The Research On Impact Of Differentially Expressed ANXA10Gene On The Gene Expression Profiles Of The Human Hepatocellular Carcinoma Cell Line (HepG2)

Objective：1、The overexpression lentiviral of human ANXA10gene was constructed andwas transfected into human hepatocellular carcinoma cell line HepG2.2、To determine the effect of overexpression lentiviral of human ANXA10on thegene expression profile of human hepatocellular cell line HepG2.The gene regulationrole of ANXA10in hepatocellular carcinoma oncogenesis and development wasilluminated simply.We want to lay a foundation for research of key genes and theirsignal transduction pathways in hepatocellular carcinoma oncogenesis anddevelopment and to seek new pathways and potential ideal target for gene therapy ofHepatocellular Carcinoma.Methods：1、In vitro experiments, the over expression of lentiviral ANXA10withMOI=10was transfected into human hapatoma cell line HepG2,divide into Lentiviralvectors group (LV-GFP-ANXA10) and HepG2group.The over expression oflentiviral ANXA10transfection efficiency was determined by observing the GFPexpression by using the fluorescence microscope.The differences of Two groups ofcells gene expression profiles were compared by Illumina-Solexa sequencing，andpart of the differences in gene expression levels was validated by quantitativereverse-transcripted polymerase Chain reaction.2、In vivo experiments, the log phase growth of HepG2cells were digestde into singlecell suspension with trypsin,and200ul HepG2single cell suspension(1×107cells/ml) were implanted right armpit. About1week after inoculation, the HepG2cellsbegin to form tumors.On the twenty-four day,the tumor diameter was about1-2cmsize. Nude mice were sacrificed with the method of dragging the neck,and remove thetumor of nude mice.The tumor was cut into1mm3size,and the tumor tissue blockswere inoculated to16female nude mice (6-8weeks old) right axillarysubcutaneous.After one week,the tumor diameter was about0.3-0.5cm size,the micewere randomly divided into two groups and named the experimental group (the LV-ANXA10group) and the blank control group respectively. Each nude mice of theexperimental group was given the lentiviral ANXA10with2×10~7TU (100ul),andevery nude mice of the blank control group was given100ul of PBS with theconcentration of0.01mol/L;Injections was repeated after3hour,four weeks later,16nude mice were sacrificed with the method of dragging the neck,and remove thetumor of nude mice.The expression level of partial differential gene protein whichwas validated by quantitative reverse-transcripted polymerase Chain reaction wasfinally examined by Immunohistochemistry.Results：1、The overexpression of ANXA10gene influenced the gene expressionprofiles of human hepatocellular carcinoma cell line HepG2.By analysis of thebiological information, a total of118genes were up-regulated and12genes weredown-regulated in the experimental group compared with the blank control group.2、The gene of VEGF、NDRG1、ATG4b was down-regulated expression and thegene of HIF-1α was up-regulated expression at the mRNA level in the experimentalgroup compared with the blank control group as the fluorescent quantitative PCRindicated.The difference was statistically significant (P <0.05).And the results areconsistent with the digital gene expression profiling analysis.3、The gene of VEGF、NDRG1、ATG4b was down-regulated expression and the geneof HIF-1α was up-regulated expression at the protein level in the experimental groupcompared with the blank control group as the Immunohistochemistry indicated withthe transplanted human hepatocellular carcinoma in nude mice.The difference wasstatistically significant (P <0.05).And the results are consistent with the fluorescentquantitative PCR analysis.Futher more,the gene of HIF-1α、VEGF、NDRG1、ATG4bwere differentially expression between transfection overexpression lentiviralANXA10of human hepatoma cell line HepG2and human hepatoma cell line HepG2.Conclusions：1、The overexpression of ANXA10gene influenced the gene expression profilesof human hepatocellular carcinoma cell line HepG2.2、The overexpression of ANXA10gene maybe inhibit the expression of VEGF、NDRG1、ATG4b and promte the expression of HIF-1α,which were the cytokines ralated to the oncogenesis and development of hepatocellular carcinoma.