Preliminary Study On The Regulation Of Inflammatory Factor Expression In Goat Mammary Epithelial Cells By LF Via MiR-224-5p/ADAM17

Goat milk has a very high nutritional value.It contains proteins,carbohydrates,various vitamins and trace elements that the human body needs.Lactoferrin(LF),as an important component of goat milk,has a variety of biological functions,such as antibacterial,anti-inflammatory and antioxidant effects.LF can regulate the immune system.In this experiment,based in milk goat mammary epithelial cells transfected with lactoferrin overexpression vector,differentially expressed mi RNAs were obtained by high-throughput sequencing technology analysis,target gene prediction websites were used to predict target genes,mi R-224-5p and target gene A Disintegrin and Metalloprotease17(ADAM17)related to immune regulation were selected to explore their effects on the immune regulation of GMECs,hoping to increase immunity and provide a theoretical basis for the study of immune regulation of goat body.The main results obtained are as follows:1.Compared with the pc DNA3.1 group,a total of 22 differential mi RNAs were identified in the LF overexpression group,including 15 up-regulated mi RNAs and 7down-regulated mi RNAs,of which chi-mi R-224-5p was significantly up-regulated;mi R-224-5p has a binding site with the 3’ UTR of ADAM17;fluorescence quantitative PCR detection showed that the m RNA expression of ADAM17 was significantly decreased after transfection of mi R-224-5p mimic,and the m RNA expression of ADAM17 was significantly increased after transfection of mi R-224-5p inhibitor(P < 0.01);it was found that mi R-224-5p mimic could significantly reduce the m RNA expression of TNF-α and IL-8(P <0.05),and mi R-224-5p inhibitor significantly increased the m RNA expression of TNF-α,IL-6 and IL-8 Expression level(P < 0.01).2.Primers were designed according to the predicted sequence of goat ADAM17 gene in Gen Bank,and the CDS sequence of goat ADAM17 gene was cloned.The sequence length is2478 bp,encoding 825 amino acids,and the protein molecular weight is 92.92 KDa.The results of homology analysis showed that ADAM17 of dairy goat had the closest relationship with sheep and cattle,with homology of 99.15% and 97.14% respectively,and the farthest relationship with rats and mice,with homology of 85.13% and 85.25% respectively.3.By transfecting pc DNA-ADAM17 vector in GMECs,it was found that the expression levels of TNF-α,IL-6 and IL-8 were significantly increased(P < 0.01);transfection of si-ADAM17 could significantly reduce the m RNA expression levels of TNF-α,IL-6 and IL-8(P < 0.01);the m RNA expressions of ADAM17 and TNF-α in breast epithelial cells transfected with mi R-224-5p inhibitor and si RNA-NC were significantly increased(P < 0.01),and the m RNA expressions of IL-6 and IL-8 were also increased(P < 0.05);the m RNA expressions of ADAM17,TNF-α,IL-6 and IL-8 were significantly decreased in transfected mi R-224-5p inhibitor and si RNA-ADAM17(P < 0.01).In conclusion,this experiment screened the mi R-224-5p with increased expression in the milk goat mammary epithelial cells overexpressing LF,and successfully cloned the CDS region of the target gene ADAM17.The regulation of inflammation-related cytokines through its target gene ADAM17 has laid an important theoretical foundation for analyzing the immune regulation mechanism of dairy goats.