The Expression Of NOB1 In Esophageal Cancer And Its Effect On The Proliferation And Drug Resistance Of Esophageal Cancer Cells

Esophageal cancer is one of the malignant tumors with high incidence in the world. It has the characteristics of insidious onset and fast development, thus most of the patients are found in advanced period and the prognosis is poor. The prognosis of patients with esophageal cancer has close relationship with early diagnosis. Because of the lack of effective means of early diagnosis, the majority of patients have been in advanced period when go to a doctor for the first time. The five-year survival rate is between 15%~25%. With the development of translational medicine, looking for specific tumor marker is the key to early diagnosis, which provide opportunities for radical surgery. The molecular targeted therapy has become another choice for tumor patients, except for surgery, radiotherapy and chemotherapy, and has provided a new research direction for individualized treatment as well. Therefore, it is necessary to explore valuable molecular markers and molecular targets so as to provide clues for the early diagnosis and individualized treatment of esophageal cancer.NOB1 encoded protein contains a PIN domain and a zinc ribbon protein domain. NOB1 plays an important role in the biological processes, such as cell cycle, tumorigenesis and multi-drug resistance by regulating transcription. The results of RT-PCR analysis show that NOB1 expression is positive in esophagus, lung, liver, spleen, kidney, colon and other tissues of human. It has been found that NOB1 was involved in the occurrence of breast cancer, cervical cancer, ovarian cancer, colon cancer, liver cancer, thyroid cancer, glioma, leukemia, etc. This study aims to detect the NOB1 expression in different grade and stage of esophageal cancer tissues, and to evaluate its effect on the growth and drug resistance of esophageal cancer cells, in order to provide some guidance for new molecular diagnosis and therapy of esophageal cancer.Objective 1. To evaluate the expression of NOB1 in patients with esophageal squamous cell carcinoma, and to analyze the correlation between the NOB1 expression and clinical pathological characteristics. 2. To evaluate the relation of NOB1 expression and the prognosis of patients. 3. To evaluate the expression of NOB1 protein in esophageal carcinoma cells and normal esophageal epithelial cells. 4. To evaluate the expression of NOB1 m RNA in esophageal cancer cell lines. 5. To establish the esophageal cancer cells with decreased expression of NOB1. 6. To evaluate the effect of NOB1 on proliferation of esophageal squamous cell cancer cells. 7. To evaluate the effect of NOB1 on drug resistance of esophageal squamous cell cancer cells.Method 1. The samples of cancer tissues and adjacent non-neoplastic tissues were collected from 164 cases of esophageal squamous cell cancer patients who have received operation in Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an. 2. The NOB1 expression was detected in cancer tissues and adjacent non-neoplastic tissues by immunohistochemistry. 3. The electronic medical record system, including follow-up table, was established. And the patients were follow-up by telephone. 4. The expression of NOB1 protein was detected in esophageal cancer cells and normalesophageal epithelial cells using western blot. 5. The expression of NOB1 m RNA was detected in esophageal cancer cells by real time PCR. 6. The esophageal cancer cells with decreased expression of NOB1 were established using lentiviral transfection technique. 7. The effect of NOB1 on proliferation of esophageal squamous cell cancer cells was evaluated using MTT assay and clone formation assay. 8. The effect of NOB1 on drug resistance of esophageal squamous cell cancer cells was evaluated using MTT assay, flow cytometry assay and Annexin/PI staining assay. 9. The Student ’s t test, Pearson χ2 test and Fisher’s test were used to evaluate the correlation of NOB1 expression with age, gender, tumor grade, depth of invasion, and lymph node metastasis of esophageal squamous cell carcinoma. 10. The Kaplan-Meier survival curves and log-rank test were used for survival analysis. The COX regression model was used for prognosis analysis. Statistics analysis of data was performed by statistical software SPSS 16.0, P<0.05 was considered statistically significant.Result 1. In 164 cases of esophageal squamous cell carcinoma tissue, NOB1 protein was expressed in the cytoplasm; 2. The results of immunohistochemistry showed that the expression of NOB1 in esophageal cancer tissues was higher than that of noncancerous tissues. The expression of NOB1 was strong positive(+ +) in 49 cases(29.9%), positive(+) in 52 cases(31.7%), and negative(-) in 63 cases(38.4%). 3. The results of PCR showed that the NOB1 m RNA expression level was high in 8 cases of esophageal cancer tissues(0.9±0.023) as compared with that of the adjacent tissues(0.3 ± 0.004). 4. The expression level of NOB1 in esophageal cancer patients was significantly related with the tumor size and differentiation, but had nothing to do with age, sex, and lymph node metastasis.5. The survival of patients with positive expression of NOB1 was significantly worse than that of patients with negative expression. The higher the NOB1 expression was, the worse the prognosis was. 6. The results of western-blot showed that the NOB1 protein expression level was high in esophageal cancer cells EC109 and EC9706 as compared with that of normal esophageal epithelium cell line HEEC. 7. The results of quantitative real-time PCR showed that the NOB1 m RNA expression level was high in esophageal cancer cells as compared with that of HEEC cells. The data were consistent with that of western-blot. 8. The expression level of NOB1 was inhibited in esophageal squamous cell cancer cell line EC9706 by lentiviral transfection. Down-regulation of NOB1 might inhibit the proliferation of esophageal squamous cell cancer cells. 9. Down-regulation of NOB1 might inhibit the drug resistance of esophageal squamous cell cancer cells.Conclusion 1. The expression of NOB1 was significantly increased in esophageal squamous cell carcinoma as comapred with that of adjacent non-neoplastic tissues, and its expression level was significantly related with the differentiation and prognosis of esophageal cancer. 2. Down-regulation of NOB1 might inhibit the proliferation and drug resistance of esophageal squamous cell cancer cells. 3. NOB1 might be a valuable prognostic marker and a potential gene therapeutic target in esophageal squamous cell carcinoma.