The Expression And Role Of SOX2 In Esophageal Squamous Cell Carcinoma

【Background】Esophageal cancer is a malignant tumor originated from esophageal squamous epithelium and columnar epithelium. It is the sixth leading cause of cancer-related death in the world. Squamous cell carcinoma is predominant worldwide, although the incidence of adenocarcinoma of the esophagus has been increasing in recent years. The causes of esophageal cancer are still unclear. Transcription factor SOX2 is located in chromosome 3 and is characterized by a highly conserved high-mobility group (HMG) domain. SOX2 protein binds to specific DNA sequences by its HMG domains to active or repress target genes as a transcription factor. SOX2 was paid to much attention because of its critical roles in stem cell biology, early embryonic development, organogenesis, neural differentiation. Recently, the roles of SOX2 have been studied in human cancers, such as esophageal cancer, gastric cancer, colon cancer, lung cancer, breast cancer and so on. However, the study of SOX2 in esophageal squamous cell carcinoma is unclear. We analyze the combined sites of Snail in SOX2 promoter region by bioinformatics. We propose whether SOX2 play key roles in esophageal cancer through regulating Snail? As we all know, Snai directly represses E-cadherin expression to trigger the epithelial-mesenchymal transition (EMT) with a zinc-finger structure. Therefore, Snail play a key role in EMT regulation. In order to understand the roles of SOX2 in the tumorigenesis and development of esophageal cancer, we explore the expression correlation between SOX2 and Snail and its function in esophageal squamous cell carcinoma.【Objectives】1. To explore the expression correlation of SOX2 and Snail in esophageal squamous cell carcinoma tissues and cell lines.2. To construct and identify RNA interference lentivirus vector of SOX2 gene and to package,purify into lentivirus particle.3. To investigate the role of SOX2 in tumorigenesis of esophageal squamous cell carcinoma.【Methods】1. The expressions of SOX2 and Snail in esophageal squamous cell carcinoma tissues, paired adjacent non-cancerous tissues,lymph nodes metastasis cancer tissues were observed by immunohistochemistry. The relationships between SOX2 and Snail expression with various clinicopathological features were analyzed statistically.2. The expression and location of SOX2 and Snail in esophageal cancer cell line EC109, EC9706 were observed by Western blot and immunofluorescence.3. Two SOX2 short hairpin RNA (shRNA) vectors were constructed and identified. They were transfected into 293T cells by Lipofectamine2000 and the interference efficiency was detected by Western blot. Then, we constructed SOX2 shRNA lentivirus vector with GFP and packaged into lentivirus particle .4. The EC109 and EC9706 cells of stable infected SOX2 shRNA lentivirus were separated by Fluorescence-Activated Cell Sorting.5. The proliferation of infected SOX2 shRNA lentivirus cells was obversed by Wst-1 assay and plate colony formation assay.【Results】1. The expressions of SOX2 and Snail in esophageal squamous cell carcinoma tissues and cell lines.1) The positive expression ratio of SOX2 was 51% (29 of 57 patients) in esophageal squamous cell carcinoma and was significantly higher than 16% (9 of 57 patients) in paired adjacent non-cancerous tissues, which demonstrated that SOX2 was up-regulated in esophageal cancer tissues (P<0.05). In another tissue array, the positive ratio of SOX2 and Snail was 45%(18/45 patients)and 68%(27/40 patients)in primary cancer and there are 28%(11/40 patients), 65%(26/40 patients)respectively in paired lymph nodes metastasis cancer tissues. The expressions of SOX2 and Snail have a positive correlation in esophageal cancer and paired lymph nodes metastasis cancer tissues (P<0.05) .2) The expressions of SOX2 and Snail showed a negative association of with age, gender, tumor differentiation and TNM stage of patients(P>0.05).3) SOX2 and Snail were co-expression in EC109, EC9706 and located in nuclei by immunofluorescence. 2. We successfully constructed and identified two SOX2 short hairpin RNA (shRNA) vectors and testified the interference efficiency. Then, we constructed a SOX2 shRNA lentivirus vector with GFP and packaged into lentivirus particle.3. The role of SOX2 in tumorigenesis of esophageal cancer .1) We infected SOX2 shRNA lentivirus into EC109 and EC9706 cells. GFP positive cells were sorted using Fluorescence-Activated Cell Sorting (FACS). Western blot results demonstrated that we successfully obtained SOX2 down-regulated stable cell lines.2) The fluorescence intensity of SOX2 and Snail expression was decreased in infected SOX2 vshRNA EC9706 cells compared with that in negative control cells.3) The slow growth rates were found in SOX2 down-regulated EC109 and EC9706 cells by Wst-1 assay .4) Interfering SOX2 in EC109 , EC9706 cells accompanied with reduced colony formation ability.【Conclusion】1. SOX2 and Snail over-expressed in esophageal squamous cell carcinoma tissues and lymph nodes metastasis cancer tissues. And there is a positive relationship between the expressions of SOX2 and Snail.2. Down-regulating SOX2 expression suppressed esophageal cancer cell growth by Wst-1 assay and plate colony assay.