Construction Of Twist Gene Plasmid And Research On Relationship Between Twist Gene And EMT In Colon Carcinoma Cells

Objective:To construct steadily and highly expressing Twist gene eukaryotic expression plasmid vector, study the role of Twist gene on Epithelial-Mesenchymal Transition (EMT)of malignant carcinoma cells.Methods:1. Twist gene was amplified by PCR from pCDNA4/myc-His A-Twist,and subcloned into pTracer-CMV/BSD to construct pTracer-CMV/BSD-Twist through double enzyme digestion and directional cloning.2.SW480 cell lines were cultured, The recombinant plasmid confirmed by DNA sequencing was transfected into SW480 cell line by the cationic liposome transfection, divided into pTracer-CMV/BSD-Twist/SW480 group (containing the target gene), pTracer-CMV/BSD/SW480(not contain the target genes),and negative control.3. SW480 cell lines were collected after culturing 48hours The mRNA expression level of Twist gene, Ecadherin gene and vimentin gene of three group cells were detected by real-time quantitative PCR and the protein expressions level of detected by Western bolt technique.Results:1.pTracer-CMV/BSD-Twist were successfully constructed and Confirmed by double digestion and DNA sequencing.2. After transfected for 48hr,the brightness and concentrated gree fluorescence protein of pTracer-CMV/BSD-Twist/SW480 and pTracer-CMV/BSD/SW480 was shown by inverted fluorescence microscope. 3. The results of Twist, Ecadherin, Vimentin gene mRNA and protein expression:①The expression levels of Twist gene mRNA and protein of pTracer-CMV/BSD-Twist group were higher than that of pTracer-CMV/BSD group and negative control.respectively, the difference was statistically significant (P <0.01), The expression levels of Twist gene mRNA and protein of pTracer-CMV/ BSD group and negative control were no significant differences(P>0.05).②the expression levels of Ecadherin gene mRNA and protein of pTracer-CMV/BSD-Twist group were lower than that of pTracer-CMV/BSD group and negative control. respectively,the difference was statistically significant (P<0.01), while the expression levels of Ecadherin gene mRNA and protein of pTracer-CMV/BSD group and negative control were no significant differences(P>0.05).③the expression levels of Vimentin gene mRNA and protein of pTracer-CMV/BSD-Twist group were higher than that of pTracer-CMV/BSD group and negative control. respectively,the difference was statistically significant (P<0.01), while the expression levels of Vimentin gene mRNA and protein of pTracer-CMV/BSD group and negative control were no significant differences(P>0.05).4. At expression level of mRNA and protein of twist gene were negatively related to expression of Ecadherin gene(P <0.01), and positively related to expression of Vimentin gene(P<0.01).Conclusions:pTracer-CMV/BSD-Twist were successfully constructed. After transfected into SW480 cells,there are higher expression of Twist and Vimentin,lower expression of Ecadherin at the same time. This phenomenon shows that Twist gene may inhibit Ecadherin expressionand promote the expression of Vimentin,in order to achieve EMT process。So Twist gene can enhance metastatic ability of malignant carcinoma cells.