The Expression And Regulatory Mechanism Of MMP11 And ZEB2 In Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer is a common malignant tumor. The main pathological type includes adenocarcinoma and squamous cell carcinoma. The squamous cell carcinoma is more common in China, accounting for about 90% of esophageal cancer. The incidence in male patients is higher than female patients, aged more than 40 years of age. The prognosis is poor due to the characteristics of esophageal anatomy. The whole survival rate is less than 10%. The 5-year survival rate is about 20%-40%. China is one of the highest mortality countries because about 15 million people died of cancer each year, accounting for 23.53% of whole cancer mortality. Early diagnosis is hard. The patients often lose the best time for treatment once clinical symptoms appear. Esophageal cancer is a disease of multi-gene, multi-factor and multi-stage, but the exact mechanism of its occurrence and internal development has not yet been elucidated. Given its survival rate and poor prognosis, early identification of esophageal cancer is particularly important.Epithelial-mesenchymal transition (EMT) is one of the main mechanisms to promote tumor invasion and metastasis. EMT is a polar epithelial cell can be converted into a freely moving cell during stromal cells, the main features of which lie in epithelial cells losing the original transition characteristics and expressing characteristics of interstitial cells. In tumor metastasis, cancer cells by EMT implement the surrounding tissue infiltration, then the cancer cells by mesenchymal-epithelial transition (MET) form ultimately the metastases similar with the primary tumor in planting site. Matrix metalloproteinases (MMP) are a group of Zn2+ dependent protease family including 28 members, involved in extracellular matrix degradation. The present study suggests MMP is involved in the remodeling of vascular basement membrane and extracellular matrix degradation. Endothelial cell invasion can be transferred to surrounding tissue and promote tumor angiogenesis. It is this process that leads to a higher rate of distant metastasis, as well as tumor invasion and metastasis. ZEB (Zinc finger E-box binding protein) is E-box zinc finger binding protein family members including ZEB1 (zinc-finger E-box binding homeobox 1) and ZEB2 (zinc-finger E-box binding homeobox 2). ZEB2 can induce cancer cells to have highly invasive and highly metastatic ability of mesenchymal phenotype changes, so that the tumor cells lose epithelial cell markers then obtained mesenchymal cell markers. Another study shows ZEB2 inhibits epithelial markers such as intercellular cadherin, claudin, connexin, desmoplakin and other target genes by binding to E-box target gene promoter region, then results in the loss of epithelial cell polarity and reduces inter-cell connections. At the same time, ZEB2 overexpression can promote interstitial marker vimentin, fibronectin expression and MMP. EMT, thereby, promotes tumor cell invasion and metastasis.Hedgehog signaling transduction pathway includes the ligand Hh, membrane receptor Ptch and Smo, downstream transcription factor Gli, negative regulatory factor Hip, Fu inhibitory factor (SuFu) and other components. After transmembrane protein Ptch and Smo receptor complex formation binds Hedgehog, Smo conformational changes, then it delivers its suppression on Ptch. Smo goes into the cytoplasm, activating transcription factor Gli. The latter goes into the nucleus, starting Gli1, Wnt and EGF gene expression. By combining Gli and SuFu, Gli prevent activation of downstream target genes, resulting in inhibition of the Hedgehog transduction signaling pathway. The study found abnormal activation of the Hedgehog transduction signaling pathway is in a variety of tumors including basal cell carcinoma, small cell lung cancer, medulloblastoma, prostate cancer, stomach cancer, breast cancer and pancreatic cancer and so on. Studies have shown that, Glil is raised by MMP11 involved in the growth, invasion and metastasis of breast cancer cells. Another study has shown Hedgehog transduction signaling pathway promotes EMT occurrence and development by regulating ZEB2 and Gli1.Is EMT involved in the occurrence and development of esophageal cancer? It is still not clear whether MMP11 and ZEB2 participate in the process; whether the Hedgehog signaling pathway regulates the process of esophageal cancer. It is also unknown how the specific regulatory mechanism is. This section aims to detect Gli1, MMP11, ZEB2 expression in esophageal carcinoma, and to explore the correlation with clinical factors by immunohistochemical method.ObjectiveTo observe the expression of Gli1, MMP11 and ZEB2 in esophageal squamous cell carcinoma, paracancerous tissues and normal tissues, and to investigate the relationship between them and the patients’ age, gender, clinical staging and so on. So that to seek a new idea for the clinical treatment of esophageal squamous cell carcinoma.MethodsTo observe the expression of Gli1, MMP11 and ZEB2 proteins by immunohistochemical method in 72 cases of esophageal squamous cell carcinoma and paracancerous tissues (more than 5cm away from the edge of tumor) and 18 cases of normal esophageal tissues. To compare the relationship between the expression of Gli1, MMP11 and ZEB2 protein and the different clinical stages of esophageal cancer tissues, paracancerous tissues and normal tissues by statistical software SPSS20.0.Results1. In esophageal squamous cell carcinoma, tumor-adjacent tissues and normal tissues, the positive expression rate of Glil are 55.6%,13.9% and 11.1% respectively, and there is significant difference between groups(P<0.05). The expression of Gli1 protein in tumor-adjacent and normal tissues is lower than that in esophageal squamous cell carcinoma. The difference is statistically significant (P<0.05). No difference is found between tumor-adjacent and normal tissues about the expression of Glil protein (P>0.05).2. The expression of Gli1 is not only much lower in T1-T2 than that in T3-T4 (P<0.05) but also much lower in clinical stage Ⅰ-Ⅱ than that in Ⅲ-Ⅳ(P<0.05). The expression of Gli1 is higher in nodal metastases group than that in group without nodal metastases (P<0.05).No correlations aer found between Glil protein and age, gender, tumor location, tumor diameter, the degree of differentiation(P>0.05).3. In esophageal squamous cell carcinoma, tumor-adjacent tissues and normal tissues, the positive expression rate of MMP11 are 69.4%,25.0% and 22.2% respectively, and there is significant difference between group(P<0.05). The expression of MMP11 protein in tumor-adjacent and normal tissues is lower than that in esophageal squamous cell carcinoma. The difference was statistically significant (P<0.05). No difference is found between tumor-adjacent and normal tissues about the expression of MMP11 protein(P>0.05).4. The expression of MMP11 is not only much lower in T1-T2 than that in T3-T4 (P<0.05) but also much lower in clinical stage Ⅰ-Ⅱ than that in Ⅲ-Ⅳ(P<0.05). The expression of MMP11 is higher in nodal metastases group than that in group without nodal metastases (P<0.05). No correlations are found between MMP11 protein and age, gender, tumor location, Tumor diameter, the degree of differentiation(P>0.05).5. The positive expression of ZEB2 protein is 60.5% in esophageal squamous cell carcinoma,14% in paracancerous tissues, and 8.3% in normal esophageal tissues, the difference is statistically significant between these groups (P<0.05). The expression of ZEB2 protein in esophageal squamous cell carcinoma is higher than that in paracancerous tissues and normal tissues. The difference is statistically significant (P<0.05). No difference is found between paracancerous and normal tissues about theexpression of ZEB2 protein (P>0.05).6. The positive expression rate of ZEB2 protein in stage Ⅰ-Ⅱ is far lower than that of stage Ⅲ-Ⅳ (P<0.05), and T1-T2 is far lower than that of T3-T4 (P<0.05). Group without nodal metastases is far lower than that of group with nodal metastases (P<0.05). But no correlations are found between ZEB2 protein and age, gender, tumor location, tumor diameter, the degree of differentiation (P>0.05).Conclusion1. Glil, MMP11 and ZEB2 is not associated the age, independent of sex, tumor location, diameter, degree of differentiation of esophageal squamous cell carcinoma, but the invasion and metastasis.2. Glil, MMP11 and ZEB2 is involved in the development process of esophageal squamous cell carcinoma, and up-regulation of Glil, MMP11 and ZEB2 may be one factor in promoting invasion and metastasis of esophageal squamous cell carcinoma.3. The joint detection of Glil, MMP11 and ZEB2 might help to determine the degree of malignancy of esophageal squamous cell carcinoma. It might provide a theoretical basis for clinical diagnosis and treatment.BackgroundThe occurrence and development of EMT is associated with a variety of proteins, genes, micro-environment and so on. It is also related to the regulation of a plurality of signal transduction pathways, including the P13K/AKT signaling pathway, TGF-β signaling pathway, Wnt signaling pathway and Notch signaling pathways. And a variety of growth factors, such as epidermal growth factor, insulin-like growth factor, transforming growth factor-β, fibroblast growth factor and hepatocyte growth factor, regulate the signal pathway. The intracellular signaling pathways are activated after getting the receptors on the cell surface of epithelial. The signal transduction pathway ultimately regulates epithelia-mesenchymal transition.Cyclopamine is an extract from the mountain hellebore steroidal alkaloids. After specifically binding to Smo, it leads to inactivation of the Hedgehog signaling pathway. The growth of various tumor cells is significantly inhibited in human tumor cell lines or mouse xenograft after transplanting cultured tumor biopsy by cyclopamineThis section detects the expression of Hedgehog signal pathway and EMT in esophageal cancer cells by using cyclopamine. To observe the correlation between these two after Hedgehog signal transduction pathway is inhibited.ObjectiveTo study the expression level of Gli1mRNA, MMP11mRNA, ZEB2mRNA in human esophageal cancer cells after cyclopamine is addicted. To observe the correlation of Hedgehog and EMT in esophageal cancer cell and verify the treatment of cyclopamine.Methods1. MTT tests the time-dose-response curve and the IC50 of cyclopamine.2. To detect the expression levels of Gli1mRNA, MMP11mRNA and ZEB2mRNA in EC9706 cells after cyclopamine is addicted by Real-time PCR.Results1. The proliferation of EC9706 cells is inhibited and the growth state is changed after cyclopamine is addicted. There is positive correlation between concentration of cyclopamine and inhibition rate. The IC50 is 14±1.87μmol/L2. The expression levels of Gli1mRNA, MMP11mRNA and ZEB2mRNA in EC9706 cells are downregulated by cyclopamine.Conclusion1. The proliferation of EC9706 cells is inhibited by cyclopamine.2. There may be some kind of correlation between Hedgehog signal transduction pathway and MMP11, ZEB2 in proliferation of EC9706 cells.3. Cyclopamine may have a therapeutic effect on esophageal cancer.