Expression Of The EZH2 Protein In The Carcinoma Of Esophagus And Its Significance

Background and ObjectiveThe human homolog of the Drosophila Enhancer of zeste gene (EZH2), as a novel gene, was identified to map on human chromosome 21 and that may contribute to the phenotype of Down syndrome (DS) in 1996. But further studies showed that this is a pseudogene, and that EZH2 actually maps to chromosome 7q35 within the critical region for malignant myeloid disorders. Sequence analysis indicates that EZH2 encodes a 746-amino-acid polypeptide that shows 60.5% identity to the Drosophila E(z) protein and contains a trithorax-like domain and a DNA-binding motif. The EZH2 gene is associated with growth and regrulation of cell, which mediates gene transcription repression in eukaryotic chromatin and increases cell proliferation. At the genetic level, EZH2 protein as a repressor can repress lots of the genes, especially for the metastasis-suppressing genes, which shows that EZH2 can promote tumor cells invasion and metastases. The esophageal cancer is the most common malignant tumor of the esophagus worldwide. 70% cases happened in China, The incidence of esophageal cancer is high in areas Shantou and Chaozhou of China, there is no relative paper on the expression and significance of the EZH2 protein in the carcinoma of esophagus at present. This study is to observe the EZH2 expression and change in the carcinogenesis and development of the esophageal cancer, then investigated the relation between EZH2 protein expression and the differentiation, stage, invasion and metastases of esophageal cancer. Materials and Methods1. The expression of EZH2 in the human immortalized esophageal epithelial cell line (SHEE) and the malignant transformation cell line (SHEEC) , derivated from SHEE, was observed by the immunocytochemistry, western blotting and flow cytometric analysis.2. The expression and distribution of EZH2 were performed on the tissue microarrays by using Streptavidin-Peroxidase technique and a polyclonal antibody against EZH2. Tissue microarray of esophageal squamous cell carcinoma are composed of the 14 samples of histological normal tissue, 16 samples of the correponding precancer tissues and 42 samples of esophageal cancer tissues, which were 10 cases grade I, 12 cases grade II and 20 cases grade III. The samples of 10 well differentiated adenocarcinoma, 13 moderately differentiated, 20 poorly differentiated adenocarcinoma and 37 normal samples were included in esophageal adenocarcinoma tissues microarray. There were 23 cases invasing the muscular layer and 29 cases mantle layer, or 30 cases metastases and 22 cases no metastases in the 52 esophageal squamous carcinoma tissues which were collected from the Sugical Pathology files at Medical College of Shantou University. We studied EZH2 protein expression by immunohistochemistry method, then analyzed that the relationship among the different grades, stages, invasive and metastatic esophageal cancer. EZH2 protein expression in the normal tissues, adjacent to esophageal cancer and esophageal cance were further dectected by western blotting.Results1. The immunocytochemical staining results showed SHEE cells and SHEEC cells expressed EZH2 protein. EZH2 protein positive staining was observed mainly in the nucleus, the staining was also observed in the cytoplasmic regions of some cells. Comparion of the intensity of EZH2 stainig between SHEE cells and SHEEC cells, the median stainging intensity for SHEE cells was stronger than that of SHEEC cells. Western blotting analysis indicated that total protein and nuclear protein of SHEE cells and SHEEC appeared the specific bands in 90ku position respectively. The comparison of the optical density of the two cells manifested that the total protein band of SHEE cells was much stronger than that of SHEEC cells (P <0.05). But therewas no significant difference on bands of the nuclear protein in SHEE cells and SHEEC cells. The results of flow cytometric analysis also explained that two cell lines overexpressed the EZH2 proteins, there was no significant difference of the fluorescence intensity in the two cell lines. The positive ratio of two cells expressing EZH2 protein were highly, the value of SHEE cells exceeded SHEEC cells.2. The immunohistochemistry results of tissue microarrays and esophageal tissue's paraffin slices showed EZH2 protein expressed in esophageal cancer tissues. The positive reactions were the brown, mainly in the nucleus. The parts of the normal tissues and correponding precancer tissues were the weak staining. The intensity analysis of positive products showed the esophageal cancer tissues were strikingly stronger than normal tissues and correponding precancer tissues, The difference of EZH2 protein expression was significantly (P<0.05). EZH2 protein expression existed differences among the different tissue types and the clinic pathological grades. For example, The median stainging intensity of esophageal squamous cell carcinoma was much higher than that of esophageal adenocarcinoma; the pathological grade I was lower than grades II, and grades II was same as grade III in the EZH2 protein expression, so the EZH2 protein expression was negative correlation with the degree of differentiation. However, it was curiously that the EZH2 protein expression was positive correlation with the degree of differentiation in esophageal adenocarcinoma. The EZH2 protein expression was associated with stages of esophageal cancer. The higher stages were higher EZH2 protein levels, more malignant degree. The majority of invasive and metastatic carcinomas had strikingly increased EZH2 expression, compared with carcinomas without invasion and metastases, which had significant differences in the statistics P<0.05. Western blot analysis displayed that EZH2 protein expressed in esophageal cancer tissue, correponding precancer tissue and normal tissues respectively. There were the bands of different density in molecular weight 90ku. The positive signals were detected weakly in molecular weight 90ku in normal tissues, and stronger signals were found in the correponding precancer tissue. Of the three bends, the strongest signals were detected in the esophageal cancer P<0.0\. Conclusion1. EZH2 protein overexpressed in the carcinoma and expressed weakly in the correponding precancer tissue or normal tissue, mainly in the nucleus. The correponding precancer tissue increased EZH2 protein expression, compared with the normal tissue. EZH2 protein expressed in the early carcinogenesis of esophageal cancer, then with the progression of the esophageal cancer invasion and metastases, the expression can be upregulated strikingly. EZH2 protein expression was also related with the tissue types possibly. EZH2 protein can offer novel predictors of esophageal cancer behavior at the time of diagnosis and prognostic knowledge, monitoring the progression of the tumor and determine the turnover of disease.2. EZH2 protein in the SHEE and SHEEC cells overexpressed mainly in the nucleus, We hypothesized that EZH2 protein overexpressing in the nucleus explained that they were inducted from human embryonic esophageal epithelial and still retained the embryonic primitive character. Further investigation in this area may be warranted wheather existed the other reasons.