Preliminary Study On The Mechanism Of MicroRNA-183 Targeting MAPK1 Gene Regulating Lactation In Buffalo Mammary Epithelial Cells

As a characteristic species in southern China,buffalo as an agricultural service animals have the the advantages of resistance to rough feeding,moist heat and adaptability.In addition,its fleshy taste is good,and dairy products also have high nutritional value.This study uses Guangxii Pulling buffalo as the research object,the microRNA-183-targeted MAPK1 gene(ID:102396206)was used to regulate the lactation of buffalo mammary epithelial cells,in order to improve the yield and quality of buffalo dairy products.The mammalian mammary gland is an organ that can still proliferate and differentiate after adulthood,and its metabolic activity is regulated by various factors.miRNA is a 18-22 nt non-coding small RNA that is widely involved in many important biological processes.The mainly function of miRNA is post-translational modification.There have been many reports that miRNAs have important regulation on the process of mammalian lactation.The role of the mechanism of lactation from the level of miRNA is also a relatively new field.Previous studies screened for differentially expressed miRNAs in buffalo before and after lactation.The online prediction software was used to predict different miRNA target loci.The target gene of miR-183 is a MAPK1 gene closely related to various biological processes.The literature found that its differential expression may be closely related to changes in milk fat content in buffalo and proliferation of mammary epithelial cells.To verify the predicted results of the target gene,we found that by overexpressing the expression of miR-183 and interfering with miR-183,and interfering with the expression of the target gene MAPK1,we found:The miR-183 have different expression in different lactation periods and between different tissues.RNA was extracted by sampling breast tissue from three different lactating buffalo,and extracted by sampling different tissues from one lactating buffalo.Quantitative PCR results showed that miR-183 had the lowest expression during lactation,and compared with other tissues miR-183 has the lowest expression in the mammary gland,which also means that miR-183 is closely related to the lactation process.Search the basic information of buffalo miR-183 through the miRbase online prediction website and predict the target gene to be MAPK1 gene.The wild type and mutant double fluorescein reporter vectors were constructed by using the predicted 3'UTR targeting region of MAPK1.The vector was transformed into DH5a expanded culture and transfected into 293t cells.It was confirmed that miR-183 and MAPK1 gene were targeted to each other.Studies of MAPK1 have shown that inhibition of MAPK1 expression by siRNA results in significant effects of buffalo mammary epithelial cell proliferation.Studies on miR-183 have shown that the use of miR-183 mimic and inhibitors affects the expression of MAPK1,which affects the proliferation of mammary epithelial cells and the secretion of milk fat and milk proteins,and there is no significant impact to cell apoptosis.The above results indicate that miR-183 has a significant regulatory effect on the proliferation and secretion of mammary epithelial cells by regulating the target gene MAPK1,which is closely related to the development and lactation of buffalo mammary gland.