Study On The Expression Of TPX2 In Esophageal Squamous Cell Carcinoma Tissue And The Relationship Between The Invasion Metastasis

Esophageal cancer (esophagealeareinoma, EC) is one of the most common malignancies of digestive tract with the biological characteristics of high aggressive and metastatic potential. The invasion and metastasis is the leading cause of death. Esophageal cancer has a distinct geographic distribution characteristic, China is a high incidence of esophageal cancer and even more pronounced in Henan Province of China. With the improvement of surgical technique, radiotherapy, chemotherapy, as well as the promotion of evidence-based medicine methodology and supportive care, the survival time and life quality of patients have improved greatly. However, the five-year survival rate is 20-35%. Its effectiveness has not been satisfactory. Therefore, to study invasion and metastasis of esophageal cancer mechanisms, and to improve diagnosis and treatment of esophageal cancer has become one of the hot oncology.TPX2 target protein (targeting for Xklp2), also known as repp86 (restricted expressed proliferation-associated protein), is a nuclear proliferation-related protein participated in the function of spindle microtubule of mitotic and regulated by the cell cycle. In vivo and in vitro experiments showed that TPX2 over-expression induced centrosome amplification and leading to DNA aneuploid and polyploidy, which may related with carcinogenesis and metastasis. It suggests that an excessive amount of TPX2 disrupt the normal mitosis and physiological activation of its own channels. TPX2 may play a role in carcinogenesis and malignant progression. However, In addition to our pre-published articles, there is no report yet regarding TPX2 and esophageal cancer.In order to better understand the association between TPX2 and esophagus squama carcinogenesis and metastasis, we investigated TPX2 protein and mRNA expression in the clinical tissue samples from esophagealeareinoma, atypical hyperplasia and normal esophageal mucosa by using immunohistochemistry and RT-PCR. To further study the mechanisms, using siRNA-TPX2 of Chemical synthesis transfected EC9706 cells by using liposomes. The effect of TPX2 down regulation on tumor cell proliferation, invasion was evaluated by using immunohistochemistry, western blot, RT-PCR, flow cytometry, cell proliferation inhibition test and boyden chamber invasion assay. Finally, we transplanted tumor cells in nude mice to further evaluate the impact of TPX2 RNA interference on tumor growth and invasion. To discussed the TPX2 in esophageal squamous cell carcinoma Systematically from the body, outside and its impact on the invasion and metastasis, to provide new ideas and theoretical basis for targeted gene therapy of esophageal cancer.1. Expression of the TPX2 in esophageal squamous cell carcinoma, atypical hyperplasia tissue and normal esophageal mucosaTPX2 expression was examined in the clinical samples from 62 cases of esophageal squamous cell carcinoma,31 cases atypical hyperplasia tissue and 62 cases of normal esophageal mucosa by using immunohistochemistry and RT-PCR. TPX2 positive staining mainly located in the nuclei of esophageal squamous cells. TPX2 protein level was very low in normal esophageal mucosa, increased dramatically in adjacent dysplasia and reached the highest level in esophageal squamous cell carcinoma. The positive rates were 4.8%,51.6%,85.5%, respectively (P<0.05). Furthermore, TPX2 protein level were correlated with tumor lymph node metastasis and invasion depth of carcinoma (P<0.05). RT-PCR results showed that no or weak expression of TPX2 mRNA in normal esophageal epithelium. The positive rate of TPX2 mRNA expression was 65.0% in esophageal cancer, which was significantly higher than in atypical hyperplasia (35.5%) (χ2=20.037, P<0.001). The quantification of TPX2 mRNA was 0.879±0.046 in carcinoma and 0.627±0.084 in adjacent dysplasia tissues (P<0.05). TPX2 mRNA level was related with invasion and metastasis of esophageal cancer (P<0.05). However, there was no relation between TPX2 protein or mRNA expression level and age, gender of patients or the location, clinical classification of cancer.2. Effect of TPX2 gene silencing on esophageal carcinoma EC9706 cell lineTPX2 siRNA of Chemical synthesis was transfected into esophageal cancer EC9706 cells by using Lipofectamine2000 transfection medium. EC9706 cell line were divided into blank control (nothing added), negative control (non-specific vector added) and siRNA interference group (the most effective interference plasmid added). The expression of TPX2 protein and mRNA were detected by using RT-PCR and Western Blot at different time after transfection. The cell viability was determined by MTT assay. Apoptosis was observed by using TUNEL labeling and fluorescence microscope. Cell proliferation was observed by checking the cell growth curve. Cell cycle was observed by using flow cytometry assay. Boyden chamber experiment was used to evaluate the invasion ability. The results showed that both TPX2 mRNA and protein expression decreased significantly after TPX2siRNA transfection compared with the negative and blank controls (P<0.05), which was more pronounced at 72h. The EC9706 cells growth was inhibited at 48h and more obvious at 72h after transfection. There was no difference with cell growth between negative control and blank control group. The number of apoptotic cell death detected by TUNEL labeling increased significantly in the siRNA group compared with negative or blank control group (P<0.05). The flow cytometry assay showed that cell division was inhibited after siRNA transfection. The number of cells at G1. phase decreased, but at S phase increased and cell cycle arrest in S phase. Boyden chamber invasion test showed Matrigel gluey's cells decrease significantly in siRNA group compared with negative or blank control group (P<0.05).3. Effect of TPX2 siRNA on the transplanted tumor growth in nude miceNude mice were injected with EC9706 cells or the cells treated either with siRNA interference or unrelated siRNA subcutaneously. The volume of transplanted tumor was measured at 5 weeks after transplantation. TPX2 mRNA and protein expression of tumor tissue was determined by using RT-PCR and immunohistochemistry. The volume of tumor in the siRNA interfered EC9706 cell transplanted group was much smaller than the other two groups (P<0.05). TPX2 protein and mRNA expression level are much lower in siRNA interference group compared with other two groups (P<0.05).4. Cloning of human TPX2 gene and construction of eukaryotic expression vectorHuman TPX2 gene was amplified by RT-PCR based on the pQE-70-TPX2 prokaryotic expression vector. The PCR product and pcDNA3.1 plasmid were digested by using restriction enzyme SphI and BamHI and then it was connected with T4 DNA Ligase ligation and transformed E. coli JM109 competent cells. Transformants were verified by PCR and positive clones were picked to expand cultivation and plasmids were collected. After enzyme digestion with SphⅠand BamHⅠ, DNA sequence of human TPX2 gene was confirmed by using gene sequencing and BLAST analysis. The eukaryotic expression vector pcDNA3.1-TPX2 was constructed successfully, which set up the platform for further research of TPX2 biological function and its related genes.Conclusions1. TPX2 highly expressed in esophageal squamous cell carcinoma in both protein and mRNA level, which was correlated with tumor lymph node metastasis and the depth of infiltration. It suggests that the increased TPX2 expression could be an early event of carcinogenesis.2. In vitro experiments showed that Transfection of siRNA-TPX2 in EC9706 cells inhibited TPX2 expression in both protein and mRNA level.In the mean time, the tumor cells proliferation and invasion ability was inhibited and more cells arrested in S phase and died with apoptosis.3. In vivo experiments showed that siRNA-TPX2 interference inhibited the transplanted tumor growth in nude mice which correlated with decrease of TPX2 expression in both protein and mRNA level. 4. The TPX2 eukaryotic expression vector, pcDNA3.1-TPX2, was constructed successfully, which set up the platform for further research of TPX2 biological function and its related genes.