Research On The Influence Of Twist Gene In Invasion And Metastasis Of Colon Cancer

In this research we will be mainly focusing on the expression of Epithelial Mesenchymal Transition (EMT) markers Twist, E cadherin and Vimentin after upregulation and downregulation of the twist genes in SW480, HT29and HCT116colon cancer cell lines in vitro. And evaluate the expression of Twist, E-cadherin and Vimentin gene in primary tumor and metastases site by making an in vivo model with Nude mice by.Part Ⅰ Influence of Twist gene in invasion and metastasis of colon cancer cell lines in vitro.Objective:To explore the expression of Twist, E-cadherin and Vimentine in SW480. HCT116and HT29colon 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 lines.Methods:Twist mRNA transcription level and protein expression level was evaluated after culturing the cell lines SW480, HCT116and HT29by Real time-PCR and Western blot. Extraction and purification of recombinant plasmids pTracer-CMV/BSD-Twist, pTracer-CMV/BSD, pGenesil1.2-Twist-shRNA and pGenesil1.2-shRNA. The recombinant plasmids were transfected into SW480. HCT116and HT29cell lines by the cationic lipid transfection. For each transfected cell lines they were divided into5experiment groups:(1) Upregulation Experiment group:pTracer-CMV/BSD-Twist;(2) Upregulation Control group: pTracer-CMV/BSD;(3) Down regulation Experiment Group: pGenesil1.2-Twist-shRNA;(4) Downregulation Control group:pGenesil1.2-shRNA; and (5) Negative control group:nontransfected group. After48hours of transfection, transfection efficacy was confirmed by Flowcytrometry, mRNA transcription level of Twist gene, E-cadherin gene and vimentin gene were detected by real-time quantitative PCR, protein expressions level were detected by Western bolt technique in all the five experiment groups. Proliferation and viability assay of the plasmid transfected 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:1. In between three cell lines SW480, HCT116and HT29, Twist mRNA transcription levels and protein expression levels were found to be higher in HCT116�'SW480�'HT29cell lines with a statistical significance.2. Successful extraction and purification of recombinant plasmids pTracer-CMV/BSD-Twist, pTracer-CMV/BSD, pGenesil1.2-Twist-shRNA and pGenesill.2-shRNA was done.3. After48hours of transfection with the above mentioned plasmids in three cell lines the, cells were visualized with green fluorescence protein under fluorescence microscope.4. Flowcytrometry result showed the transfection efficacy of pTracer-CMV/BSD-Twist in SW480, HCT116HT29as21.6%,22.3%and22.7%respectively. Also the transfection efficacy of plasmid pGenesil1.2-Twist-shRNA in SW480, HT29and HCT116were21.2%and23.4%,30.3%respectively.5. After transfection of pTracer-CMV/BSD-Twist plasmid in SW480, HCT116and HT29cell lines the mRNA transcription and protein expression level of Twist and Vimentin were induced (P<0.05), whereas E-cadherin was inhibited (P<0.05). But comparison in between each plasmid transfected cell lines the difference was found to be statistically insignificant (P>0.05). Correlation analysis showed, Twist and Vimentin mRNA transcription and protein levels were positively correlated (P<0.01), but negatively correlated (P<0.01) with E-cadherin.6. After transfection of pGenesil1.2-Twist-shRNA plasmid in SW480, HCT116and HT29cell lines, the mRNA transcription and protein expression level of Twist and Vimentin was prominently inhibited in HCT116cell line(P<0.05), but transcription and expression level of E-cadherin was not statistically significant (P>0.05) before and after the transfection. Before and after the transfection of pGenesill.2-Twist-shRNA, all the parameters in SW480and HT29cell lines were statistically insignificant (P>0.05). Correlation analysis showed that only HCT116cell line’s mRNA transcription and protein expression levels of Twist and Vimentin were positively correlated (P<0.01).7. The proliferation and viability of three cell lines were not affected by the transfection of recombinant plasmid. After transfection of pGenesil1.2-Twist-shRNA plasmid in HCT116cell line the migration and invasion ability was significantly inhibited (P<0.01).Conclusion:1. Expression of Twist is high in HCT116human colon cancer cell line in comparison to SW480and HT29.2. Upregulation of Twist gene expression in three cell lines can induce high expression of Vimentin molecules and Low expression of E-cadherin molecules. Hence promoting the EMT molecular events enhanced metastatic ability of tumor cells.3. Interfering Twist gene can effectively silence Twist gene expression in HCT116cell line, inhibit Vimentin expression, reverse epitheial-mesenchymal transition, promote mesenchymal-epithelial transition, and effectively inhibit colon cancer cell migration and invasion.PART Ⅱ Influence of Twist gene in invasion and metastasis of nude mice colon cancer model in vivoObjective:To evaluate the expression of Twist and EMT markers in nude mice colon cancer liver metastases models.Method:Making xenogenic spontaneous liver metastases at the heterotopic (spleen) site for colon cancer liver metastases model by intrasplenic injection with SW480, HCT116and HT29human colon cancer cell lines that are post transfected with plasmid pTracer-CMV/BSD-Twist and pGenesil1.2-Twist-shRNA respectively. The experimental groups were devided according to the intrasplenic injection of the following as:(1) Plasmid pTracer-CMV/BSD-Twist transfected cell line;(2) Plasmid pGenesil1.2-Twist-shRNA transfected cell line;(3) Non transfected cell line and (4) Control:PBS injected. After30days liver and spleen specimen were collected. And the mRNA transcription level of Twist gene and Vimentin gene were detected by real-time quantitative PCR where as the protein expressions level was detected by immonohistochemistry of the specimens.Result:Xenogenic spontaneous liver metastases at the heterotopic (spleen) site for colon cancer liver metastases model was successfully made by intrasplenic injection of SW480, HCT116and HT29human colon cancer cell lines post transfected with plasmid pTracer-CMV/BSD-Twist and pGenesill.2-Twist-shRNA respectively. Twist and Vimentin mRNA transcription level were found higher in the liver then spleen (P<0.05) with injection of three cell lines transfected with pTracer-CMV/BSD-Twist. The mRNA transcription level of Twist and Vimentin gene was comparatively low in the liver than spleen injected with pGenesil1.2-Twist-shRNA plasmid transfected cell lines (P<0.05). But in comparison to non transfected cell lines only HCT116cell line appeared to have prominent inhibited mRNA transcription level of Twist and Vimentin then cell lines.Conclusion:RNA interference Twist gene can effectively silence the Twist gene expression in HCT116cell line in vivo. Hence inhibiting the invasive and metastatic potential capability in nude mice colon cancer liver metastases model.