| Objective To delineate the effect ofHBV X Gene (HBX) expression levels and Arsenic trioxide (As2O3) on apoptosis and p53 expression and activity of cell line HepG2 by use of shRNA-mediated RNA interference (RNAi)Methods HBV X gene fromplasmid pEcob6 was amplified by PCR assay with specific primers. With the characteristics of multiple clone sites in pCEP4 and pcDNA3.1 (+), pGEM-T Easy Vector was selected to construct middle vector pEasy-X.. After enzyme digestion, specific fragments were linked to constructeukaryotic expression vectors pCEP4-X and pcDNA3.1 (+)-X vectors. HepG2 Cells were:infected by plasmid lipofecmatin 2000with pCEP4-X and pcDNA3.1 (+)-X.. Positive cell clones was selectedby Hygromycin and Neomycin, After propagating Culture, HBX expression was identified by PCR, RT-PCR and Western Blot. Sequence-specific shRNA expression vector pXi-1,pXi-2 and sequence-unrelated control pXi-3 were constructed by cGFP-marked pRNAT-U6.1/Neo. They are transfected into HepG2-pCEP4-X,. GFP positive cell clones was selected by Hygromycin andNeomycin. After propagating culture, HBX expression levels was identified by RT-PCR and Western Blot. HepG2 and HepG2-pCEP4-X, were treated with 2umol/L As203 for 36 hours, with corresponding untreated cells as controls. The cell cycle and apoptosis of HCC were detected by flow cytometry,caspase-3 activity was assayed by Caspase-3/CPP32 colorimetric assay kit,total level and the relative activity absorbance of p53 was detected by modified ELISA kit. ShRNA expression vector, pXi-1,pXi-2 and pXi-3 were transfected into HepG2-pCEP4-X, and the cell cycle and apoptosis ratio and caspase-3 activity and p53 expression and activity were retested.Results HBV X gene was succeesfully cloned from pEcob6 by PCR assay and the amplified gene products were successfully inserted into pEasy, pCEP4 and pcDNA3.1 (+) vectors, The successful insertion of HBX gene into pCEP4-X and pcDNA3.1 (+)-X vectors was confirmed by enzyme digestion, PCR assay and sequencing. Positive cell clones with HBX transferred resistant to Hygromycin and Neomycin was selectively cultured. PCR, RT-PCR and Western Blot identification demonstrated expression of HBX. HepG2-pCEP4-X cells were infected with ShRNA expression vector pXi-1, pXi-2 and pXi-3, RT-PCR and Western Blot demonstrated that sequence specific pXi-1, pXi-2 could significantly inhibited HBX expression, while sequence-unrelated control, pXi-3, did not produce any effect on HBX expression. The apoptosis ratios of cells treated with 2.0umol/L As2O3 for 36 hours was significantly higher, but and ratio of cells transfected with shRNA expressing vectors was higher too. The apoptosis ratios were grossly significantly different among various groups with different HBX expression levels (P＜0.05), the ratios were down-regulated in groups with expression of HBX, and the down-regulation was removed after HBX expression was repressed by shRNA. The cells in G1 phase were down-regulated in groups treated with 2.0umol/L As2O3 for 36 hours (P＜0.01), and cells in G2/M phase were up-regulated (P＜0.01). But cells in G1 phase or in G2/M phase or in S phase revealed no significant difference despite different levels of HBX expression. OD405nm which reflected the relative activity of caspase 3 in cell lysate were grossly significantly different among various groups (P＜0.01), but activity of caspase 3 revealed no significant difference despite treated with As2O3 or not (P＞0.05). Activity of caspase 3 was significant difference among As2O3 treated cell groups with different HBX expression levels (P=0.000), and the activity was down-regulated in groups with expression of HBX, and the down-regulation was removed after HBX expression was repressed by shRNA. Total p53 level was up-regulated and its relative activity ratio was enhanced by As2O3 in HepG2 and HepG2- pCEP4-X. The total p53 level induced by As2O3 was further up-regulated by HBX expression, while its relative activity was significantly suppressed. The suppression was removed after HBX expression was suppressed by shRNA.Conclusion 1. Two eukaryotic expression vectors of HBV x gene with different selection characteristics were successfully constructed.2. Two HCC cell lines with steady expression of HBV x gene and different selection characteristics were successfully constructed.3. Specific shRNA could substantially inhibit HBV X gene expression in HepG2-pCEP4-X.4. Treatment with 2.0 umol/L As2O3 for 36 hours could induce apoptosis in HepG2, and As2O3 could block the cells proliferation in G2/M phase, but whether it was corrected with the activity of caspase 3 in cell need farther studies.5. After treatment with As2O3, cell apoptosis ratio and caspase 3 activity of HepG2-pCEP4-X were lower than those of HepG2. Repression of HBX expression by shRNA could remove the difference. The response was unrelated with cell cycle. induced by As2O3 was down-regulated in HepG2 with expression of HBX, and the relative activity of caspase 3 was down-regulated too, the down-regulation could be removed after HBX expression was repressed by shRNA, and the effect did not correct with cell cycle.6. As203 could up-regulate p53 expression and enhance its activity.7. HBV X expression could up-regulate p53 gene expression level induced by As2O3, while it suppressed the activity of p53, the down-regulation could be removed after HBX expression was repressed by shRNA.
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