The Investigation Of Mechanism By Which P44 Regulating Cell Cycle In Hepatoma Cells In Vitro

Objective:To explore the effects of P44 on the cell cycle of the hepatoma cells .Methods:Human XPD gene was transfected into Hepatoma SMMC-7721 cells by LipofectAMINE and selected in medium containing G418 , 800μg/ml for SMMC-7721-pEGFP-N2/XPD ; SMMC-7721-pEGFP-N2; Cell lines for comparison were matched on the same genetic background and passage .After stably transfection,the antisense oligonucleotides of P44 were added to treat the stably transfected cells. Cells were randomly divided into 6 groups: Group①, control group, Group②, transfected with blank plasmid pEGFP-N2, Group③, transfected with recombinant plasmid pEGFP-N2/XPD, Group④, transfected with ASODN complementary to translation initiation site of pEGFP-N2/XPD, Group⑤, transfected with ASODN complementary to translation terminal site of pEGFP-N2/XPD, Group⑥, transfected with ASODN complementary to translation Exon5 site of pEGFP-N2/XPD. The expressions of wild-type P44,XPD,cdk7,cdk2,c-myc and cdc25A were detected by RT-PCR and Western blot. The cell growth and the cell cycle were examined by MTT and Flow Cytometry.Results:The P44 and XPD mRNA expression levels of group③are significantly higher than those of group①and②(both P<0.01), Western blot indicated that the changes of P44 and XPD protein expression levels are consistent with those of their mRNAs respectively; while the mRNA and protein expression levels of cdk7,cdk2,c-myc and cdc25A were all decreased. MTT method showed that the hepatoma cells grow slowly, FCM showed that the number of the cells arrested at the G1 stage of group③higher than those of group①and②.After the blockage of P44 gene expression , the expression of XPD mRNA and protein were decreased . the XPD mRNA and protein expression levels of group④,⑤and⑥are significantly higher than those of group③(all P<0.01); The mRNA and protein expression levels of cdk7, cdk2, c-myc and cdc25A are upregulated. MTT method indicated that cells grew fast. FCM showed that the numbers of the cells arrested at the G1 stage of group④,⑤and⑥were all lower than that of group③.The expressions levels of cell cycle regulatory genes including cdk7, cdk2, c-myc and cdc25A were markedly decreased,the hepatoma cells grew slowly; after the blockage of P44 gene expression, the expression of XPD mRNA and protein were decreased, whereas the expression levels of the cell cycle regulatory genes mentioned above were enhanced,and the hepatoma cells grew faster.Conclusion:There are interaction between P44 and XPD, which can regulate cell cycle.