A Investigation Of Silencing Twist Gene By Small Interfering RNA Enhanced Chemosensitivity In Caski Cells

Background: The embryo development gene Twist, belonging to the family ofbasic helix-loop-helix structure, is a highly conserved transcription factor. It has thecharacteristics of the oncogene, not only involes in tumorigenesis, development, butalso relates with poor chemosensitivity and drug resistance of tumor cells closely.Poor chemosensitivity and drrug resistance is a major obstacle in chemotherapy,andthe key issues of poor clinical efficacy as well. It will have far-reaching significanceto improving clinical efficacy of chemotherapy, if we clarify the reasons and thepossible mechanisms for them. Therefore, this project is mainly to study therelationship of the Twist gene and paclitaxel-resistance of cervical cancer. Meanwhile,it will bring new means for improving efficacy of chemotherapy and the clinicaltreatment in cervical cancer.Objective: To research cervical cancer cells’ proliferation, cloning, apoptosis, thecell cycle of cervical cancer cells by silencing Twist gene, and then to reveal whethersilencing the Twist gene could increase sensitivity to paclitaxel in cervical cancercells.Methods:1. The Caski cells and Hela cells of cervical cancer were cultured in vitro.Semi-quantitative PCR and fluorescence quantitative PCR were used to screenappropriate cell lines.2. The siRNA1and siRNA2targeting Twist gene and a negative control siRNA weredesigned and synthesized, we use fluorescence quantitative PCR to detect theexpression of Twist mRNA and choose the optimal siRNA.3. Caski cells were transfected with si-Twist/nc-Twist respectively usingLipofectamineTM2000, then it was treated with0.001、0.01、0.1、1、10umol/LTaxol, MTT assay, colony formation assay and flow cytometry were employed toevaluate cell proliferation, colony forming ability and apoptosis/cell cycle separately.Results:1. The expression of Twist gene was higher in Caski cells than in Hela cells (p<0.05). 2. Inhibition rates of the Twist mRNA in group siRNA1and group siRNA2were20.3%,38.2%,33%;24%,68.6%,50.8%. siRNA2is better than siRNA1in silencingTwist gene(p<0.05). And there was no further decrease of the Twist mRNA after48h.3. Contrast by the group of NC-Twist, the growth inhibition ratio of si-Twist wasobviously increased(p<0.05) and the growth inhibition effect was dosage dependent.At the same dosage, the growth inhibition ratio was higher in the24-48h points thanin other points (p<0.05).IC50of si-Twist group was significantly lower than other twogroups (p<0.05).4. On1umol/L Taxol, cloning formation rate of si-Twist RNA group was lower thanNC-Twist group (p<0.05).5. On1umol/L Taxol, apoptosis rate of si-Twist RNA group was signifan-cantlyhigher than NC-siRNA group (p<0.05) and the Caski cells was mainly arrested in thephase of G0/G1.Conclution:1．The Twist gene is high expression in Caski cells of cervical cancer.2．Silencing Twist gene, it could inhibit Caski cells’ proliferation, cloning andpromote apoptosis to Taxol, thereby it enhanced chemosensitivity in cervicalcancer.