Research Of Regulation And Molecular Mechanism Of MiR-99a-3p On Apoptosis In Goat Mammary Epithelial Cells

Milk and dairy products are an important source of nutrition for young animals and humans.Mammary gland epithelial cells（MECs）are the site of milk synthesis and secretion,and their number and activity determine the amount of milk produced by the mother.Apoptosis is one of the important factors affecting the number of MECs.Apoptosis of MECs exhibits periodic changes during the periodic development of the mammary gland to remove low activity and a part of unwanted cells to maintain the stability of mammary gland function.Therefore,studying the mechanism of MECs apoptosis is important for maintaining mammary gland tissue homeostasis and promoting lactation function of mammary gland.miRNAs have functions involved in the regulation of apoptosis.A study found that miR-99a-3p was highly expressed during the degeneration of mammary gland tissue during the dry period of dairy goats,and it may be involved in the regulation of mammary gland involution during dry milk stage in dairy goats.The previous study of our group showed that miR-99a-3p can increase the level of Ca²⁺in goat mammary gland epithelial cells（GMECs）.A certain concentration of Ca²⁺may induce cell apoptosis.Therefore,it is speculated that miR-99a-3p may play a regulatory role in the occurrence of GMECs apoptosis,but related research has not been reported.In this study,the GMECs were used.Firstly,the effect of miR-99a-3p on GMEC apoptosis was explored,and subsequently,bioinformatics prediction and molecular cell biology experiments confirmed the molecular mechanism of miR-99a-3p targeting GPR19 to regulate apoptosis of goat mammary epithelial cells.The results obtained are as follows:（1）.After transfection of miR-99a-3p mimic and inhibitor into GMECs,q RT-PCR and Annexin V-FITC/PI staining combined with flow cytometry showed that overexpression of miR-99a-3p inhibited GMECs apoptosis（p<0.05）,inhibition of miR-99a-3p expression promotes GMECs apoptosis（p<0.05）;indicating that miR-99a-3p inhibits the apoptosis of GMECs.（2）.Through Target Scan and miRWalk database screening,GPR19 is one of the target genes of miR-99a-3p.And the targeted relationship between miR-99a-3p and GPR19 was demonstrated by dual luciferase activity detection assay;（3）.After overexpression of GPR19,the expressions of pro-apoptotic proteins Bax and cleaved Caspase3 in GMECs were up-regulated（p<0.05）,and after interference with GPR19expression,the expressions of pro-apoptotic proteins Bax and cleaved Caspase3 in GMECs were down-regulated（p<0.05）;the results showed that GPR19 promotes apoptosis of GMECs（4）.After co-transfection of miR-99a-3p inhibitor and Si-GPR19,inhibition of GPR19expression in GMECs rescued the increased expression of Bax and cleaved Caspase3 by transfection of miR-99a-3p inhibitor（p<0.05）,indicating that miR-99a-3p regulates apoptosis of GMECs via GPR19.（5）.After treatment of GMECs with U0126（MAPK/ERK1/2 inhibitor）,U0126 alleviated Bax expression caused by overexpression of GPR19（p<0.05）,and the results showed that GPR19 induced apoptosis of GMECs through MAPK/ERK1/2 signaling pathway.In summary,this study elucidated the molecular mechanism for miR-99a-3p can target GPR19 and through MAPK/ERK1/2 signaling to inhibit GMECs apoptosis,accumulating data to reveal the molecular regulatory mechanism of GMECs apoptosis.