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Objective To construct wtp53/junB fusion gene and its eukaryotic expression vector in order to provide the basis for further application of polygene union therapy in hepatocellular carcinoma. Methods Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and gene recombination techniques were used to construct the eukaryotic vector of pEGFP-C1-wtp53/junB fusion gene, which carries the enhanced green fluorescent protein (EGFP). The transfection of pEGFP-C1-wtp53/junB in hepatoma HepG2 cells was detected by the location of green fluorescence. Results The DNA sequence of wtp53/junB fusion gene was successfully cloned into the pEGFP-C1 plasmid and the sequence was the same as what we expected. Green fluorescence located on cell nucleus proved that pEGFP-C1-wtp53/junB was transfected into HepG2 cell line successfully. Conclusion We successfully constructed the eukaryotic vector of pEGFP-C1-wtp53/junB fusion gene, which carries the EGFP, and transfects it into human hepatoma cell nucleus. It may lay the basis for studying the synergetic effect of wtp53 and junB in hepatocellular carcinoma.
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Objective To analyze the human cellular repressor of E1A-stimulated genes (hCREG)using bioinformatics tools and predict the promoter position and transciption factor binding sites.Methods Complete coding hCREG sequences were obtained from Genbank.hCREG was analyzed with First EF software in order to obtain the promoter sequence of hCREG.Moreover,transcription factor binding sites of hCREG was convinced by Motif software.Subsequently,the transcription factor of hCREG by bioinformatics tools was related between hCREG and smooth muscle cells differentiation observed by both Western blot and immunoflourescence.Results The promoter of hCREG was located in -109~-359 bp of up-stream of transcriptional site,which was predicted with 945bp length.Transciption factor binding sites of hCREG was predicted by Motif software which was found with 80 transciption factors.The expression of hCREG and smooth muscle ?-actin(SM ?-actin)increased in HITASY after 72 hours with serum deprivation detected by both Western blot and immunoflourescence.Meanwhile,the results showed that the expression of wtp53 increased significantly.These results suggested that transcription factor wtp53 might regulate the expression of hCREG and promoted dedifferentiated phenotype of VSMCs.Conclusion The transcriptional information of the proximal promoter of hCREG obtained.As up-stream regulational factor of hCREG,wtp53 can up-regulate the expression of hCREG and may play a vital role in the process of VSMCs differentiation and phenotype modulation.
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Objective:To investigate the influence of p33~ING1b and wt-p53 on the biological activities of pancreatic cancer cells. Methods: Sense and anti-sense cDNAs of p33~ING1b were cloned into pcDNA3 eukaryotic expression vector separately, and the recombinant plasmids were solely or co-transfected with wt-p53 into pancreatic cell line SW1990. Flow cytometry was used to analyze the changes of cell cycle, Western blotting was used to evaluate the expression of p33~ING1b and p53 protein, and MG-P-MY staining was applied to observe cell apoptosis. Results: SW1990 cells solely transfected with sense p33~ING1b plasmid or co-transfected with wt-p53 and sense p33~ING1b plasmid expressed more p33~ING1b and had more apoptosis than the antisense p33~ING1b plasmid and mock transfected cells did (P