Effects Of E2F-1Overexpression In Human Gastric Cancer Cell Line SGC-7901of Proliferation And Its Mechanism

Objective Explore the effect of Application of E2F-1gene reversetranscription of recombinant lentiviral overexpression vector in human gastriccancer cell line SGC-7901on proliferation, growth,cell cycle and the molecularmechanism.Methods The log phase growth of human gastric cancer SGC-7901cellswere inoculated in culture plate and divided into three groups. Humans gastriccancer SGC-7901cells were infected by carrying E2F-1recombinant lentiviralparticles (LV-E2F-1-GFP),as a experimental group (LV-E2F-1-GFP group);Control Lentiviral Particles (LV-GFP) infected with human gastric cancerSGC-7901cells, as a negative control group (LV-GFP group); blank controlgroup was cultured conventionally, without any treatment. CCK-8were used todetect cell proliferation activity, each group was detected by flow cytometrycell cycle distribution, semi-quantitative reverse transcription-polymerase chain reaction (PT-PCR) and Western blot to detect cells Skp2, Bax,Bcl-2, CylinD1and Survivin mRNA and protein expression. The Measurementdata was expressed by mean+-standard deviation (+-s) Multi-group comparisons were analyzed with one-way anova,two group comparison wasanalyzed with SNK-q test,rates were compared by chi-square test,when p<0.05it has statistical diffrentcesResults CCK-8were used to detect cell proliferation activity,experimental group, negative control group and blank control group, theirabsorbance values(A450) at each time were:0h:0.223±0.020、0.229±0.027、0.226±0.018respectively;24h:0.360±0.024、0.490±0.040、0.484±0.049respectively;48h:0.480±0.057、0.631±0.019、0.631±0.014respectively;72h:0.740±0.020、0.939±0.052、0.911±0.043respectively;96h:0.816±0.128、1.197±0.047、1.232±0.065respectively. Compared with LV-GFP group andblank control group, LV-E2F-1-GFP group of human gastric cancer cell lineSGC-7901proliferation activity was significantly lower (F=0.419，P <0.05),The difference was statistically significant. The cell cycle was detected by flowcytometry, experimental group, negative control group and blank controlgroup, Their cell number total proportion in cell division cycle of each phasewere as follow: G0/G1phase were6.389±1.954%、4.781±0.635%、4.784±1.484%respectively;G2/M phase were1.182±0.123%、1.190±0.711%、1.178±0.918%respectively;S phase were2.429±1.832%、4.029±1.307%、4.038±1.231%respectively; Compared with negative control group and blankcontrol group,in experimental group the G0/G1phase proportion increased(χ2=7.175、7.546，P <0.05), while S phase proportion reduced, the differencewas statistically significant. The relative expression of mRNA of experimentalgroup, negative control group and blank control group respectively were:E2F-1was0.599±0.018、0.483±0.001、0.507±0.008，Bax were0.786±0.000、0.610±0.150、0.588±0.024， Bcl-2were0.264±0.003、0.441±0.003、0.432±0.007; Skp2were0.395±0.009、0.501±0.005、0.517±0.010; Survivin were0.472±0.050、0.628±0.010、0.641±0.003，CylinD1were0.394±0.018、0.693±0.005、0.714±0.006，Compared with negative control group and blankcontrol group, In experimental group,the relative mRNA expression of Skp2,Bcl-2, CylinD1and Survivin Obviously decreased, the relative mRNAexpression of E2F-1and Bax obviously increased (F=11.637、36.567、17.154、14.294、12.669、135.211，P <0.05), the difference was statistically significantIn The experimental group, negative control group and blank control group, thegene protein relative expression of human gastric cancer SGC-7901cellsrespectively were:E2F-1:1.242±0.176、0.432±0.000、0.545±0.215; Bax:0.967±0.059、0.469±0.894、0.363±0.037; Bcl-2:0.087±0.008、0.149±0.012、0.144±0.005; Skp2:0.144±0.001、0.363±0.002、0.321±0.002; Survivin:0.517±0.016、0.801±0.46、0.665±0.011; CylinD1:0.576±0.041、0.773±0.016、0.777±0.007，Compared with negative control group and blank control group, Inexperimental group,the relative protein expression of Skp2, Bcl-2, CylinD1and Survivin Obviously decreased, the relative protein expression of E2F-1and Bax obviously increased (F=186.183、84.552、2.327、10.546、5.439、22.682，P <0.05), the difference was statistically significant.while Compared withnegative control group and blank control group, the difference was notstatistically significant (P>0.05).Conclusion Lentivirus-mediated overexpression of E2F-1geneinhibits cell proliferation and growth of gastric cancer, so that the cell cyclearrest in the G0/G1phase. And its mechanism may be related to theoverexpression of E2F-1gene in gastric cancer cells which leads to the downregulated expression of gene Survivin,Skp2,Bcl-2,CylinD1,and the up regulatedexpression of gene Bax.