Research On The Role Of Twist Gene In Invasion And Metastasis Of Colon Cancer Cell Lines In Vitro

Objective:To explore the role and mechanism of Twist gene in Epithelial-mesenchymal transition (EMT) of human colon cancer cell lines, by up-regulating and down-regulating the twist expression, respectively; and then study the effect of twist on invasion and metastasis of malignant tumors. This research is also aimed at furnishing experimental evidence for further research on gene therapy for colon cancer.Methods:The recombinant high-expressed twist plasmid (pTracer-CMV/BSD-Twist) and empty vector plasmid (pTracer-CMV/BSD) were used to transfected into the HT29cell line, which express Twist in relatively low level. To obtain cell lines transfected stably, selection was initiated using Blasticidin. RT-PCR and Western blot were used to validate the gene upregulation efficiency of Twist in colon cancer cell line HT29. Meanwhile, pGenesill.2-Twist-shRNA, which expressed short hairpin RNA that were targeted against Twist, and empty vector plasmid (pGenesill.2) were used to transfected into the HCT116cell line, which express Twist in relatively high level. The cell lines transfected stably were obtained using G418screening. RT-PCR and Western blot were used to examine the gene downregulation efficiency of Twist in colon cancer cell line HCT116. The mRNA transcription and protein expression level of EMT makers (E-cadherin, N-cadherin and Vimentin) were detected by RT-PCR and Western bolt respectively in the above cell lines transfected stably. The ability of migration and invasion of was evaluated by transwell insert migration and invasion assay for the post-transfected cell.Results:1. These plasmid was successfully transfected into the HT29and HCT116cell lines, and the stably transfected cell lines was successfully obtained by screening.2. Compared with the groups of cell lines with nontransfected and transfected with empty plasmid, the mRNA transcription and protein expression levels of Twist were induced in HT29cell line transfected with high-expressed twist plasmid, and the difference was found to be statistically significant (P<0.05); however, the mRNA transcription and protein expression levels of Twist were inhibited in HCT116cell line transfected with the plasmid expressing short hairpin RNA, and the difference was found to be statistically significant (P<0.05).3. After up-regulating expression of Twist gene in HT29cell line, the mRNA and protein expression level of E-cadherin significantly decreased (P<0.05), whereas the mRNA and protein expression level of N-cadherin and Vimentin were induced (P<0.05). However, after down-regulating expression of Twist gene in HCT116cell line, the mRNA and protein expression level of E-cadherin significantly increased (P<0.05), whereas the mRNA and protein expression level of N-cadherin and Vimentin were inhibited (P<0.05).4. Up-regulation of Twist gene expression in HT29led to significantly increased migrated and invaded cells though the insert in comparision with those of groups nontransfected and transfected with empty plasmid, as the Transwell migration and invasion assay result showed. However, down-regulation of Twist gene expression in HCT116led to significantly decreased migrated and invaded cells though the insert in comparision with those of groups nontransfected and transfected with empty plasmid, as the Transwell migration and invasion assay result showed.Conclusion:1. Up-regulation of Twist gene expression in colon cancer cell line HT29can cause low expression of epithelial marker (E-cadherin) but high expression of mesenchymal markers (N-cadherin and Vimentin). Hence promoting the EMT molecular events enhanced the migration and invasion ability of tumor cells in vitro.2. Down-regulation of Twist gene expression in colon cancer cell line HCT116can induce the expression of epithelial marker (E-cadherin), but inhibit the expression of mesenchymal markers (N-cadherin and Vimentin), which reverse EMT and promote mesenchymal-epithelial transition, as well as effectively inhibit colon cancer cell migration and invasion in vitro.3. RNA interference can effectively silence Twist gene expression in colon cancer cell line HCT116, and thus this study provide experimental evidence for the application of RNA interference technique in gene therapy for colorectal cancer in the future.