The Effcet Of Hepatitis C Virus Genotype 1b Core Protein On The Expression Of Plk1, CDK1 In Huh7 Cells

Objective:In this study we mainly investigated the effect of hepatitis C virus genotype 1b core protein on the expression of Plk1, CDK1 in Huh7 cells,and explored the relationship between HCV core and cell cycle, and its possible mechanism.Methods:The recombination vectors of pcDNA3.1(-)core) was transfected into Huh7 cells using Lipofectamine2000, simultaneously, untransfected and transfected with empty vectors Huh7 cell as control group;pcDNA6.2-GW/EmGFP-miR-core and pcDNA3.1(-)core co-transfected into Huh7 cells by Lipofectamine 2000, simultaneously, untransfected,transfected with miR-negative, co-transfected core and miR-negative as control group. The expression of mRNA and protein of HCV core, Plk1 and CDK1 was detected by RT-PCR and Western blot. MTT assay was used to test the effect of HCV core on Huh7 cells proliferation. The cellcycle was analysed by Flow cytometry.Results:1. RT-PCR and Western blot analysis showed HCV core was detected successfully in transfected Huh7 cells.2. The relative expression of mRNA and protein of Plk1, CDK1 were higher in Group Huh7/core than those in Group Huh7, Huh7/ empty vector(P<0.05), there were no significant differences between Group Huh7 and Group Huh7/ empty vector(P>0.05); The relative expression of mRNA and protein of Plk1, CDK1 were higher in Group Huh7/core+miR-negative than those in Group Huh7, Group Huh7/miR-negative and Group Huh7/core+miR-core(P<0.05), there were no significant differences between the other groups(P>0.05).3. MTT assay was used to detect the Huh7 cells with one to five days after transfection, the rate of cells growth were faster in Group Huh/core than those in Group Huh7 and Group Huh7/ empty vector(P<0.05), while the rate of cells growth between the other groups were no significant differences(P>0.05); the rate of cells growth were faster in Group Huh7/core+miR-negative than those in Group Huh7,Group Huh7/miR-negative and Group Huh7/core+miR-core(P<0.05),there were no significant differences between the other groups(P>0.05).4. The levels of G0/G1 phase, S phase were lower in Group Huh/core than those in Group Huh7 and Group Huh7/ empty vector, but the rate ofG2/M phase were significantly higher in Group Huh/core than those in Group Huh7 and Group Huh7/ empty vector(P<0.05), in addition, the cells of G0/G1 phase, S phase or G2/M phase among the remaining groups were no significantly different(P>0.05). The levels of G0/G1 phase, S phase were lower in Huh7/core+miR-negative than those in Group Huh7, Group Huh7/miR-negative and Group Huh7/core+miR-core,but the rate of G2/M phase were significantly higher in Group Huh7/core+miR-negative than those in Group Huh7, Group Huh7/miR-negative and Group Huh7/core+miR-core(P<0.05), there were no significant differences between the other groups(P>0.05)Conclusion:HCV core protein promote the cell to enter G2/M phase by up-regulating the expression of Plk1 and CDK1 in Huh7 cells, which can accelerate the cell cycle progression and promote cell proliferation.