Study The Mechanism Of Change In Chemosensitivity In The Low Expression Of Twist Gene Caski Cell Lines

The first part Constructing the Twist gene pRNAT-U6.1/NeoshRNA eukaryotic expression vector and establish stable transfectioncell lineObjective:By constructing the Twist gene pRNAT-U6.1/Neo shRNA eukaryoticexpression vector,establish a stable low expression of Twist gene in cervical cancercell line Caski,to explore the Twist gene on cervical cancer drug resistance experimentpreparation.Method:1.Construct four pairs of recombinant SL36-l,SL36-2, SL36-3and SL36-4whichcontain efficient targeting Twist gene. Then the recombinant vectors were identifiedby enzyme digestion and gene sequencing.2.pRNAT-U6.1/Neo-Twist-shRNA plasmid was transfected into human cervicalcancer caski cells by liposome transfection. After form monoclonal were collected，then fluorescence microscopy was used to observe the green fluorescent protein（GFP）expression.3.G418-resistant clones of Caski cells were obtained and expand culture.4.The mRNA expression of Twist in monoclonal cell lines Caski-Twist-shRNA wasmeasuered by RT-PCR, identification interference effect and screen the shRNA withthe most interference efficiency.Results:1. Enzyme digestion and gene sequencing revealed that restructuring vector pRNAT-U6.1/Neo-Twist-shRNA was successfully constructed.2.The recombinant vector was transfected to cervical cancer cells by Lipofectamine2000, and observed by fluorescence microscopy.3.RT-PCR determine the Twist mRNA expression after stable transfection pRNAT-U6.1/Neo-Twist-shRNA plasmid showed that, the ratio of amplificationproduct of Twist （489bp） to β-actin.There was no statistically difference betweenCaski-nontransfection group and Caski-shRNA-NC group cells （P>0.05）,WhileCaski-Twist-shRNA1group,Caski-Twist-shRNA2group, Caski-Twist-shRNA3groupand Caski-Twist-shRNA4group showed significantly lower thancaski-nontransfection and caski-shRNA-NC group （P<0.05）. It demonstrates thelow expression of Twist and stable interference cells （Caski-Twist-shRNA group） wassuccessfully constructed. Caski-Twist-shRNA2group showed significantly lower thanCaski-Twist-shRNA1group,Caski-Twist-shRNA3group and Caski-Twist-shRNA4group （P<0.05）. After follow-up experiments applications shRNA2transfected group.Conclusion:1.The recombinant vectors pRNAT-U6.1/Neo-Twist-shRNA was successfullyconstructed.2.Establish a stable and low expression of the Twist gene department of cervicalcancer caski cells. The second part:Investigate the role and mechanism of Twist incervical cancer to paclitaxel resistanceObjective: Discussion the Twist gene with paclitaxel synergistic effect on tumorcells.Method:1. In the study, the cells were divided into three groups: Untransfected group（Caskigroup）,negative control group（Caski-shRNA-NC group）and experimentalgroup（Caski-Twist-shRNA2group）, paclitaxel treatment with differentconcentrations（250,500,1000,2000,4000nmol/L）, observed the morphologicalchanges by inverted phase cintrast microscope, through MTT colorimetricdetection the proliferation of cells in each group after48hours.2. Westemblot was used to detect the protein expression of PI3K and AKT ofcervical cancer cells in caski group,caski+Taxol,Caski-shRNA-NC+Taxol and Caski-Twist-shRNA2+Taxol. Analysis of pRNAT-U6.1/Neo the Twist-shRNA2plasmid transfection for caski cells resistant to paclitaxel PI3K and AKT proteinexpression.Results:1. MTT assay showed that, the experimental group of the proliferation inhibition ratecompared with Caski group and Caski-shRNA-NC group increased significantly（P<0.05）.IC₅₀ of the experimental group compared to Caski group andCaski-shRNA-NC group significantly decrease （P<0.05）,optimal concentration ofpaclitaxel is1000nmol/L.2. Western-blot assay showed that the protein expression of PI3K decreasedsignificantly in caski-Twist-shRNA2+Taxol group compare with that of caski,caski+Taxol and caski-shRNA2+Taxol group， while the expression of AKTdecreased significantly compared with that of caski, cask+Taxol andcaski-shRNA-NC+Taxol group （P<0.05）.There is no significant difference in theexpression of PI3K and AKT between caski+Taxol group andcaski-shRNA-NC+Taxol group（P>0.05）.Conclusion:Regulation of Twist drug resistance of cervical cancer to paclitaxel by PI3K/AKTsignal pathway.