Molecular Mechanism Study Of CDK2 On The Proliferation And Apoptosis Of Goat Mammary Epithelial Cells

Cyclin-dependent kinase 2(CDK2)belongs to the CDK serine/threonine kinase family.CDK2 combines with Cyclin E and Cyclin A to form a complex.It promotes the G1/S phase of the cell cycle by phosphorylating many substrates such as retinoblastoma protein(Rb),and is an important regulatory factor in the process of the cell cycle.Early laboratory studies found that EGF could promote the proliferation of goat mammary epithelial cells,which might be related to the up-regulation of CDK2 expression level through ERK/AKT pathway through EGFR.In order to study the role and mechanism of CDK2 in breast development of dairy goats,we constructed pc DNA 3.1-CDK2 over expression vector using mammary epithelial cells as experimental material,used si RNA to interfere with CDK2 gene,and used RT-PCR,Western blot,CCK-8,EDU and flow cytometry to study the regulatory effect of CDK2 on GMECs,providing an experimental basis for regulating breast development and lactation.The results are as follows:1.It was found that over expression of CDK2 in goat mammary epithelial cells could significantly improve the proliferation efficiency of mammary epithelial cells(P<0.01)and promote the synthesis of DNA replication(P<0.01).On the contrary,the efficiency of interfering CDK2 cell proliferation was significantly reduced,and the synthesis of DNA replication was reduced(P<0.01);Flow cytometry results showed that after CDK2 over expression,the number of cells in G1 phase phase was significantly reduced,and the number of cells in S phase was significantly increased(P<0.01),which proved that CDK2 over expression could promote the transformation of cells from G1 to S phase.The proportion of cells in G1 phase phase after CDK2 interference was significantly higher than that in the control group(P<0.01),while the proportion of cells in S phase and G2 phase phase decreased significantly(P<0.01),indicating that interference with CDK2 would prevent cells from G1 to S phase,causing cycle arrest.Up regulation of CDK2 can promote cell proliferation,but in the detection of cell apoptosis,it was found that over expression of CDK2 significantly increased the proportion of apoptotic cells(P<0.05),while interference with CDK2 decreased the proportion of apoptotic cells(P<0.05).In summary,CDK2 has a positive regulatory effect on the proliferation and cell cycle of goat mammary epithelial cells,but at the same time,high expression of CDK2 leads to an increase in apoptotic cells.Successfully constructed pc DNA 3.1-CDK2 eukaryotic over expression vector and designed and synthesized si RNA that effectively interferes with CDK2 CDS region encoding.2.It was found that the level of CDK2 protein is positively correlated with the same family proteins CDK1 and CDK4.At the same time,CDK2 positively regulates the expression of downstream proliferation related genes Rb,p-Rb,and PCNA.To further investigate the regulatory relationship between CDK2 and CDK1,CDK4 kinases,CDK1 and CDK4 were interfered with and over expressed,respectively.The level of CDK2 protein also changed in the same direction,indicating a positive synergistic effect between CDK2 and CDK1,CDK4,promoting cell proliferation together.The detection of apoptosis related signaling pathways showed that after CDK2 interference,the expression of tumor suppressor protein P53,pro apoptotic protein Bax,and the activation splicing level of key apoptotic effector molecule Caspase3 were significantly reduced(P<0.01),while over expression of CDK2 significantly increased the expression levels of P53,Bax,and cleared Caspase3(P<0.01),indicating that abnormal high expression of CDK2 may activate the P53-Bax-Caspase3 signaling pathway,thereby increasing cell apoptosis.In summary,this experiment preliminarily studied the role of CDK2 in the proliferation and apoptosis of mammary epithelial cells in dairy goats,laying a foundation for further research on the regulatory mechanism of breast development.Research has found that CDK2 positively regulates the proliferation of goat mammary epithelial cells and promotes the transition of the cell cycle to the G1-S phase.CDK2 promotes the proliferation of milk goat mammary epithelial cells by regulating the expression of downstream proteins Rb,p-Rb,and PCNA;At the same time,there is a positive correlation between the expression of CDK2 and CDK1,CDK4.The high expression of CDK2 in mammary epithelial cells of dairy goats showed an increase in cell apoptosis rate,which may be related to the activation of the P53-Bax-Caspase3 signaling pathway.