The Research On Impact Of ANXA10on Gene Expression Of VEGFA, AKT3, PIK3CA In Human Hepatocellular Carcinoma Cell Line (hepG2)

Objective：1.The overexpression recombinant lentiviral of human ANXA10gene withenhanced green fluorescent protein (EGFP) was constructed, then transfected intohuman hepatocellular carcinoma cell line (HepG2).2.The effects of overexpression lentiviral of human ANXA10gene on geneexpression profiles of human hepatocellular carcinoma cell line (HepG2) wasexamined by Transcriptome sequencing,the effect of the biological behavior ofANXA10on HepG2,related genes and signaling pathways was illuminated initiallyso as to lay a foundation for the study on ANXA10in the formation of primary livercancer.Methods：The overexpression lentiviral of human ANXA10gene with enhanced greenfluorescent protein (EGFP) was constructed.In vitro cultured HepG2are divided intothree groups: the experimental group, empty carrier group and blank control group.LV-ANXA10,negative vector lentiviral and an equal amount of Polybrene weretransfected into each group respectively(lentiviraland negative vector transfected withMOI=10).Transfection after72hours,fluorescence inverted microscope was used toobserve the green fluorescent protein (EGFP) expression, and thus to determine thetransfection efficiency of ANXA10. Transfection after96hours and120hours, RNAof three groups of cells was extracted respectively.The differences of three groups ofcells gene expression profiles at the same time point and different time points werecompared by Transcriptome sequencing,and part of the differences in gene expressionlevels was validated by quantitative reverse-transcripted polymerase chain reaction.Results：1.The overexpression lentiviral of human ANXA10gene may influenced thegene expression profiles of human hepatocellular carcinoma cell line(HepG2).Through analyzing of information of Transcriptome sequencing,69geneswere found to change significantly compared with blank control group andexperimental group96hours after the transfection,26genes were up-regulated and43 genes were down-regulated.53genes changed significantly compared with emptycarrier group and experimental group,25genes were up-regulated and28genes weredown-regulated.12genes changed significantly compared with empty carrier groupand experimental group,8genes were up-regulated and4genes were down-regulated.However,742genes were found to change significantly compared withblank control group and experimental group120hours after the transfection,110genes were up-regulated and632genes were down-regulated.313genes changedsignificantly compared with empty carrier group and experimental group,168geneswere up-regulated and145genes were down-regulated.36genes changedsignificantly compared with empty carrier group and experimental group,24geneswere up-regulated and12genes were down-regulated.Furthermore,Through studyingof information of Transcriptome sequencing,it can be found that the expression ofVEGFA, AKT3and PIK3CA were downregulated compared with empty carrier groupand blank control group96hours and120hours after the transfection, whereas thesechanges did not exist in negative control group and blank control group.2.The gene of VEGFA,AKT3,PIK3CA in experimental group was down-regulated expression at the mRNA level in the experimental group compared withempty carrier group and the blank control group as the fluorescent quantitative PCRindicated.The difference was statistically significant (P <0.05).And the results areconsistent with the Transcriptome sequencing analysis.But the empty carrier grouphad no significant difference compared with the blank control group(P>0.05).Thisstudy demonstrated that the expression of VEGFA,AKT3,PIK3CA existed differencein experimental group(LV-GFP-ANXA10),empty carrier group (Negative vectorgroup) and blank control group(HepG2cell group).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 VEGFA,AKT3, PIK3CA during the formation of liver cancer.