LncRNA TCONS_00055666调控奶山羊乳腺脂类代谢的分子机制

Goat milk is a very nice product of the future of industrialization. The investigation of the regulatory mechanisms of lipid metabolism in dairy goat mammary gland is very important for the study of the mechanism of lactation, improvement of goat milk and reformation of milk supply. LncRNAs play vital roles in animal organ development however the roles of lncRNAs on dairy goat mammary gland development and lactation are not fully understood yet. In our preliminary study, we have found that lncRNAs can regulate lipid metabolism in goat mammary gland while the underlying mechanisms are unknown. In this project,dairy goat mammary gland will be used as a model to investigate the molecular mechanisms of lncRNAs in regulation lipid metabolism. Knockdown or overexpression of the special lncRNA TCONS_00055666 or/and knockdown/overexpression its target gene A in the dairy goat mammary gland cells will be applied to explore the roles of lncRNA in lipid metabolism. The impacts of the interaction of TCONS_00055666 and target gene A on TCONS_00055666 binding protein and the proteins involved in lipid metabolism will be explored. Then, knockdown or overexpression of TCONS_00055666 in the cultured goat mammary tissue (3D culture) will be used to validate the role of TCONS_00055666 in lipid metabolism. The findings from this project will provide fundamental knowledge for the understanding of the lactation process, the increase in the value of goat milk and the improvement in dairy goat breeding.

羊奶具有良好的产业化前景，探究奶山羊乳腺脂类代谢调控机制对研究乳腺泌乳机理、改善乳品质、优化乳业供应等有重要意义。LncRNA在调节动物重要经济器官的生长发育中起到重要作用，但在奶山羊乳腺发育及乳脂代谢方面鲜有报道。我们发现lncRNA能够调控奶山羊乳腺脂类代谢，但机理尚不清楚。本项目以奶山羊乳腺为模型，探究lncRNA参与调节乳腺脂类代谢的机理，挖掘参与调节的关键lncRNAs，并利用体外细胞降低或超表达实验验证TCONS_00055666或（和）关联基因A在乳腺脂类代谢过程中的作用，探究其互作关系及对TCONS_00055666结合蛋白、脂类代谢过程重要调节蛋白的影响。此外，利用体外培养的乳腺组织通过降低或超表达TCONS_00055666验证其在乳腺脂类代谢中的作用。该研究将为进一步揭示奶山羊乳腺泌乳的调控机制、提高奶山羊乳用价值奠定理论基础，为奶山羊乳质改善和品种选育提供科学指导。

羊奶具有良好的产业化前景，探究奶山羊乳腺脂类代谢调控机制对研究乳腺泌乳机理、改善乳品质、优化乳业供应等有重要意义。在前期实验挖掘出可能参与奶山羊乳脂调控的多个关键lncRNAs的基础上，本研究通过在体外对乳腺上皮细胞中lncRNA TCONS_00055666进行抑制或超表达实验来验证其在乳腺脂类代谢过程中的作用，并探究其调控机制。. 对体外培养的乳腺上皮细胞进行慢病毒转染，使lncRNA TCONS_00055666超表达和抑制其表达，通过油红染色和甘油三酯检测发现，超表达lncRNA TCONS_00055666乳腺细胞中脂滴含量升高，抑制表达乳腺细胞中脂滴含明显降低。对转染慢病毒后的山羊乳腺上皮细胞进行高通量测序分析发现ABCA1和FABP的表达量在抑制组中降低；对脂类代谢相关基因PPARγ、SREBF1、FASN的表达进行检测发现PPARγ和SREBF1蛋白水平在超表达组中表达量升高，在抑制组中表达量降低。在此基础上，我们对lncRNA是否通过影响miRNA来影响脂类基因的蛋白表达进行了验证。对转染慢病毒后的山羊乳腺上皮细胞进行small RNA-seq分析发现了差异表达的miR-30a和miR-199；通过转染miRNA后分析发现miR-30a和miR-199对脂类代谢基因的表达有抑制趋势，而lncRNA TCONS_00055666高表达对这种抑制趋势有减弱趋势，进一步研究发现PPARγ作为miRNA与mRNA之间的介导因子，即lncRNA TCONS_00055666通过影响miR-30a/miR-199对PPARγ的作用，从而影响脂类代谢。综上所述，lncRNA TCONS_00055666参与调控奶山羊乳腺上皮细胞内脂类代谢，可作为未来改善乳品质、优化乳业供应的潜在因子在育种工作中加以应用。