Apoptosis Of Hepatoma Cells Induced By Trail With Survivin Promoter

BackgroundLiver cancer is one of the most common malignant tumors,currently the main treatments are surgery,intervention and radiotherapy,while the gene therapy has become a new treatment of liver cancer.In the study of tumor gene therapy,the key is to rise the therapeutic targeting.Using the promoter sequence expressing in most tumors to induce the specifical expression of target genes in tumor cells is an effective way of tumor gene therapy.Survivin is a new member in IAP family.The most important feature of Survivin is the selectivity of distribution,which is highly expressed in embroys and a variety of tumor cell lines,but not expressed in normal tissues.Therefore,the researchers think survivin promoter can be used as a tool for tumor therapy:to activate specific expression of the downstream anti-gene in tumor cells,and avoid damage to normal cells.TRAIL(tumor necrosis factor related apoptosis inducing ligand),also known as Apo2L(ligand),is a member of TNF superfamily found by Wiley.TRAIL can induce tumor cells and transform cell apoptosis but has no effect to the majority of normal cells,which indicates that Trail has a wide application prospect in tumor therapy.In this study,we intend to construct the recombinant plasmid containing Survivin340 promoter and Trail cDNA gene, evaluate the difference in the expression of Trail protein between tumor cells and normal cells,as well as apoptosis of tumor cells induced by the recombinant plasmid.ObjectiveTo clone human Trail cDNA genes and Survivin promoter gene,construct recombinant plasmid pcDNA3.1/SurP/Trail.To detect the expression of Trail protein induced by Survivin340 promoter in different cells and the apoptosis of hepatoma carcinoma cells,which were transfected with recombinant plasmid pcDNA3.1/SurP/ Trail.Methods1,Primer designAccording to the TRAIL and Survivin340 promoter gene sequence,we separately designed two pairs of primers.Trail gene sense primer:5'-TCCG CTCGAGCCTCACTGACTATAAAAGA-ATAGAG-3 ' containing restrict ion site Xhoâ… ;anti-sense primer:5'-CCCAAGCTTGGGTTAGC-CAACTAA AAAGGCC-3' containing a Hindâ…¢restriction site.The expected fragment length of PCR amplification product is 955bp.Survivin340 gene promoter sense primer 5'-GAA GATCTTC-TCAAGTGATGCTCCTGCCTA-3 'containing the restriction site Bglâ…¡, the anti-sense primer 5'-TCCGCTCGAGTGTAGAGATGCGGTGGTCCT-3' containing the Xhoâ… restriction site.The expected fragment length of PCR amplification product is 423bp.2,Construction of pcONA3.1/TrailTotal RNA was isolated from HepG2 cells.Reverse transcription-polymerase chain reaction(RT-PCR) was performed in accordance with the manufacturer' instructions.The PCR conditions were as follows:pre-denaturation at 94℃2 min, then denaturation at 94℃15 sec,annealing at 53℃30 see,and extension at 68℃75 sec,for 32 cycles,with the final extension at 68℃for 5 min after the last cycle of amplification.The PCR product was digested with Xhoâ… and Hindâ…¢restriction enzymes and subcloned into Eukaryotic expression vector pcDNA3.1(-) between Xhoâ… and Hindâ…¢sites.The reconstruction plasmid was amplified and purified, detected by digesting with Xhoâ… and Hindâ…¢restriction enzymes,and sequenced.3,Construction of pcDNA3.1/SurP/Trail Cloned Survivin340 gene promoter using plasmid pluc340(preserved in our laboratory) as a template,PCR amplification conditions were as follows: pre-denaturation at 94℃15s,denatured at 94℃30s,annealing at 56.5℃30s,and extension at 68℃75s,for 30 cycle,with the final extension at 68℃5min.The PCR product and recombinant vector pcDNA3.1/Trail were separately digestied with restriction endonuclease Bglâ…¡and Xhoâ… ,the product was purified after digestion and connected T₄ ligase.The new reconstruction plasmid named pc DNA 3.1/SurP/Trail,then sequenced.4,Liposome transfectionAt the day before transfection,HepG2 cells and 3T3 cells were transferred to 6 well culture plates,with the concentration of 2×10⁵/ml.When about 80%of cells fused,the recombinant plasmid pcDNA3.1/SurP/Trail was transfected into HepG2 and 3T3 cells by lipofectamine-LTX.After 72 hours extracted total protein of cells, and detected the differences in expression of Trail protein between two groups of cells,by Western Blot method.5,Identify the activity of recombinant plasmidHepG2 cells and 3T3 cells were washed with PBS buffer,resuspended in RIPA cell lysis buffer and PMSF for 30 min,then 12000×g centrifugated for 30min, collected supernatant.The total amounts of protein were determined by the BCA assay.A total of 50μg of sample protein was then mixed with 2×sample buffer, denatured at 96℃for 10min,and separated on 12%SDS-PAGE.Proteins were transferred to PVDF membranes(Milipore).Membranes were blocked with 5%BSA in TBST for 2hr and then incubated with anti-trail antiserum diluted 1:1000 at 4℃overnight.The membranes were washed three times with TBST(TBS with 0.1% Tween-20) and then incubated for 1hr with 1:2000 dilution of horseradish peroxidase-conjugated anti-rabbit antibody.The bound antibody complexes were detected by the enhanced chemiluminescence(ECL) system.6,Detect the expression of Trail mRNA by semi-quantitative RT-PCRHepG2 cells were divided into 3 groups,cultured in 6-well plates.The first group was transfected recombinant plasmid pcDNA3.1/SurP/Trail,and defined as the recombinant plasmid group;the second group transfected pcDNA3.1(-) empty vector, defined as the blank plasmid group;the third group not transfected with any plasmid, defined as non-transfected group.After transfection 48 hours,extract the cell total RNA of each hole,then detected the expression of Trail mRNA of each group by semi-quantitative RT-PCR.7,Analysis of apoptosis by double labeling flow cytometryHepG2 cells were divided into 3 groups(the same as Items 2.6).Transfected with recombinant plasmid pcDNA3.1/SurP/Trail and pcDNA3.1(-) for 48 hours,then collected all cells in each group,and analysed the rate of apoptosis of cells in each group.8,Statistical AnalysisUsing SPSS13.0 software to analyze,the results were expressed in mean±SD. Comparison of two groups was used independent sample test,three groups used one-way analysis of variance(one-way ANOVA),inter-group multiple comparisons used Bonferroni method of analysis,and when P＜0.05,the difference was considered statistically significant.Results1,Construction of recombinant plasmid pcDNA3.1/TrailObtained 955bp DNA by RT-PCR.The PCR products were detected by 1%(w/v) agarose gels analysis(Fig.1).The recombinant plasmid pcDNA3.1/Trail was indentified by Xhoâ… and Hindâ…¢digestion,and detected by 1%(w/v) agarose gels analysis(Fig.2).2,Construction of recombinant plasmid pcDNA3.1/SurP/TrailWe successfully cloned Survivin340 gene promoter(435bp) and constructed a new recombinant plasmid pcDNA3.1/SurP/Trail.The amplified products were detected by 1%(w/v) agarose gels analysis(Fig.3A).The recombinant plasmid pcDNA3.1/SurP/Trail was indentified by Bglâ…¡and Xhoâ… digestion,and detected by 1%(w/v) agarose gels analysis(Fig.3B). 3,Identify the activity of recombinant plasmidThe recombinant plasmid pcDNA3.1/Trail was separately transfected into two cell lines:HepG2 hepatoma cells and 3T3 mouse embryonic fibroblast cells(as a control),the western blot results showed that there was no difference in expression of Trail protein in the two cells(Fig6).Compared the means of the two indepent groups by the independent sample t-test and the difference was significant(t=-0.580, P=0.593).Anthor recombinant plasmid pcDNA3.1/SurP/Trail was separately transfected into the same cell lines:HepG2 cells and 3T3 cells,the results showed that there was difference in expression of Trail protein in the two cells(Fig7). Compared the means of the two indepent groups by the independent sample t-test and the difference was significant(t=13.677,P=0.000).4,Detect the expression of Trail mRNA by semi-quantitative RT-PCRRT-PCR products of three different cells were detected through agarose gel electrophoresis(1%agarose gel electrophoresis 80V,time:30min) and the results showed the expression of Trail mRNA in the first group transfected with pcDNA3.1/SurP/Trail was significantly higher than the other 2 controlled groups (Figure 8).The three groups were compared by One-way analysis of variance (One-way ANOVA),and inter-group multiple comparisons used Bonferroni method. The difference between the recombinant plasmid group and blank plasmid group(P =0.001) was statistically significant.The difference between the recombinant plasmid group and non-transfected group(P=0.000) was statistically significant.While between the blank plasmid group and the non-transfected group(P=1.00) there was no difference.5,Analysis of apoptosis by double labeling flow cytometryThe apoptosis rate of the group transfected with recombinant plasmid pcDNA3.1/SurP/Trail was significantly higher than the other two groups.Figure 9. Among the three groups was compared by One-way analysis of variance(One-way ANOVA),and inter-group multiple comparisons used Bonferroni method.The difference between the recombinant plasmid group and blank plasmid group(P =0.000) was statistically significant.The difference between the recombinant plasmid group and non-transfected group(P=0.000) was statistically significant.While between the blank plasmid group and the non-transfected group(P=1.00) there was no difference.ConclusionIn this study,through molecular cloning techniques,we successfully cloned the Trail cDNA gene and Survivn promoter gene,and we constructed the recombinant plasmid pcDNA3.1/SurP/Trail,through western blot we know that the recombinant plasmid can significantly induce proteins Trail expression in tumor cells than in normal cells,also the recombinant plasmid pcDNA3.1/SurP/Trail can induce apoptosis of hepatoma cells.This experiment explores a new approach for tumor gene therapy.The results are expected to enhance the therapeutic effect of clinical liver cancer,the survival rate of patients and improve the quality of life for humanity;a new type of gene therapy vectors can be used for gene therapy,expected to declare patents and transfer technology,which will have a direct economic benefits.