Research Of Mechanisms By Which S100A4 Regulates The Development Of Emt Of Esophageal Squamous Cell Carcinoma

Esophageal squamous-celled carcinoma is one of the deadliest malignant tumor among human squamous-celled carcinoma. The prognosis is poor and efficacy of treatment is limted. In China, there are 200 000-300 000 new cases, which attribute to over 50% new cases worldwide. Especially, Lin county and Ci county are the area with high rates of new cases. Hence, esophageal squamous-celled carcinoma is one of the tumors that threatens the health and life of the people of our country. Although the perioperative and post-operative chemotherapy and radio therapy have improved the life span of esophageal squamous-celled carcinoma patients nowadays, the prognosis of esophageal squamous-celled carcinoma patients at advanced stage still remains poor, with 50% of patients living about 6-8 months and 5-year survival rates are about 10%-25%. The main reason is the invasion and metastasis of esophageal squamous-celled carcinoma. esophageal squamous-celled carcinoma tends to invase lymphatic and vascular more easier compared with other types of gastrointestinal solid tumors. Hence, the mechanisms of the invasion and metastasis of esophageal squamous-celled carcinoma is one of the highlights and researchers found that epithelial-mesenchymal transition(EMT) and mesenchymal-epithelial transition(MET) are the important regulating mechanisms of multiple malignant tumors including esophageal squamous-celled carcinoma.S100A4 is one of the members of calcium-binding S100 family and is involved with the interaction between cellular scaffolding and cell membrane, signal transmission, cell growth and differentiation as well as promoting cell hyperplasia and vascular development to promote the invasion and metastasis of cancer cells. Hence, S100A4 is also termed as metastasis-related gene. It has been found that S100A4 upregulation are in positive correlation with the recurrence and metastasis of breast cancer, rectal carcinoma, lung carcinoma, gastric carcinoma, esophageal carcinoma, prostate carcinoma and et al. However, the mechanisms by which S100A4 promotes invasion and metastasis of esophageal squamous-celled carcinoma remains unclear.In this study, we examined S100A4 m RNA and protein expression levels in esophageal squamous-celled carcinoma tissues and paired non-cancerous tissues using q RT-PCR technique, immunohistochemistry and western blotting method followed by analysis of correlation between S100A4 exrpession levels and clinical parameters; S100A4 expression was depleted using si RNA and the ability of invasion and metastasis of EC9706 was examined; E-cadheirn, vimentin and Snail expression were analyzed after S100A4 was knockdown; and the ability of invasion and metastasis of EC9706 was examined after Snail was depleted in the EC9706 cells.In general, this study focuses on S100A4 as a molecular target. To explore its role in the invasion and metastasis of esophageal squamous-celled carcinoma, this study is divided into three parts.Prat Ⅰ: Expression of S100A4 in the esophageal squamous-celled carcinoma tissues and the prognostic analysisMethods1. S100A4 m RNA and protein expression levels in 150 cases of esophageal squamous-celled carcinoma tissues and paired non-cancerous tissues were examined by using in situ hybridization technique and immunohistochemistry followed by analysis of correlation between S100A4 exrpession levels and clinical parameters. q RT-PCR technique and Western blotting technique were used to examine the expression of S100A4 m RNA and protein in the 10 randomly selected cases of esophageal squamous-celled carcinoma.2. Kaplan-Meier survival curve was used to analyze the relationship between the expression of S100A4 m RNA and protein and the survial time of esophageal squamous-celled carcinoma, further to analyze the role of S100A4 in the esophageal squamous-celled carcinoma and molecular mechanisms.3. All the data were analyzed by using SPSS 12.0. numeration data were analyzed by using χ2 test. Survival curve was analyzed by using Kaplan-Meier method and the log-rank test. Measurement data( x ±s) was analyzed by using the single factor analysis of variance and t test. The size of test α=0.05. Results1. S100A4 m RNA and protein mainly locate in the cytoplasm of esophageal squamous carcinoma and paired non-cancerous tissues. S100A4 m RNA and protein was significantly upregulalted in the esophageal squamous-celled carcinoma than that in the paired non-cancerous tissues(P<0.05).2. The expression of S100A4 m RNA and protein in the esophageal squamous-celled carcinoma were not relavant with patients gender, age and tumor differentiation degree(P>0.05), but were relavant with tumor size, TNM stages and lymph node metastasis of esophageal squamous-celled carcinoma(P<0.05). Part Ⅰ: Expression of S100A4 in the esophageal3. The lifespan of esophageal squamous-celled carcinoma patients with higher S100A4 m RNA and protein expression was significantly longer than that of lower expression group.Part Ⅱ: The mechanisms of S100A4 affects esophagealsquamous-celled carcinoma invasion and metastasis Methods1. S100A4 expression was depleted by using si RNA in EC9706 cells and the growth, cell cycle, invasion and metastasis of EC9706 was examined then.2. S100A4 expression was depleted by using si RNA in EC9706 cells and the E-cadheirn, vimentin and Snail expression levels of EC9706 cells was examined.3. Western blotting results were analyzed by using Gene Tools for grey value analysis(n=3). All the data were analyzed by using SPSS 12.0. numeration data were analyzed by using χ2 test. Survival curve was analyzed by using Kaplan-Meier method and the log-rank test. Measurement data( x ±s) was analyzed by using the single factor analysis of variance and t test. The size of test α=0.05.Results1. si RNA transfection depleted S100A4 m RNA expression level significantly(P<0.05).2. Depletion of S100A4 inhibited EC9706 cell growth significantly; the rate of cells treated with si RNA-S100A4 in G0/G1 phase increased significantly compared with that of the control groups; the invasion and metastasis ability of si RNA-S100A4 group decreased significantly compared with that of the control groups(P<0.05).3. Downregulation of E-cadheirn expression, upregulation of vimentin and Snail are correlated with the S100A4-depletion mediated the inhibition of EC9706 cell inhibition and ability of invasion and metastasis(P<0.05).Part Ⅲ: Mechanisms by which S100A4 regulates the EMT ofesophageal squamous-celled carcinoma through Snail Methods1. pc DNA 3.1-Snail plasmid was transfected into EC9706 cells and stable transfected cells were acquired through G418 screening, which could compromised the ability of cell growth, invasion and metastasis effect mediated by si RNA-S100A4 transfection.2. E-cadheirn and vimentin expression were examined by using q RT-PCR and western blotting method after pc DNA 3.1-Snail plasmid was transfected into EC9706 cells.3. All the data were analyzed by using SPSS 12.0. numeration data were analyzed by using χ2 test. Survival curve was analyzed by using Kaplan-Meier method and the log-rank test. Measurement data( x ±s) was analyzed by using the single factor analysis of variance and t test. The size of test α=0.05. Results1. Upregulation of Snail could reverse partly the EMT phenoma mediated by si RNA-S100A4 transfection in the EC9706 cells(P<0.05).2. Upregulation of Snail could increase the ability of invasion and metastasis of EC9706 of si RNA-S100A4 group(P<0.05).3. Upregulation of Snail could increase cell growth of EC9706 cells transfected by S100A4(P<0.05). Conclusion1. The expression of S100A4 in esophageal squamous carcinoma cell line and paired non-cancerous tissues were examined; S100A4 may play an important role in the growth, invasion and metastasis of esophageal squamous-celled carcinoma and may become a molecular marker of metastasis and prognosis of esophageal squamous-celled carcinoma.2. S100A4 may be involved with the regulation of cell growth, cell invasion and metastasis. Because S100A4 mediated esophageal squamous-celled carcinoma cell invasion and metastasis, it is expected to provide the molecular target therapy for esophageal squamous-celled carcinoma.3. Mechanims of S100A4 regulating the EMT progress of esophageal squamous-celled carcinoma is through Snail.