The Research On The Invasion And Migration Ability Of Twist Gene In Human Colon Cancer Cells In Vivo

Objective:To explore the impact of migration and invasion ability of colon cancer cells by Twist gene expression and investigate the role of Twist gene in inducing Epithelial-Mesenchymal Transition in human colon cancer cell line by establishing colon-to-liver metastases model in BALB/c nude mice.Method:1. HT-29human colonic cancer cell line was cultured, screened by blasticidin. Then the stably transfected colon cancer cell line HT-29was achieved.2. The liver metastases model was established in BALB/c nude mice by intrasplenic injection of HT-29colonic cancer cells. The experiment groups were devided into three groups:(1) Blank control group:non-transfected cells group;(2) Experiment group:plasmid pTracer-CMV/BSD-Twist stably transfected cells group;(3) Negative control group:plasmid pTracer-CMV/BSD transfected cells group. Remove the mice spleen and liver specimens completely5weeks later after the tumor cells injection. The mRNA and protein expression level of Twist, N-cadherin, Vimentin and E-cadherin was determined and analyzed by fluorescence quantitative real time PCR, Western blot, and immunohistochemistry staining method respectively in mice spleen and liver tumor specimens.Result:1. The colon carcinoma liver metastases model in BALB/c nude mice was established successfully. The pathological findings were in line with the features and characteristics of human poorly differentiated colon adenocarcinoma which was caused by metastasis process of cancer cells from colon to liver.2. The expression of Twist, N-cadherin, Vimentin gene at mRNA transcription (9.9082±0.9137,2.431±0.1636,3.0310±0.2026) and protein level (0.9916±0.0508,0.9417±0.1025,0.7231±0.0218) in the pTracer-CMV/BSD-Twist group was significantly higher than that of the pTracer-CMV/BSD and the negative control group in mice spleen and liver respectively with statistical significance difference (P<0.05). The expression of E-cadherin at mRNA transcription (0.4531±0.0536) and protein level (0.71±0.0613) in pTracer-CMV/BSD-Twist group was significantly lower than that of the pTracer-CMV/BSD and the negative control group in mice spleen and liver tumor specimens respectively without statistical significance difference (P>0.05). The difference between pTracer-CMV/BSD group and negative control group had no statistical significance (P>0.05).3. The mRNA expression level of twist gene in metastasis liver tumor specimens was higher than that in splenic specimens with statistical significance (P<0.05).4. Immunohistochemistry staining results demonstrated the numbers of positive stained cells of Twist, N-cadherin, Vimentin in pTracer-CMV/BSD-Twist group was significantly higher than that of the pTracer-CMV/BSD and the negative control group (P<0.05). The positive results of stained cells of E-cadherin in pTracer-CMV/BSD-Twist group was significantly lower than that of the pTracer-CMV/BSD and the negative control group (P<0.05).Conclusion:1. The colon cancer liver metastasis model in nude mice was successfully established. The tumorigenicity of the mice in this study is in high security and stability. And this model is a realistic animal model of simulating human colon cancer metastasis process with better value in basic and clinical study for the mechanism of human colon cancer liver metastases pathological process. It is a simple and operable preparation technique.2. Twist gene was up-regulated effectively in stably-transfected HT-29colonic cancer cell line, which was able to up-regulate N-cadherin and Vimentin expression and down-regulate E-cadherin expression respectively, could promote EMT and reverse MET in human colon cancer cell line HT-29.3. The mRNA transcription level of Twist in metastasis tumor was higher than in primary spleen tumour. It demonstrated that Twist gene expression could promote tumor metastasis.4. Promoting Twist gene expression effectively in vivo can increase the ability of migration and invasion in colon cancer cells in this study. Transcription factor Twist plays a critical role in the mechanism of epithelial-mesenchymal transition, and it could be a therapeutic target in the treatment of malignant tumors in gene therapy, and it may be useful for better understanding the mechanism of invasion and metastasis in colorectal cancer.