| The mammary gland is the lactating organ of dairy goats and the study on the regulation mechanism of mammary gland at the molecular level is a research hotspot at domestic and foreign.Numerous studies have shown that mi RNAs play an important role in the regulation of mammary gland development and lactation performance.Previous research found in the laboratory,miR-3880 was found to be differentially expressed during breast development and lactation of dairy goats by sequencing.However,there has been no experimental study on mi R-3880 regulating milk gland development and lactation in dairy goats.In this study,the target gene of miR-3880 was verified by bioinformatics,RT-qPCR,Western blot and dual luciferase vector.The regulation of target gene OGR1 on dairy goat mammary epithelial cells was studied by siRNA interference technology.The aim of this study was to elucidate the mechanism of miR-3880 and OGR1 regulating the growth and lactation of goat mammary epithelial cells.This study may provide experimental basis for lactation regulation of dairy goats.The main findings are as follows:1.The regulation of mi R-3880 to OGR1 gene in mammary epithelial cells of dairy goat.The bioinformatics software was used to predict that the 3’UTR region of OGR1 contains a targeted binding site for miR-3880.By constructing dual luciferase reporter vector with OGR1-3’UTR,then we make miRNA mimics and reporter vectors were co-transfected into 293 T cells and detect the luciferase activity.The results showed that,miR-3880 mimics transfection group significantly inhibited the luciferase activity of the wild-type vector,but had no significant effect on the luciferase activity of the mutant vector compared with the NC group.Detecting the effect of miR-3880 on the expression of target gene OGR1,the results showed that compared with NC group,miR-3880 significantly down-regulated the expression of target gene OGR1 at mRNA and protein levels.Based on this,we determined that OGR1 is the target gene of miR-3880.2.The effect of OGR1 interference on the apoptosis in mammary epithelial cells of dairy goat.We use the siRNA technology interferes the expression of OGR1,synthesizes the siRNA mimic of OGR1 gene(siOGR1),and transfects siOGR1 into mammary epithelial cells.The results showed that OGR1 significantly inhibited MEC apoptosis.At the same time,OGR1 interference significantly inhibited the expression of Bcl-2 and Bax protein and up-regulated the protein ratio of Bcl-2/Bax;OGR1 also significantly down-regulated the expression of Fas apoptotic gene protein.3.The effect of OGR1 interference on the proliferation in mammary epithelial cells of dairy goat.The CCK8 method was used to detect the proliferation of dairy goat mammary epithelial cells.We found that compared with the NC group,OGR1 interference significantly promoted the proliferation of mammary epithelial cells.4.The effect of OGR1 interference on the synthesis of β-casein and triglyceride in mammary epithelial cells.The synthesis of β-casein and triglyceride in mammary epithelial cells was detected by ELISA and TG kit,and the results showed that interference the gene of OGR1 significantly increased the synthesis of beta-casein and triglyceride in mammary epithelial cell compared with NC group.Simultaneously,we detected the expression of AKT and mTOR phosphoprotein.The results showed that interference the gene of OGR1 significantly up-regulated the phosphorylation level of AKT and mTOR.In summary,miR-3880 inhibits the expression of the target gene OGR1 in goat mammary epithelial cells;mi R-3880 promotes the proliferation of mammary epithelial cells by the target gene OGR1,and by upgrading the phosphorylation of AKT and mTOR,activating AKT/mTOR signaling pathway,in turn,increases the synthesis of beta-casein and triglycerides. |
