Construction Of PCDF-Twist Eukaryotic Expression Vector And Its Effection On Colon Cancer Cells

Objective：To construct the pCDF-Twist lentiviral expression vector and aquire highexpression of Twist in colon cancer cell line-HT29.To explore the effects of Twist on theinvasion and metastasis of colon cancer cells. To detect the expression of Twist in differentstaged colon cancers.Methods：RT-PCR method was used to clone the gene sequence of human Twist, afterenzyme digestion, inserted it into pCDF plasmid to construct pCDF-Twist lentiviraleukaryotic expression vector. pCDF-Twsit lentivirus was used to infect HT29colon cancercells to aquire Twist over-expression cell line.Western blot was used to detect theexpression of Twist, E-cadherin, Vimentin and other related proteins. Scratch assay wasused to research the influence of Twist on HT29cell migration.48cases of coloncarcinoma specimens were colleced from surgical resection. Immunohistochemistry wasused to detect the expression of Twist in different staged colon cancers.Results：1. The pCDF/Twist plasmid was confirmed by restriction enzyme digestion, PCR, DNAsequencing. Fluorescence images displayed the lentivirus of pCDF-Twist had beenconstructed successfully2. The Western Blot results demonstrated that pCDF-Twist lentivirus infection cellsincreased the expression of Twist and Vementin,but decrease expression of E-cadherin 3. The scratch assay showed increased migration ability was aquired in HT29cellsinfected with lentivirus of pCDF-Twist.4. Immunohistochemistry assay indicated the expression of Twist in colon cancer tissueswas higher than nomal tissues and tumor staging was positively related to theexpression of Twist in colon cancers.Conclusion： pCDF-Twist eukaryotic over-expression lentivirus is constructedsuccessfully and over-expression of Twist in HT29cell line is aquired. Twist can promotethe processes of invasion and metastasis in colon cancers...