Studies On The Molecular Mechanisms That CVB3 VP1 Arrests Hela Cells In G1/S Phase

Objective: To further explore functions of the interaction between CVB3 VP1 and the host protein MAT1, and check the expression level of CDKs(CDK4 、 CDK2), the corresponded Cyclin(Cyclin D1、Cyclin E1), the cell cycle inhibitory gene p21 and Rb to investigate the molecule mechanism that CVB3 VP1 arrested Hela cells in G1 phase.Methods: 1. The VP1-D8(3044-3319bp)and VP1-D4(2459-2926 bp, control)directionally inserted into vector p Bud CE4.1. Then the recombinant plasmid p Bud CE4.1-VP1-D4 and p Bud CE4.1-VP1-D8 were identified by restriction endonucleases digestion as well as sequencing; 2. The expression of MAT1 in Hela cells transfected by p Bud CE4.1-VP1-D4, p Bud CE4.1-VP1-D8, p Bud CE4.1-VP1 and p Bud CE4.1 for 24 h were detected by Western blot; 3. The effection of p Bud CE4.1-VP1-D4, p Bud CE4.1-VP1-D8, p Bud CE4.1-VP1 and p Bud CE4.1 on cell cycle were detected by flow cytometry; 4. The m RNA expression of CDK4, Cyclin D1, CDK2, Cyclin E1, CDC25 A and p21 in Hela cells transfected by p Bud CE4.1-VP1-D4, p Bud CE4.1-VP1-D8, p Bud CE4.1-VP1 and p Bud CE4.1 for 24 h were detected by Quantative Real-Time RT-PCR; 5. The m RNA expression of CDK4, Cyclin D1, p21 and CDK2 in Hela cells infected by CVB3 at 3 h, 6 h and 9 h were detected by Quantative Real-Time RT-PCR; 6. The expression of Rb, p-Rb Ser795, p-Rb Ser780 and p-Rb Ser807/811 in Hela cells transfected by p Bud CE4.1-VP1-D4, p Bud CE4.1-VP1-D8, p Bud CE4.1-VP1 and p Bud CE4.1 for 24 h were detected by Western blot;Result:1. VP1-D4 and VP1-D8 were successfully inserted into the vector p Bud CE4.1 confirmed by double enzyme-digestion and sequencing; 2. The reusults of Western blot shown that MAT1 were downregulated obviously in Hela cells transfected by p Bud CE4.1-VP1-D8 and p Bud CE4.1-VP1 compared to cells transfected by p Bud CE4.1-VP1-D4 and p Bud CE4.1, and statistical significance were attained; 3. Flow cytometry analysis showed: cells transfected by p Bud CE4.1-VP1-D8 and p Bud CE4.1-VP1 were obviously arrested in G1 phase, the results were statistically significant compared to the control group; 4. The reusults of Quantative Real-Time RT-PCR revealed: the m RNA of CDK4, Cyclin D1 and CDK2 in cells transfected by p Bud CE4.1-VP1-D8 and p Bud CE4.1-VP1 were decreased significantly, whereas p21 m RNA was increased, the results were statistically significant; the m RNA of CDC25 A and Cyclin E1 did not changed in four groups; 5. Statistically significant decrease in m RNA expressions of CDK4, Cyclin D1 and CDK2 in cells infected by CVB3 at 3 h were observed; p21 m RNA expression elevated gradually with infection of CVB3 at 3 h, 6 h and 9 h, the results were statistically significant; 6. The expression of p-Rb Ser780 and p-Rb Ser807/811 reduced in cells transfected by p Bud CE4.1-VP1-D8 and p Bud CE4.1-VP1, whereas Rb was increased, these differences were statistically significant; p-Rb Ser795 did not changed in four groups.Conclusions: Our results indicate: 1. Due to infection of CVB3 and the interaction between VP1-D8 and MAT1, MAT1 expression is downregulated, the activity of CAK is inhibited, then the inhibited CAK fails to phosphorylate CDK/Cyclin, and makes CDK4, Cyclin D1, CDK2 downregulated, and makes p21 upregulated; 2. Downregulated CDK2, CDK4 lead to a decrease of phosphorylation level of Rb, the genes required in G1-S transition can not be transcripted, finally induce cellsarrest in G1 phase.