Study Of SiRNA Silencing TPX2Gene In Hela Cell Line Of Human Cervical Cancer

Objective: TPX2target protein (targeting for Xklp2), is a nuclearproliferation-related protein participated in the function of spindle microtubuleof mitotic and regulated by the cell cycle. In vivo and in vitro experimentsshowed that TPX2over-expression induced centrosome amplification andleading to DNA aneuploid and polyploidy, which may related withcarcinogenesis and metastasis.In this research, using TPX2-siRNA of chemical synthesis to research theefficiency of TPX2gene and protein silence in Hela cells of cervical cancer, toprovide new ideas and theoretical basis for targeted gene therapy of cervicalcancer.Methods: With Hela cells of cervical cancer as the object of study,accroding to the principle of siRNA, We designed and synthesised four kindsof siRNA targeting TPX2gene(TPX2-siRNA-1, TPX2-siRNA-2, TPX2–siRNA-3and TPX2-siRNA-4) and one negative control siRNA (TPX2–siRNA-Neg). The ctotoxicity was evluated by methl thiazolyl tetrazoliuassay. Four hours later, tranfection efficiencies of different protocols wereevaluated by fouorescent microscopy and flow cytometer to select an optimalcondition. Based the best condition, TPX2-siRNA was transfected into Helacells with Lipofectamine2000to observe the inhibition of siRNA silencingTPX2gene and protein. The experiments were divided into6groups:Hela(blank control group), TPX2-siRNA-1, TPX2-siRNA-2, TPX2-siRNA-3, TPX2-siRNA-4and TPX2-siRNA-Neg(the negative control group).Then we tranfected them into Hela cells. Hela cells were extracted total RNAand protein of each group after48h of transfection respectively, TPX2mRNAlevels were detected by RT-PCR and Western Blotting was applied to examinethe protein expressions. Statistical analyses: The SPSS13.0statistical software package was used for all analyses. The data were expressed by means±tandarddeviation. T-test and analysis of variance used to compare two samples andvaride in this analysis. The level of significant difference is α=0.05.Results:1,The survival rate of Hela cells transfected with siRNA were alwaysabove95%, and there is no statistically significant among different groups ineach time(P>0.05). The survival rate of Hela cells transfected withLipofectamine2000in1.25μl and1.5μl were90.32%and89.39%, and withthe extension of time the survival rate decreased. The results were significantcompared with other groups(P<0.05).2,When the concentration of siRNA was20μmol/L, the ratio betweensiRNA and Lipofectamine2000was2.5:2, the transfection efficiency was thehighest[(98.33±1.53)%] after they were transfected for24hours.3,Four kinds of TPX2-siRNA in the experimental group all silence TPX2gene after transfecting Hela cells, the relative quantity of TPX2mRNA in Helacells transfected with TPX2-siRNA-4was0.089±0.008and0.507±0.056inthe blank control group and0.463±0.115in the negative group(P<0.05) andthe expression of TPX2mRNA was (82.5±0.43)%with the most silencingeffect.4, The relative quantity of TPX2protein was0.298±0.072, and0.814±0.085in the blank control group and0.809±0.055in the negativegroup(P<0.05). The expression of TPX2protein was reduced (63.4±1.05)%.Conclusion: TPX2-siRNA can induce the degradation of TPX2mRNA,and inhibit the expression of the TPX2protein levels after transfected intoHela cells successfully, which establish the foundation for study of its impacton the biological characteristics of Hela cells.