Anti-HCC Effect Of A Recombinant Vector Carrying The Buforin Ⅱ Gene Dirven By Survivin Promoter

Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors.The incidence and mortality of HCC is found increasing year by year. Theworldwide new cases of HCC are approximately560,000each year, of which nearly50%of cases occur in China. Surgical resection remains the main treatment of HCC.But5-year survival is only30%to40%for the patients who accepted radical surgery.Local regional therapy including cryoablation, radiofrequency ablation, andtransarterial embolization exhibits obvious adverse reaction. Based on the traditionaltreatment, with the development of molecular biological theory and technology, genetherapy for HCC becomes a new available selection. However, gene therapy of HCCis still in the exploratory and experimental stage, because problems including thespecific expression of therapeutic gene, the safety, effectiveness and controllness ofthe vectors need to be solved before it can be applied clinically.Survivin is the smallest member of the inhibitors of apoptosis (IAP) proteinfamily which is highly expressed in primary tumors and cancer cell lines, butundetectable in most somatic tissues and normal differentiated cells. Owing to itsmassive upregulation in human tumors, the survivin promoter has the potential to beused for cancer-specific expression of a therapeutic gene.A number of genes have been tested for their use in cancer gene therapy. Mostof these genes encode cellular proteins that are involved in apoptosis and/oranti-proliferation, such as p53, E1A, BAX, and Bik. Recently, significant progresshas also been made in the exploitation of various novel cytotoxic genes as therapeutic tools to specifically kill cancer cells, arrest angiogenesis, and suppressthe growth of different tumors. Buforin Ⅱ is a member of AMPs. The amino acidssequence of Buforin Ⅱ protein was determined as TRSSRAGLQFPVGRVHRLLRKwhich has selective cytotoxicity in60different tumor cell lines.In this study, we attempt to construct the recombinant plasmid pSUR-Buforin2,which will be driven by survivin promoter and encode Buforin Ⅱ gene, theanti-tumor effect on HCC induced by pSUR-Buforin2will be observed by in vitroexperiments. The mchanism of apoptosis and proliferation revealed by this studywill lay the foundation for further animal experiments.ObjectiveTo construct a recombinant plasmid pSUR-Buforin2encoding BuforinⅡdirven by survivin promoter, and observe its anti-tumor effect on HCC.Methods1. The eukaryotic expression plasmid pSUR-Luc and pSUR-GFP encoding greenfluorescent protein (GFP) driven by survivin promoter were constructed bymeans of RT-PCR and gene cloning technique, these plasmids were furtherconfirmed by restriction endonuclease analysis and sequencing analysis.2. The recombinant plasmid pSUR-GFP was transfected into HepG2cells and L02cells with X-trmeGENE according to the manufacturer’s instructions. Toevaluate the activity of survivin promoter, the expression of GFP was detectedby flow cytometry and observed under fluorescence microscope.3. Buforin Ⅱ gene was artificially synthesized in mammal cell preferred condonsmanner according to its amino acid sequence. The recombinant plasmid pSUR- Buforin2was generated by cloning the Buforin Ⅱ gene into the vectorpSUR-Luc and characterized by restriction endonuclease analysis andsequencing analysis.4. HepG2cells and L02cells were transfected with the recombinant plasmidpSUR-Buforin2using X-tremeGENE. Plasmid vector pSUR-Luc and cellculture medium were used as negative and blank control, respectively. RT-PCRwas used to detect the expression of Buforin Ⅱin the HepG2and L02cells.5. HepG2cells and L02cells were transfected with the recombinant plasmidpSUR-Buforin2using X-tremeGENE. MTT assay was used to evaluate theproliferation of HepG2and L02cells.6. The recombinant plasmid pSUR-Buforin2was transfected into HepG2cellsusing X-tremeGENE. Cell apoptosis induced by pSUR-Buforin2transfectionwas observed by flow cytometry analysis, DNA gel electrophoresis for DNAfragmentation, and detection of apoptosis and proliferation signaling.Results1. The pSUR-GFP plasmid was constructed successfully, which was confirmed byDNA sequencing. After pSUR-GFP was transfected into HepG2cells and L02cells, the expression of green fluorescent protein was observed in HepG2cells,but not in L02cells, and the GFP-positive HepG2cells accounted forapproximate40.9%of whole HepG2cells determined by flow cytometryanalysis.2. The pSUR-GFP plasmid was constructed successfully, which was confirmed byrestriction endonuclease analysis and DNA sequencing. RT-PCR results showedthat the survivin promoter could induce abundant expression of Buforin Ⅱgenejust in HepG2cells tranfected with pSUR-Buforin2, whereas the Buforin Ⅱ gene expression was barely detected in L02cells transfected withpSUR-Buforin2.3. Forty eight hours after pSUR-Buforin2transfection, HepG2cells showedsignificant decrease in cell viability, compared with control HepG2cells, therewas no discernable cytotoxicity of pSUR-Buforin2transfection on normal L02cells.4. Flow cytometry analysis showed that pSUR-Buforin2transfection induced muchmore apoptotic cells than pSUR–Luc transfection or normal control HepG2cells.pSUR-Buforin2transfection could induce DNA fragmentation in the HepG2cells. Apoptosis related signaling transducers like caspase-3and proliferationrelated molecules like PCNA were detected by western blotting analysis.Caspase-3was activated accompanied by obvious induction of the cleavedcaspase-3, while PCNA expression was inhibited in the pSUR-Buforin2-transfected HepG2cells.Conclusions1. In the present study, a non-viral vector was successfully constructed by insertingthe survivin promoter into pGL3-basic vector, and used to drive GFP expression.Results from the in vitro transfection experiments, and flow cytometry analysisdemonstrate that survivin promoter is specifically activated only in HepG2cellsbut not in L02cells, indicating survivin is a tumor specific promoter, andsuitable for construction of HCC gene therapy vector.2. We successfully constructed a recombinant plasmid pSUR-Buforin2thatencodes Buforin Ⅱ driven by survivin promoter for HCC-specific gene therapy.pSUR-Buforin2expressed Buforin Ⅱ specifically in tumor cells and killedtumor cells. The basic apoptotic mechanism was involved in the process of tumor cell cytolysis.