR-224 targeting FABP4 may promote biological processes such as TG synthesis and lipid droplet formation through PPARG, which lays a solid foundation for further analysis of the functional mechanism of milk lipid metabolism in dairy cows from a miRNA-mRNA perspective.
乳脂肪由乳腺产生和分泌，其主要受饮食和基因 - 分子网络调控。因此，了解乳脂肪合成的分子机制对提高牛奶品质具有实际意义。脂肪酸结合蛋白4（FABP4）是通过对先前高乳脂和低乳脂组奶牛乳腺上皮细胞的转录组分析获得的与乳脂肪代谢密切相关的候选信使核糖核酸（mRNA），其表达模式和功能仍不清楚。qRT - PCR结果表明，FABP4在乳腺组织和高乳脂组的牛乳腺上皮细胞（BMECs）中高表达。随后，通过CCK8、EdU和流式细胞术分析FABP4对BMECs的调控作用，结果表明FABP4抑制BMECs的增殖和活力并促进其凋亡。接下来，探究了FABP4对乳脂质代谢的影响。将pEGFP - N1 - FABP4转染到BMECs中，FABP4上调了乳脂质标记基因XDH、PPARG和ACSS2的表达水平，并促进了甘油三酯（TGs）、胆固醇、脂滴和β - 酪蛋白的形成。使用STRING预测鉴定出FABP4与PPARG之间有强烈的相互作用。蛋白质免疫印迹法显示FABP4与PPARG相互作用以促进PPARG表达，而干扰FABP4后观察到相反的结果。微小RNA（miRNA）的基因调控对脂肪酸代谢和合成至关重要。通过网站预测并结合前体miRNA转录组测序结果，我们推测FABP4可能是bta - miR - 224的靶基因。双荧光素酶报告基因和qRT - PCR结果表明，bta - miR - 224通过靶向FABP4的3' - UTR负调控FABP4的表达。通过探究bta - miR - 224的功能我们观察到，bta - miR - 224模拟物下调了乳脂肪标记基因AGPAT6、ACSS2和XDH的表达，并抑制了TG合成和脂滴分泌。然而，bta - miR - 224抑制剂呈现相反的结果。总之，FABP4在调节BMECs增殖和分化中起关键作用。靶向FABP4的bta - miR - 224可能通过PPARG促进TG合成和脂滴形成等生物学过程，这为从miRNA - mRNA角度进一步分析奶牛乳脂质代谢的功能机制奠定了坚实基础。
