The Feasibility And Mechanism Of Survivin Regulation Of Transcription Level By P21^CIP1 Overexpression In HepG2 Hepatoblastoma Cell

PartⅠConstruction of Eukaryotic Expression Vector pEGFP-C2-p21 and Establishment of HepG2 Cell Stably Transfected by p21^cip1 GeneObjective:To construct the Eukaryotic expression vector pEGFP-C2-p21 and identify its DNA sequence. In addition, to transfect the recombined plasmid pEGFP-C2-p21 into HepG2 cell and screen out the HepG2 cell with overexpressed wt-p21^cip1.Methods:The fragment of p21^cip1cDNA was cloned into the expressing vector pEGFP-C2, and then the constructed plasmid was verified by the restriction enzyme analysis, colony PCR and DNA sequencing. Following this, Eukaryotic vector pEGFP-C2-p21 was transfected into HepG cell by lipofectamine and positive clones were screened out by G418. At last, total mRNA of HepG2 cell was extracted to warrant further investigation.Result:(1) The p21^cip1 gene was successfully inserted into pEGFP-C2 vector and identified by DNA sequencing.(2) After transfection and G418 treatment, the single clone HepG2 cell with p21^cip1 overexpression was got at last, meanwhile the expression GFP-p21^cip1 fusion protein was found by fluorescence microscope detection.Conclusion:The Eukaryotic expression vector pEGFP-C2-p21 has been successfully constructed and it could efficiently express in HepG2 cell, it may thus provide experimental basis for further research. PartⅡThe Feasibility and Mechanism of survivin Regulation of Transcription Level by p21^cip1/waf1 Overexpression in HepG2 Hepatoblastoma Cell LineObjective:To observe the effect of cyclin-dependent kinase inhibitors Cip1/Waf1(p21) on regulatory expression of survivin transcription of human liver neoplasm cells HepG, and explore the related mechanisms.Methods:adriamycin (ADM) was used to treat HepG; eukaryotic vector pEGFP-C2-p21 was transfected into HepG by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21, p53 and survivin were detected by real-time fluorescent quantitative polymerase chain reaction(RQ-PCR); flow cytometry method was used to examine the cell phase; and revere transcription polymerase chain reaction(RT-PCR) was used to measure the levels of E2F-1 or p300.Result:⑴After treatments with ADM, the expression of p53 and p21 was increased, whereas that of survivin was reduced during 24 hours of treatmentst respectively.⑵p21 level after transfection was significantly enhanced to 2100.11-folds or 980.89-folds comparison with HepG2 ones or HepG2-C2ones; survivin level was markedly down-regulated to 0.54% or 0.59% relative to the other two groups; nevertheless that of p53 changes was not observed.⑶Overexpression of p21 resulted in G1/G0 phase arrest (F = 31.59, P < 0.01); meanwhile, E2F-1 mRNA or p300 mRNA were less expressed compared with that of the other controls (F E2F-1 = 125.28, P < 0.05; F p300 = 46.01, P < 0.01).Conclusion:p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cells, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1and p300.PartⅢInhibitory Effect of Adriamycin Combined with p21CIP1 Gene Transfection on Proliferation of Human Hepatoblastoma Cell Line HepG2Objective:Chemotherapy protocols using adriamycin (ADM) is a standard treatment for hepatoblastoma, but the treatment results became unsatisfied because of drug resistance. Recently, ADM combined with gene therapy is a developing alternative treatment for hepatoblastoma. This study was to investigate the effect of ADM combined with human P21CIP1 transfection on the proliferation of hepatoblastoma cell line HepG2.Methods:HepG2 cells were divided into empty control (no treatment), ADM group (treated with 0.5μg/mL ADM), blank control group (transfected with blank plasmid pEGFP-C2), P21 group (transfected with plasmid pEGFP-C2-P21), and combination group (ADM treatment plus P21 transfection). The proliferation of HepG2 cells was observed by MTT assay. The mRNA level of p21 and survivin were detected by real-time polymerase chain reaction (PCR).Result:After transfection, the mRNA level of p21 in P21 group was increased by 2100.11 folds of that in empty control group (P<0.05). P21 inhibited the proliferation of HepG2 cells at Day 3 and Day 4 after transfection (P<0.01). The proliferation inhibition rate was significantly higher in combination group than in ADM group and P21 group (43.92% vs. 32.97% and 35.77% at Day 3, P<0.01; 59.86% vs. 39.35% and 40.96% at Day 4, P<0.01; 51.81% vs. 33.91% and 10.68% at Day 5, P<0.01). This effect was enhanced along with the increasing time of co-treatment from Day 1 to Day 4 (r=0.91, P<0.05), and it was obvious at Day 4 (Q=1.07). The mRNA level of survivin was significantly lower in combination group than in P21 group and ADM group (P<0.01).Conclusion:P21 gene transfection plus ADM can co-effectively inhibit the proliferation of HepG2 cells and down-regulate the level of survivin mRNA, thus may be a potential therapeutic strategy against human hepatoblastoma.