The Study Of The Gene MEN1 On The Regulatory Mechanism Of Cell Cycle From Bovine Mammary Epithelial Cells

Milk yield depends on the amount and secrete activity of mammary epithelial cells,increase the number of mammary epithelial cells has important implications for improving milk production.Menin, the protein encoded by the gene MEN1,had been observed to be involved in various cell functions, such as cell division control,cell differentiation,gene transcription and maintenance of genomic integrity. Menin, is the key disease-causing protein for multipleendocrine neoplasia type 1 syndrome. It plays an induce role in the abnormal proliferation of cells in specific tissues.On the other hand, MEN1 plays an important role in regulating metabolism.Breast is an important metabolic organ.This study was designed to investigate the function of menin in cow mammary tissue and mammary epithelial cells.The study found that the expression of MEN1/menin has a change during different stages and the change of MEN1/menin can alter cell growth and proliferation in MAC-T cells. The main results were as follows:1. The expression changes of MEN1/Menin during different stages were detected by RT-PCR, WB and IHC. The results indicated that the positive point of Menin was mainly expressed in the nucleus from luminal and myoepithelial basal cells and the expression of MEN1/menin has a change during different stages.2. The eukaryotic expression vector was constructed successfully by restriction enzyme identification, PCR, and sequence analysis. The vector was transfected into mammary epithelial cells, CHO cells, and C2C12 cells, respectively, and the expression of MEN1 gene/protein was detected at 24h,48h, and 72h, respectively. The results showed that the efficiency of expression was the highest after 24h of transfection.The efficiency of overexpression was 4.41,5.65,1.93 in MAC-T cells, CHO cells, and C2C12 cells, respectively.3. The eukaryotic expression vector was transfected into mammary epithelial cells, and the results showed that MEN1 gene expression had a significant increase between the control and treated measurments after transfection (P<0.01) and reached the highest after 24h of transfection; Transfection group was 27.5 times that of the control group and the protein of menin was 4.41 times that of the control group.4. The siRNA was transfected into mammary epithelial cells in which the silencing effect on MEN1 expression was investigated by both RT-PCR and Western blot. The result showed that the sibMEN-1/2/3 more remarkablely inhibited the expression of MEN 1 on both mRNA and protein compared with the control at 24h (P<0.01).The efficiency of knockdown was 75%,71%,respectively.5.Menin can regulate the proliferation and cell cycle of bovine mammary epithelial cells by CCK-8, FCM, and RT-PCR under the condition of overexpression/knockdown.The results indicated that the overexpression of the MEN1 gene had no remarkable effects on both cell proliferation and cell cycle; knockdown of MEN1 remarkablely increased cell percent at Gl phase, decreased cell percent at S phase(P<0.05), remarkablely increased the expression of CyclinD3, CyclinB2, and CDK6 (P<0.05), and significantly decreased the expression of CyclinD1 (P<0.05).