Effects Of Hepatitis C Virus Genotype 1b NS4B On The Expression Of Cdc25C And CyclinB1 In Huh7 Cells

Objective: To investigate the effects of hepatitis C virus genotype 1b(HCV1b) nonstructural protein 4B(NS4B) on the expression of Cdc25 C and Cyclin B1 in Huh7 cells and its possible mechanism involved.Methods: The plasmid pc DNA3.1(-)HCV 1b NS4 B and pc DNA6.2-GW/Em GFP-mi R NS4 B and were transfected into Huh7 cells,named Huh7-NS4 B and mi R-NS4 B group.The third group was Huh7. The m RNA expression of NS4 B,Cdc25C and Cyclin B1 were detected by RT-PCR and the expression of NS4 B,Cdc25C,Cyclin B1 and p-ERK protein was detected by Western Blot. P-ERK inhibior U0126 was added into Huh7-NS4 B cells, named U0126 group, compared with Huh7-NS4 B cells. The m RNA and protein level of Cdc25 C,Cyclin B1 were tested by RT-PCR and Western Blot respectively.Results: 1. At 48 h after transfection,relative m RNA level of NS4 B in Huh7-NS4 B group and mi R-NS4 B group were: 0.763±0.012,0.554±0.010.Relative protein level in two groups were: 0.798±0.010,0.442±0.025. Relative m RNA and protein level of NS4 B in Huh7-NS4 B group were higher than that in mi R-NS4 B group(p<0.01); 2. At 48 h after transfection,relative m RNA level of Cdc25 C in three groups were: 0.595±0.007,0.726±0.011,0.503±0.012,and Cyclin B1 in three groups were: 0.834±0.011,1.019±0.017,0.672±0.011. Relative protein level of Cdc25 C in three groups were: 0.513±0.011,0.612±0.090,0.432±0.010,and Cyclin B1 in three groups were: 0.502±0.015,0.611±0.011,0.411±0.011.Relative m RNA and protein level of Cdc25 C,Cyclin B1 in Huh7-NS4 B group were higher than that in mi R-NS4 B group and Huh7 group(p<0.01),and mi R-NS4 B group were lower than Huh7 group(p<0.01); Relative protein level of p-ERK in three groups were: 1.238±0.009,1.349± 0.012,1.051±0.012. Relative protein level of p-ERK in Huh7-NS4 B group was higher than that in mi R-NS4 B group and Huh7 group(p<0.01),and mi R-NS4 B group was lower than Huh7 group(p<0.01). 3. Relative m RNA level of Cdc25 C in Huh7-NS4 B group and U0126 group were:612±0.013,0.411±0.0120,and Cyclin B1 in two groups were:0.985±0.008,0.886 ±0.007.Relative protein level of Cdc25 C in Huh7-NS4 B group and U0126 group were:0.914±0.017,0.464±0.008,and Cyclin B1 in two groups were: 0.914±0.007,0.464 ±0.018.Relative m RNA and protein level of Cdc25 C and Cyclin B1 were lower in the cells of U0126 group than that in Huh7-NS4 B group(p < 0.01).Conclusion: 1.HCV1 b NS4B can upregulate the expression of Cdc25C、Cyclin B1, p-ERK in Huh7 cells; 2.HCV1 b NS4B may be upregulate the expression of Cdc25 C, Cyclin B1 through activation of MAPK/ERK signaling pathway.