The Regulation Of Ets2 On Growth, Invasion And MTOR/p70S6K Molecular Signal Of Esophageal Squamous Cancer Cells

Esophageal cancer(EC),which is one of the most common lethal malignancies,is a disease of esophageal epithelial and esophageal mucosa. There are two major histologic types of EC: esophageal adenocarcinoma(EAC) and esophageal squamous cell carcinoma(ESCC). The two histologic types of EC are different in tumor pathological results and pathogenesis. EC is the ninth most common cancer and the eighth most common cause of death from cancer in the world. The EC’s characteristical distribution is regional. EAC occurs mainly in the western country, and ESCC is common in Asia countries. In China, the provinces nearby the Taihang Mountains have the higher incidence of ESCC, and Linzhou country is the highest. Despite great advances have achieved in clinic therapeutic approaches and basic research,the etiology of ESCC is not yet fully understood.The traditional methods of ESCC include surgery, chemotherapy and radiotherapy, and surgery is the main effective treatment. Due to no significant symptoms in the early stage, most patients have been in advanced stages of ESCC when they have obvious symptoms. Surrounding tissue infiltration or metastases were observed in most of ESCC patients, so the patients have lost the opportunity to surgery, and the 5-year survival rate is less than 10%. However, the poor prognosis of patients with ESCC is due to the lack of early diagnostic marker and therapeutic target.As a member of ETS oncogene family,Ets2 can regulate many downstream target genes. Through the interaction with protein or signaling pathways, Ets2 protein was involved in cell apoptosis, differentiation, proliferation, metastasis, angiogenesis, organogenesis and so on. Ets2 encoding gene is expressed in various cells and tissues, but its function is different in different cells and tissues. Abnormal expression of Ets2 has been observed in many tumors. Therefore, Ets2 may become a potential biological molecular target in tumor biotherapy. In recent years, more and more studies have showed that the expression of Ets2 closely related to the occurrence, development and prognosis of many tumors, but the underlying molecular mechanism is unclear. At present, few related studies of Ets2 on ESCC has been reported, and some studies suggested that Ets2 was overexpressed in ESCC tissues. Therefore, the study on the role and the underlying molecular mechanism of Ets2 in ESCC can provide the foundation for biological therapy of ESCC.In this study, the expression levels of Ets2 protein in Het-1A、EC1、Eca109 and EC9706 were detected by Western blot. And then, Ets2 gene was silenced by si RNA to study the function and biological effects of Ets2 in ESCC.The study mainly divided into two parts:The first part: We detected the Ets2 protein expression levels by Western blot in ESCC cell lines EC1, Eca109 and EC9706 cells.The second part: To study the function of Ets2 in occurrence and development of ESCC, the Ets2 gene expression was blocked by si RNA, and then cell proliferation, apoptosis, invasion, cell cycle, and related protein expression of the m TOR/p70S6 K signaling pathway were detected in ESCC.Method: 1. The detection of Ets2 expression in ESCC cellsWe analyzed the expression of Ets2 in Het-1A, EC1, Eca109 and EC9706 by Western Blot.2. The preliminary funcional study of Ets2 on ESCC carcinogenesisTo screen the three designed short interfering RNA(si RNA), the interference efficiency was analyzed by real-time PCR and Western blot after cell transfection of Ets2 si RNA via lipofectamine. Non-specifical si RNA transfected cells, Non-transfected cells and lipofectamine cells were set as control groups. After Ets2 was inhibited by si RNA in ESCC cells, cell proliferation was detected by Ed U and CCK-8; apoptosis was analyzed by Annexin V-FITC/PI Flow Cytometry and the protein expression of caspase-3; cell cycle was studied by Flow Cytometry; invasion was analyzed by transwell and the protein expression of E-cadherin; the activity of m TOR-p70S6 K signaling pathway was detected by Western Blot.Results: 1. The detection of Ets2 expression in ESCC cellsThe results of Western blot indicated that the expression of Ets2 in EC1, Eca109, EC9706 were higher than Het-1A cells, especially in EC9706 cells.2. The preliminary funcional study of Ets2 on ESCC carcinogenesis The short interfering RNA-- Ets2 si RNA1 could significantly decrease the Ets2 gene m RNA and protein levels after 48 h transfection. Compared with control groups, si RNA-mediated down-regulation of Ets2 can significantly inhibit the proliferation of ESCC cells. Compared with the negative control group, Ets2 si RNA group had a higher apoptosis rate and the caspase-3 protein expression level increased. The G0/G1 phase’s cell number of Ets2 si RNA group in Eca109 and EC1 cells increased significantly than in the negative control group.The number of invasion cells was lower in Ets2 si RNA-transfection group than that of the negative control group, and the protein expression of E-cadherin was higher. Moreover, the expressions of p-m TOR and p-p70S6 K were both decreased in Ets2-suppressing ESCC cells compared with that of control.Conclusion 1. The higher expression of Ets2 was observed in three ESCC cell lines; 2. Si RNA-mediated down-regulation of Ets2 can reduce the cell proliferation, induce the apoptosis, block the cells in G0/G1 phase, and decrease the invasion. 3. The expression of key factors in m TOR/p70S6 K signaling pathway were significantly downregulated in si RNA-transfected cells. It can be preliminary concluded that Ets2 transcription factor may participate in ESCC cell growth and survival via m TOR-p70S6 K signaling pathway.