Change Of Hep-2Cell Apoptosis After Annexin A5Knockdown

Objective:（1）Detect the transfection efficiency of special simall interferenceRNA(siRNA) of annexin a5in Hep-2cells.To analyze the influence of siRNAfor Hep-2cells and to assess the way of cell trasfection about ourexperctation.（2）After special siRNA of annexin a5was successfully trasfected intoHep-2cells,Detect the annexin a5expression of all groups from mRNA level;at the same time detect the protein expression of annexin a5of allgroups.To validate the special siRNA successfully knocked-down annexin a5in Hep-2cells.（3）Analyze the apoptosis efficiency of Hep-2after annexin a5successfully been knockn-down. To research the relationship between theexpression of annexin a5and apoptosis efficiency of Hep-2.Methods:（1）Through siDirect Version2.0design software, we designed specificsiRNAs against annexinA5gene, after blast with Pubmed, were beensynthesived and marked by Fluorescence by TaKaRa company. And theHep-2cell lines were cultured in RPMI-1640(10%FBS (Gibco, USA),100U/ml penicillin and100U/ml streptomycin) at37℃with5%CO2.Appropriate Hep-2cells were seeded in a6×well plate, when the cells inproliferative phase covered the plate about70%-80%or just bespread, specialsiRNA were trasfected into Hep-2by Lipofectamine2000. After trasfected4-6h, draw the mixture of trasfection and add2ml RPMI-1640with10%FBS into the plates, count one hundred cells and count the fluorescence cellsstraightly of the total one hundred cells by fluorescence microscop at leastthree times and estimate the transfection efficiency of special simallinterference RNA(siRNA).（2）After annexinA5been knocked-down, according Semi-quantitativeRT-PCR assay to detect the mRNA expression of annexinA5in special siRNAgroup wether or not significantly knocking down contrast other groups.Western blotting were applied to validate the results of RNA interference fromprotein expression.（3）The flow cytometry assay with AnnexinV-FITC/PI apoptosisdetection kit was performed to detect the change of Hep-2cell apoptosis afterannexin A5been knocked-down. Compare the expression of annexin A5inspecial siRNA group and other contrast groups; and explore the relationshipbetween annexin A5and Hep-2cells.Results:（1） After trasfection, count one hundred cells and count thefluorescence cells straightly of the total one hundred cells by fluorescencemicroscop at least three times,and count the transfection efficiency of specialsimall interference RNA(siRNA), the transfection efficiency among80%-90%.This way for siRNA knocking down annexin A5in Hep-2cells wassuccessful.（2）RT-PCR analysis showed that the relative mRNA expression ofannexin A5in siRNA group,negatice control group,Lipofectamine2000groupand blank control group were0.70±0.03,1.18±0.05,1.17±0.06and1.23±0.07.And the relative mRNA expression of annexin A5in siRNA groupwas significantly decreased than negatice control group （t=-14.77,P=0.001<0.05）,Lipofectamine2000group （t=-13.23,P=0.001<0.05） and blank control group（t=-12.99,P=0.002<0.05）；Among contrast groups differences of statistical data had no significantdeviation(tnegatice group-Lipofectamine group=0.31,P=0.77〉0.05,tnegatice group-blank group=-0.98,P=0.39〉0.05, tLipofectamine group-blank group=-1.21,P=0.30〉0.05).In Westernblotting assay,The trend of protein expression levels were consistent with themRNA expression levels of annexinA5. The relative levels of proteins insiRNA group, negatice control group, Lipofectamine2000group and blankcontrol group were shown1.21±0.03,3.88±0.06,3.87±0.02and3.95±0.08.Significant differences were found between siRNA group and contrast groups(negative control group t=-70.34, P=0.000<0.05, Lipofectamine2000groupt=-150.62,P=0.000<0.05,blank control group t=-56.32, P=0.000<0.05).Thesame situation for contrast groups, differences of statistical data had nosignificant deviation(tnegatice group-Lipofectamine group=0.46, P=0.68〉0.05, tnegaticegroup-blank group=-1.15, P=0.32〉0.05, tLipofectamine group-blank group=-1.77, P=0.21〉0.05).（3）At the flow cytometry the apoptotic rate of siRNA group, negativecontrol group，Lipofectamine2000group and blank control group were4.43±0.12,13.67±0.22,13.66±0.12and13.35±0.13. And the differencebetween the siRNA group and contrast groups(negatice control groupt=-62.50, P=0.000<0.05, Lipofectamine2000group t=-14.16, P=0.005<0.05,blank control group t=-11.47, P=0.007<0.05) was statistically significant;Differences of statistical data had no significant deviation(tnegaticegroup-Lipofectamine group=0.03, P=0.98〉0.05, tnegatice group-blank group=0.42, P=0.72〉0.05, tLipofectamine group-blank group=0.31, P=0.77〉0.05). So after RNA interference, expression of annexin A5decreased, and the results in theapoptosis inhibition of Hep-2cell.Conclusion:According to our data, we found the apoptosis efficiency of Hep-2cellssignificant decrease After annexinA5been knocked-down.All of us know thatthe change of cell apoptosis of cancer is significant for cancer cloning growth.Furthermore, cell apoptosis of cancer is also tightly associated withOncodiagnosis and therapeutics. So we considered that annexin A5mightenhance laryngeal cancer apoptosis (Hep-2) and might represent a noveltherapeutic target for laryngeal cancer.