Regulation Of MiR-142-3p To Lactation Of Dairy Goat Mammary Epithelial Cells

Mammary is the special gland of mammals, which function is lactation. In order to increase the amount of milk cows, dairy goats and other livestock, the researchers have done a lot of researches the basis of the molecular biology of breast. Mi RNA plays a vital role in the regulation of animal cell proliferation, apoptosis and other life activities. It has become a bright spot of life science research in recent years. It has found that lactation-related prolactin receptor(PRLR) presence of mi R-142-3p binding sites and miR-142-3p related to breast lactation in miRbase by Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University. The study aimed that mi R-142-3p regulate milk of lactating goat mammary epithelial cell for further study. It provides new ideas and lays basis on experiments for lactating mammary gland biology research and regulation.In the study, it utilizes dairy goat mammary epithelial cell line as a cell model.The cells were transected mi R-142-3p mimics / inhibitor.This essay uses real-time fluorescence quantitative PCR and its related pathway genes Prlr changes detected by Western blotting using the influence of miR-142-3p on PRLR signaling molecules, such as milk proteins. It uses the method of EDU cell proliferation assay, which flow cytometry was used to detect the cell cycle and apoptosis that use CCK to detect the changes of cell viability assay. At the same time, the application of UV spectrophotometric detects the changes of lactose secretion, and use the kit to detect the secretion of TG cells triglycerides.The results show that:①Detecting the expression of Prlr after treated with mi R-142-3p using qRT-PCR.When miR-142-3p was overexpressed, Prlr was down-regulated.When miR-142-3p was inhibited, Prlr was upregulated.②Western Blotting technique test results showed that the expression of PRLR, SREBP, AKT1, p-AKT1, mTOR, p-mTOR, STAT5, p-STAT5, CSN2, PPARγ, Cyclin D1 declined in dairy goat mammary epithelial cells when mi R-142-3p was overexpressed. The miR-142-3p inhibition experiment showed results that opposed to those of the miR-142-3p overexpression experiment. Overexpression and inhibition, the expression of GLUT1 changes significantly.③Immunofluorescence showed that mi R-142-3p regulated PRLR position and expression at the cellular level.④Applicating EDU deteced cell proliferation assay showed that miR-142-3p inhibited cell proliferation.⑤Flow cytometry was used to detect the cell cycle and apoptosis.The detection showed that mi R-142-3p inhibited cell cycle and promoted apoptosis.⑥Using CCK to detecte the changes of cell viability showed that mi R-142-3p inhibited cell viability.⑦TG kit detected cell triglycerides and lactose assay kit detected lactose.the results showed that miR-142-3p inhibitsed the biosynthesis of cell triglycerides. But lactose was not effect significantly.In summary, miR-142-3p reduced proteins which related lactation signal pathways in order to inhibiting the proliferation,viability and promote apoptosisof diary goat mammary epithelial cells by inhibiting the expression of PRLR and regulate negativly to to lactation of diary goat mammary epithelial cells.