The Effect And Mechanism Study Of Arsenic Trioxide Joint Canstatin Gene Therapy For HepG2Cells

Object: Human hepatoma HepG2cells for the study,observed Canstatin,a small dose of As2O3and twojoint program of HepG2cells in vitro(proliferation,adhesion,invasion,migration,apoptosis),and to explorethe underlying mechanism,with a view to As2O3beat the feasibility of a joint program of chemotherapy andthe prevention and treatment of liver cancer in clinical experimental data and theoretical basis.Methods:1. Identification of adenovirus;2.adenovirus infection to determine ways and multiplicity ofinfection;.3. Real-Time-PCR and relative quantification Canstatin mRNA and protein expression in cellsdetected by Western blot;4. CCK-8assay in HepG2cells As2O3for72h half inhibitory concentration(IC50),with an IC50of after baseline subtraction to determine the concentration of As2O3interventionsmaller doses;5. Experiment were divided into As2O3group(1/8、1/4、1/2、1)IC50、Canstatin(Ad-Canstatin-GFP)group、As2O3(1/8、1/4、1/2、1)IC50+Canstatin group、Ad-GFPempty vectorgroup and the control group.6. CCK-8assay at24h,48h,72h, respectively,in each group to detectchanges in cell proliferation; Annexin V-PE/7ADD flow cytometry in each group for24h apoptosis rate;incells homogeneous matrix adhesion and adhesive ability changes were detected by Cell counting andCCK-8assay; Transwell chamber assay variation of each group cell invasion and migration capabilities.7Relative quantitative Real-Time-PCR method to detect the effect of the cells after24h, PCNA mRNAexpression in cells, Western blot to detect the expression of PCNA、MMP-2and Caspase-3protein.Results:1PCR electrophoresis see the size of476bp bands Sequencing canstatin identify human genes;2Quantitative PCR showed cells by Ad-Canstatin-GFP transfected,canstatin initial gene copy number wasapproximately38.82±4.24times the control group;cells with GFP green fluorescence;Determined infectedwith MOI=40,3times72h for subsequent experiments Infection;3Canstatin group Canstatin proteinexpression in cells than the control group and empty vector group(P<0.05);4SPSS17.0softwarecalculated IC50of As2O3for72h was10μmol/L;5Proliferation(450nm wavelength absorbance values):①As2O3group,24h(1/8,1/4)IC50group compared with control group P>0.05,1/8IC50group72h>48h>control group(P<0.01);(1/2,1)IC50group,72h<48h<24h <control group(P<0.01), IC50group<1/2IC50group (P<0.01);②Canstatin group:24h,48h,72h <control group (P<0.01),24h lower thanAs2O3(1/8,1/4,1/2)IC50group(P<0.01),48h、72h respectively below As2O3(1/8,1/4,1/2,1)IC50group(P<0.01);③c ombined group,Canstatin with As2O3(1/8,1/4,1/2)IC50,24h,48h,72h were lowerthan during the corresponding concentrations of As2O3group (P<0.05),48h, while72h Canstatin+IC50group with IC50group P>0.05; Kim Jong law judge Canstatain and As2O3were combined effect:24h theQ value,1.15>Canstatin+1/8IC50group>Canstatin+1/2IC50group>0.85indicates that the sum effect,0.55<Canstatin+1/4IC50group<Canstatin+IC50group<0.85indicate the role of antagonist;48h the Qvalue,1.15>Canstatin+1/8IC50group>Canstatin+1/4IC50group>Canstatin+1/2IC50group>0.85indicates that the sum effect,0.55<Canstatin+IC50group<0.85indicate the role of antagonist;72h the Qvalue,1.15>Canstatin+1/4IC50group>Canstatin+1/8IC50group>Canstatin+1/2IC50group>Canstatin+IC50group>0.85indicates that the sum effect.6Apoptosis rate (24h):①As2O3groupexcept1/4IC50group outside the rest of the group were higher than control group(P<0.05);②Canstatingroup with the control group and the empty vector group P>0.05;③c ombined group was higherthancontrol group (P<0.05),and higher than the corresponding concentrations of As2O3group(P<0.05).7Adhesion,invasion and migration:①As2O3group: Adhesion of cells60min homogeneous group>30mingroup(P<0.01),IC50group>1/2IC50group>1/4IC50group>control group(P<0.01); Matrix adhesion(450nm wavelength absorbance)IC50group<1/2IC50group<1/4IC50group<group(P<0.01); Invasion(cell number)IC50group<1/2IC50group<1/4IC50group<1/8IC50group<control group(P<0.01);Migration(cell number)IC50group<1/2IC50group<1/4IC50group<control group(P<0.01).②Canstatingroup with the control group and the empty vector group P>0.05;③Canstatin each combination groupwith the control group and empty vector group P<0.01, but with the corresponding concentrations of As2O3 group P>0.05.8PCNA mRNA expression:①As2O3IC50group<1/2IC50group<control group(P<0.05);②Cansatain group with the control group and the empty vector groupP>0.05;③Canstatin eachcombination group were lower than in the control group(P<0.01), with the corresponding concentrations ofAs2O3group P>0.05.9Western blot detection of protein expression:①PCNA protein expression,As2O3IC50group<1/2IC50group<1/4IC50group<1/8IC50group<control group(P<0.01); MMP-2proteinexpression,As2O3IC50group <1/2IC50group <1/8IC50group <control group (P<0.01),1/2IC50groupwith1/4IC50group P>0.05; Caspase-3protein expression,As2O3IC50group>1/2IC50group>1/8IC50group>control group(P<0.01),1/4IC50compared with control group P>0.05.②Cansatain groupPCNA,MMP-2protein expression than the control group and the empty vector group(P<0.05), Caspase-3protein expression is higher than in the control group and the empty vector group (P<0.01);③Joint Group:PCNA expression and MMP-2protein were lower than Canstatin group(P<0.05), the expression ofCaspase-3protein were higher than Canstatin group (P<0.01).Conclusion:1As2O3(5,10μmol/L)with a more stable HepG2cells inhibited proliferation, adhesion, invasion, migration,promote apoptosis, the role and function of the concentration with time increasing their proliferation,adhesion inhibition enhanced, probably with As2O3down PCNA and MMP-2protein expression, increasedexpression of Caspase-3-related; but its concentration is too small(1.25,2.5)μmol/L, the above effect ofinstability or render the opposite effect.2Canstatin can downregulate the expression of PCNA protein in HepG2cells, and inhibit the proliferationof HepG2cells; although can upregulate the expression of Caspase-3protein and reduced expression ofMMP-2protein, but on apoptosis, adhesion, invasion and migration behavior did not affect.3Canstatin additive effect combined with As2O3inhibited the proliferation of HepG2cells was strongerthan As2O3, and Canstatin with low concentrations of As2O3(1.25μmol/L,2.5μmol/L)jointly presentedwith high concentrations stronger than As2O3(10.0μmol/L)combined group, may be associated withreduced expression of PCNA, MMP-2protein expression of Caspase-3protein upregulated related;Canstatin combined with As2O3inhibited HepG2cell adhesion, invasion and migration behavior andpromote apoptosis, but no significant difference between As2O3group.