奶山羊乳腺上皮细胞PTEN/CREB通路调控脂肪酸代谢的分子机理研究

Goat milk is rich in short- and medium-chain fatty acids and unsaturated fatty acids, which has a good health care and can be effective in preventing certain metabolic diseases of humans. Lipid metabolism is of great importance in the ruminant lactation mammary gland that affecting the fatty acid composition in goat milk. Previous study indicated that PTEN and CREB interact with each other in goat mammary epithelial cells (GMEC). CREB is an important upstream regulator of lipid metabolism. Thus, PTEN/CREB signaling pathway may play a pivotal role in the transcriptional regulation of fatty acid metabolism. The GMEC is purified from biopsy mammary gland samples of goat at peak-lactation period. Using transcriptome analysis to identify the effects of PTEN on the gene network involved in lipid metabolism and the downstream target genes. The milk fatty acid composition, mRNA expression and protein levels of genes will also be assessed in GMEC following the treatment with overexpression or knockdown of PTEN/CREB. The promoter region of target genes will be cloned and bioinformatics analysis will be used to identify the main transcription factor binding sites. Combining with site-specific mutagenesis, EMSA and ChIP, key transcriptional regulatory elements within target gene promoters will be identified for the function analysis and validation. The comprehensive function analyses of PTEN/CREB signaling pathway in the transcriptional regulation of fatty acid metabolism in dairy goat mammary gland will be performed, which will provide theoretical and experimental evidences for further studies on molecular mechanism of mammary gland fatty acid metabolism and goat milk fatty acid composition manipulation.

山羊奶富多种不饱和脂肪酸，具有独特的营养价值和保健功能。反刍动物泌乳期乳腺脂肪酸代谢是影响乳中脂肪酸组成的重要生理过程。研究表明，PTEN和CREB在山羊乳腺细胞中存在相互作用，CREB是脂代谢的重要上游调控因子。因此PTEN/CREB通路可能在脂肪酸代谢过程中起重要调控作用。本课题通过采集泌乳期奶山羊乳腺组织，分离培养乳腺上皮细胞，转录组测序研究PTEN/CREB信号通路对脂代谢相关基因的调控网络，寻找下游关键靶基因；过表达或干扰PTEN/CREB分析脂肪酸组成、脂肪酸代谢相关基因mRNA及蛋白水平变化；克隆关键靶基因启动子2-3个，利用生物信息学预测潜在的转录因子结合位点，结合定点突变、EMSA以及ChIP技术对靶基因启动子的调控元件进行筛选鉴定；综合分析PTEN/CREB信号通路在奶山羊乳腺脂肪酸代谢转录调控的关键功能，为乳腺脂肪酸代谢机理和羊奶脂肪酸组成调控研究提供理论和实验依据。

奶山羊乳腺脂肪酸代谢受诸多因素的影响。PTEN是一个具有抑癌作用的肿瘤抑制因子，PTEN低表达可以升高CREB的磷酸化水平。本研究以奶山羊乳腺上皮细胞为研究对象，探讨PTEN/CREB通路对奶山羊乳腺细胞脂质代谢及脂肪酸组成的影响。结果发现，PTEN过表达后，细胞中甘油三酯含量显著下调，乳脂代谢关键调控因子SREBP1的表达量显著下调，脂肪酸合酶FASN、脂肪酸去饱和酶SCD1表达量显著下调，而脂解基因LIPE表达量显著上调，脂肪酸氧化基因ACOX1和CPT1B显著上调。细胞中C16:1脂肪酸占总脂肪酸的比例显著降低。反之，干扰PTEN促进了细胞中脂质积累。随后，在乳腺上皮细胞中过表达CREB，发现SREBP1的表达量显著上调，甘油三酯合成基因DGAT1和LPIN1显著上调，细胞中C16:1，C18:1 n-9，C20:1脂肪酸占总脂肪酸的比例显著升高，C16:1 和C18:1的去饱和指数显著上调，同时细胞内甘油三酯的含量显著上升；反之，干扰CREB抑制了细胞中脂质的积累。而当干扰CREB并过表达PTEN，发现过表达PTEN并干扰CREB与单独过表达PTEN相比，SREBP1和FASN的表达量没有显著变化，表明PTEN直接通过CREB调控奶山羊乳腺乳脂代谢。通过对PTEN过表达奶山羊乳腺细胞进行RNA-Seq分析，在转录组水平上筛选出了与脂肪酸代谢相关的90个DEGs。包括FoxO信号通路和脂肪酸延长通路2个与脂肪代谢直接相关的通路。同时发现FABP3基因的表达水平显著下调，而FABP3参与长链脂肪酸的转运过程，因此选取FABP3为下游靶基因进行转录调控研究。进一步研究发现CREB干扰后下调了FABP3的启动子活性，突变CREB结合位点后消除了干扰CREB对FABP3启动子的下调作用，表明PTEN/CREB信号通路通过CREB的结合位点参与调控FABP3启动子活性。这些结果明确了PTEN/CREB信号通路对奶山羊乳腺细胞脂肪酸代谢的调控机制，为调控羊奶脂肪酸组成和改善羊奶风味、提高羊奶品质具有重要意义。