The Study On The Role Of Twist Gene Down Regulation And Bcl-2in Cervical Cancer Caski Cell Lines To Chemotherapy Sensitization

Objective:This experiment aims to silence the expression of Twistgene, and to observe the expression of Bcl-2protein in cervical cancercaski cells, to analyze the influence of susceptibility to chemotherapy bychemotherapy by silencing Twist gene in cervical cancer caski cells.Methods: Bioinformatics methods were used to analysize the vectorwhich down regulation Twist gene level;it was proved that theinteferenced sequences of Twist gene were constructed successfully.Therecombined vectors were transfected into cervical cancer caski cells byusing lipofectamin2000. And obtained reconstructed cell linesCaski-Twist–shRNA with low expression of Twist gene. The expressionof Twist-mRNA of each group was detected by RT-PCR.The cells weretreated48hours with different concentrations (0.25,0.5,1,2,4umol/l) oftaxol, the growth inhabition of cells is tested by MTT, then the bestinhibitory concentration was selected. The expression of Bcl-2protein ineach group cells were detected by Western blot.Results：1. The reconstracted cell lines Caski-Twist–shRNA with lowexpression of Twist gene were successfully established. 2. In the four different vectors of transfection groups,the expression ofTwist-mRNA of four groups were lowed,The mRNA expression of Twistgene in Transfection group2was significantly lower than Transfectiongroup1, Transfection group3and Transfection group4(p<0.05), whichsuggest the transfection efficiency of transfection group2was best.Compared with blank control group and negative control group, there aresignificant difference for the expression of Twist mRNA in Transfectiongroup1, transfection group2, transfection group3and transfection group4(p<0.05). Blank control group and the negative control group have nosignificant difference (p>0.05).3. The inhibition rate of the three group cells were accordinglyincreased with the increase of the paclitaxel concentrations. And thesurvival rate of transfected groups cells were lower than the blank controlgroup and negative control group. The IC50value of the blank controlgroup, negative control group and experimental group were5.86,1.06and6.80μmol/L,respectively. And the IC50value of transfected groupswere significantly higher than the negative group and blank control group(P<0.05).4. The expression of Bcl-2protein in transfection group2wassignificantly lower than negative control group and blank control group(p<0.05). The expression of Bcl-2protein had no signifinant differencebetween the negative group and blank control group (p>0.05). Conclusions:1.The down-regulated expression of Twist gene,enhance thesensitivity of cervical cancer cells to paclitaxel.2. The down-regulation of Twist gene expression can enhance thesensitivity of cervical cancer cells to paclitaxel, it may be associated withthe reduce the expression of Bcl-2protein.