Expression Of VEZT In Gastric Cancer Tissues And Construction Of VEZT Over-expression Vector

Background and objective:Gastric cancer is one of the malignant tumors, which has influenced human physical and mental health for many years. At present, its incidence is the second highest of tumor in the world and the death rate is the third highest in the world. Gastric cancer is the result of various gene mutation and is a complicated process involving in many factors and multi stage. Over the years, many scholars have done a lot of researches in the pathogenesis and prognosis of gastric cancer, but its mechanism has not been fully elucidated, and no one index can predict the survival period of patients with gastric cancer better. Down-regulation of the tumor suppressor gene is an important cause of gastric cancer. Therefore, up-regulated the expression of tumor suppressor genes provides an important idea for the development of new anti-cancer drugs. The VEZT gene, discovered by our research group recently, is a candidate tumor suppressor gene associated with gastric cancer. its protein is a transmembrane protein, consists of three parts, including the short film outside, transmembrane portion and the longer inner film, and the film inside is connected to myosin Ⅶa. Myosin Ⅶa and VEZT protein are connected to E-cadherin. VEZT protein, whose function is similar to that of E- cadherin, can enhance the adhesive interactions between epithelial cells, and also plays an important role in the polarity and integrity of epithelial tissue. In our previous study, VEZT gene mRNA expression were detected lower in 5 gastric cancer cell lines and gastric cancer tissue specimens compared with GES-1 and the adjacent normal gastric mucosa by QRT-PCR. The results of this study showed that VEZT gene can be used as a candidate tumor suppressor gene associated with gastric cancer, and one of the possible potential anti-tumor therapeutic targets. However, the location of VEZT protein in gastric cancer cells and the relationship between the clinical pathological characteristics, prognosis and VEZT expression have not been fully elucidated. In this study, the location of VEZT protein and E-cadherin in gastric cancer cells were detected. The expression of VEZT protein in gastric cancer tissue and adjacent normal tissues were explored. The relationship between VEZT expression and clinical pathological features and prognosis were also analyzed. In order to make a further study of biological function of VEZT in the development of gastric cancer, the VEZT over-expression eukaryotic vector has been constructed, which provides a basis for the further study of the role of VEZT in the development and treatment of gastric cancer.Methods:The location of VEZT protein and E-cadherin in SGC-7901 gastric cancer cells were detected using immunofluorescence staining. The expression of VEZT was detected in 119 cases of gastric carcinoma and their corresponding normal tissues using immunohistochemical staining method. The relationships between the VEZT expression levels and its clinicopathological characteristics and prognosis were also investigated. And the VEZT cDNA was extracted from 293T cells and amplified by polymerase chain reaction (PCR). After the target gene and pEGFP-N1 were purified, the target gene was inserted into the pEGFP-N1 vector to construct recombinant plasmid vector using DNA recombinant technique. Then the recombinant plasmid was identified, respectively, by the 1% enzyme electrophoresis and DNA sequencing.Results:1. VEZT protein was mainly expressed in the membrane and cytoplasm of gastric cancer cells and E-cadherin protein was mainly expressed in the membrane of gastric cancer cell. They were partially overlap.2. The positive expression rate of VEZT in gastric carcinoma tissue was 30.25%, which was significantly lower than the corresponding adjacent normal tissues of 60.50%(P<0.05).3. VEZT positive expression had significantly associated with tumor differentiation, TNM stages and lymph node metastasis (P＜0.05), but had no relationship with age and tumor size of patients (P>0.05).4. VEZT expression was significantly associated with the prognosis of gastric cancer analyzed by single factor analysis (P＜0.01).5. The target fragment, consistent with the theoretical value, was verified by gel electrophoresis and the gene sequence was confirmed having no mutation by DNA sequencing.Conclusions:1. VEZT protein was mainly expressed in the membrane and cytoplasm of gastric cancer cells and partially connected with E-cadherin.2. The expression of VEZT in gastric cancer tissues is low, which is negtively related to differentiation, TNM stages and metastasis of gastric cancer. The positive expression of VEZT was an independent protective prognostic factor in patients with gastric cancer.3. The VEZT over-expression plasmid vector was successfully constructed.