Research On The Influence Of The Invasion And Metastasis Of Colon Cancer Cells HCT116by Silencing Twist Genes In Vivo

ObjectiveTo explore the role of Twist gene in EMT phonomenon and the ininfluence on formation, development and metastasis of colonic carcinoma by making Twist gene silent and observing its impact on formation and metastasis of colonic carcinoma HCT116cell line as the E-cadherin and Vimentin mRNA transcription and protein expression in vivo.MethodMaking xenogenic spontaneous liver metastasis model at the heterotopic (spleen) site for colonic liver metastasis model by intrasplenic injection with HCT116cell line that are common and post transfected plasmid1.2and recombinant plasmid1.2-Twist-shRNA respectively in nude mice. The experimental groups were devided according to the intrasplenic injection.The following as:A. blank control group (blank cell line); B. negative plasmid control group (transfected plasmid1.2cell linr);C. experimental slienced group (transfected plasmid1.2-Twist-shRNA) Observing the weight, diet and nutrition of nude mice everyday, after30days of intrasplenic injection, collecting liver and spleen specimens and observing the growth of tumor. And the mRNA transcription level of Twist, E-cadherin and Vimentin gene were detected by real-time quantitative PCR where as the protein level was detected by Western blot of the specimens.Result1. It was successfully to make Xenogenic spontaneous liver metastasis at the heterotopic (spleen) site for colonic cancer liver metastasis model in nude mice, there is no significant difference found in the size of tumour in spleen and in liver in experimental silenced group than blank group and negative plasmid control group, but the mount of metastasis tumour in liver decreased.2. The mRNA transcription level of Twist and Vimentin was comparatively significantly lower in experimental silenced group than blank group and negative plasmid control group by49.3%and36.5%respectively(p<0.05), but the mRNA transcription level of E-cadherin was significantly higher for 2.49times (p<0.05); The protein expression level of Twist and Vimentin was comparatively lower in experimental silenced group than blank group and negative plasmid control group by45.1%and32.9%respectively (p<0.05), but the protein expression level of E-cadherin was significantly higher for2.27times (p<0.05).3. The mRNA transcription level of Twist in liver tumour was significantly higher than in spleen tumour by1.26times (p<0.05)(p<0.05); The protein expression level of Twist in liver carcinoma was higher than primary carcinoma in spleen by1.24times.Conclusion:1. It is feasible and effective to make Xenogenic spontaneous liver metastasis at the heterotopic (spleen) site for colonic cancer liver metastasis model.2. Silencing on Twist gene of HCT116cell line could decrease the tumour metastasis, Twist gene has no definite influence on the growth of tumour.3. Silencing on Twist gene of HCT116cell line could decrease the mRNA transcription level and the protein expression level of Twist, Vinmentin respecttively while increase the mRNA transcription level and the protein expression level of E-cadherin.4. The mRNA transcription level and the protein expression of Twist in metastasis tumour was higher than in primary tumour, Twist could promote tumour metastasis.5. Twist gene of HCT116cell line was inhibited successfully in vivo, that could prove that twist gene can reverse EMT phenomenon to some extent, and comfirm it is possible to treat colonic carcinoma by inhibiting Twist gene signal.