Gene Of MEN1 Involved In Regulation Milk Protein Synthesis In Dairy Cow Mammary Epithelial Cells

The target of breeding researchers is to improve lactation performance and lactation production of dairy cow.Research has shown that MEN1 gene can affect the function of the pituitary gland,inhibition of prolactin promoter activity and the catalytic activity of ER? to participate in regulation of mammary gland development.MEN1 gene is named by its mainly causative disease,multiple endocrine neoplasia type 1.Menin is encoded by MEN1 gene that can interact with many important transcription factors,epigenetically regulating the expression of its target gene and induced cell phenotypes,also joined different intracellular signaling pathways,widely regulating body development and metabolic stability.But the function of MEN1 gene in regulating the cow mammary gland lactation is rarely reported.This study intended to research the role of MEN1 gene in lactation,especially in milk protein synthesis.As the important signal pathway of milk protein synthesis,studying MEN1 gene regulation function in PI3K/Akt/mTOR and JAK2-STAT5 pathways,has great significance to study the synthesis of milk protein.Therefore,dairy cows mammary gland epithelial cells line MAC-T were used as models to research the function of MEN1 expression.Recombinant plasmid pEGFP-C2-bMEN1 was constructed and transient transfection into cell for MEN1 gene overexpression experiments;RNAi method was used to transfect MEN1 siRNA in the MEN1 gene inhibition.QRT-PCR and Western blotting were used to detect the mRNA and protein levels of gene involved in lactation-related signaling pathways and the mRNA levels of CSN3.At the same time,genes involved in lactation-related signaling pathways and MEN1 mRNA expression at different development stages of dairy cows mammary gland tissue were detected.By the combination of in vitro and in vivo experiments to investigate the relationship of MEN1 gene expression and mammary glands development and lactation,which provides basic data for the study of dairy cows mammary gland development and lactation mechanisms,and the theoretical support for milk production and milk quality of the artificial regulation.The results show that:(1)Recombinant plasmid pEGFP-C2-bMEN1 was successfully expression in the MAC-T cells,the mRNA and protein levels were significantly increased(P<0.01)after transfection 24h;(2)Overexpression of MEN1 gene down-regulated expression of AKT,mTOR,S6K1,4E-BP1 and STAT5 in mRNA levels which were involved in milk protein synthesis-related signaling pathways;meanwhile,the overexpression of MEN1 genealso can down-regulate expression of AKT,mTOR,and S6K1 in protein levels(P > 0.05).While,the MEN1 gene inhibition experiment showed the opposite results of the gene overexpression experiment.(3)Under the condition of MEN1 gene overexpression,the mRNA detection of CSNK gene was significantly decreased(P<0.05);while the lower expression of MEN1 gene can significantly increase the expression of mRNA level of CSNK gene(P <0.01).(4)Compared with dry period,the detection results revealed that the levels of mRNA and protein expression for MEN1 were decreased in the lactation period;however,the mRNA expression of different casein genes included CSNB(P > 0.05),CSNAS1,CSNAS2 and CSNK(P<0.05)were increased in the lactation period.At the same time,in the lactation period,AKT(P<0.05),mTOR,S6K1,4E-BP1 and STAT5(P>0.05)which were involved in milk protein synthesis-related signaling pathways also increased.This result is consistent with the experimental results in mammary gland epithelial cells.In summary,we showed that MEN1 gene can negatively control the expression of genes which were involved in PI3K/Akt/mTOR and JAK2-STAT5 signaling pathways,and negatively control the synthesis of milk protein.