Influence Of Twist Gene In Invasion And Metastasis Of Colon Cancer Cell Lines In Vitro

Objective:To evaluate the expression of Twist, Ecadherin and Vimentine in colon cancer cell lines, by inducing twist gene with endogeneous twist plasmid transfection and by inhibiting (knockdown) twist gene with exogenous ShRNA twist plasmid transfection, hence finally study the role of Twist gene on Epithelial-Mesenchymal Transition (EMT) in above colon cancer cell linesMethods:Colon cancer cell lines SW480, HCT116and HT29were cultured and the levels of Twist mRNA and protein were tested by RT-PCR and Western blot. Extrac-tion and purification of recombinant plasmids pTracer-CMV/BSD-Twist, pTracer-CMV/BSD, pGenesill.2-Twist-shRNA and pGenesill.2-shRNA. HCT116cell lines cultured, the recombinant plasmid were transfected into HCT116cell lines by the cationic lipid transfection respectively. Transfection efficacy of the plasmid in each cell lines was confirmed by Flowcytrometry. For transfected cell lines experiment groups were divided into:(1) Experiment group:pTracer-CMV/BSD-Twist;(2) Con-trol group:pTracer-CMV/BSD;(3) Experiment Group:pGenesill.2-Twist-shRNA;(4) Control group:pGenesill.2-shRNA; and (5) Negative control group:nontransfected group. After48hours of transfection the mRNA expression level of Twist gene, Ecadherin gene and vimentin gene were detected by real-time quantitative PCR where as the protein expressions level were detected by Western bolt technique in all the five experiment groups. Proliferation and viability assay of the plasmid transfect-ed cells was analyzed by MTT proliferation assay. Migration and invasive analysis was done by transwell insert migration and invasion assay for the post transfected cell.Results:After48hours of transfection, cells were visualized with green fluorescence protein under fluorescence microscope. The levels of Twist mRNA and protein in HCT116were higher than HT29and SW480. We selected HCT116cell line, which had high expression level Twist for the further experiment.1) After transfection of pTracer-CMV/BSD-Twist plasmid inHCT116, the mRNA transcription and protein expression levels of Twist and Vimentin were induced (P<0.05), whereas E-cadherin was inhibited (P<0.05).2) After transfection of pGenesil1.2-Twist-shRNA plasmid inHCT116, the mRNA transcription and protein expression levels of Twist and Vimentin were prominently inhibited (P<0.05), but transcription and expression level of E-cadherin was not statistically significant before and after the transfec-tion(P>0.05).3) Hence it was seen that the mRNA and protein levels of Twist gene were negatively correlated to expression of Ecadherin gene (P<0.01) and positively correlated to expression of Vimentin gene (P<0.01) respectively.4) The proliferation and viability of HCT116were not affected by the transfection of recombinant plasmid.5) Transwell migration and invasion assay of pGenesill.2-Twist-shRNA transfected group was inhibited significantly (P<0.01). And increased migration and invasive ability of pTracer-CMV/BSD-Twist group can be seen (P<0.01).Conclusion:1) Expression of Twist is higher in HCT116in comparison to SW480and HT29.2) In HCT116, up-regulating the level of Twist gene is compared with low level of Ecadherin and high level of Vimentin. It is mean Twist gene may promote the procedure of EMT through Ecadherin and Vimentin and increase the ability of HCT116cancer cell migration and invasion.3) RNA inference for Twist gene is able to down-regulate the expression of Twist gene in HCT116. Hence it may reverse EMT and inhibit colon cancer cell migration and invasion.