The Effects Of XPD On The Expressions Of DNp73and GADD45β In SMMC-7721Hepatoma Cells

Aim：To evaluate the mRNAs and proteins expressions of GADD45beta and DNp73genes in human SMMC-7721hepatoma carcinoma cells that have been transfectedwith XPD. And observe the impact of XPD on the SMMC-7721cell proliferation andapoptosis.Methods：1. SMMC-7721hepatoma cells were cultured in PRIM-1640supplementedwith10%fetal calf serum at37℃and5%CO2.2. pEGFP-N2-XPD and pEGFP-N2were transfected into SMMC-7721cells byLipofectamine2000, respectively. SMMC-7721cells were divided into four groups:SMMC-7721-pEGFP-N2-XPD（XPD group), SMMC-7721-pEGFP-N2(N2group),Lipofectamine (Lip group) and blank control group（Blank group）. Fluorescentmicroscope was employed to observe the transfection results.3. The PCR primers of those genes（XPD, DNp73, GADD45beta）weredesigned and produced by Invitrogen company. At48hours after transfection, RNAsand proteins were extracted; Then the expressions of XPD, DNp73andGADD45βwere detected by RT-PCR and Western blotting, respectively.4. Band Leader3.0and Quantity One were used to analysis the results ofRT-PCR and Western blotting.5. The cell proliferation was assessed by MTT assay.6. The changes in cell apoptosis were evaluated by flow cytometry.7. Statistical software SPSS13.0was used to analysis the datas.Results:1. Green Fluorescent Protein (GFP) was expressed in SMMC-7721-pEGFP-N2-XPD group cells and SMMC-7721-pEGFP-N2group cells. In contrast, GFP wasundetectable in Lip group and Blank group cells by fluorescent microscope.2. Compared with SMMC-7721-PeGFP-N2group、liposome group and Blankgroup，the expreesion of DNp73mRNAs reduced obvioussly in SMMC-7721- Pegfp-N2-XPD group（P<0.01）. However，the expressions of XPD and GADD45βmRNAs were enhanced significantly in SMMC-7721-Pegfp-N2-XPD group(P <0.01). There was no statistical diffirences of XPD, DNp73and GADD45βmRNAsexpressions among Blank group, Lip group and N2group（P>0.05）.3. Western blotting was used to observe the protein expressions of XPD、GADD45βand DNp73. And the resualts suggest that the protein expression trends ofthem were consistent with their mRNAs expression trends.4. The proliferation of SMMC-7721cells was markedly inhibitedby MTT (P <0.01).5. The apoptosis of SMMC-7721cells was increased after transfected by XPDby flow cytometry (P <0.01).Conclusion：XPD as an tumor suppressor gene can inhibit HCC proliferation and promotethem apoptosis, one of the mechanism maybe to regulate the expressions ofoncogenes and tumor suppressor genes.We found that the expression ofGADD45βincreased and the expression of DNp73decreased with the overexpressionof XPD, which suggests that they may play an important role in the process of XPDinhibiting the carcinogenesis of HCC.