| Objective Gastric carcinoma is one of the most common carcinomas in China. In the world, the mortality of gastric cancer is the second common among all of the malignant tumors. In recent years, although there are many methods to treat it, they are not satisfied. To explore the molecular mechanism of carcinogenesis and progression might improve the traditional treatment, such as surgery, radiotherapy, chemotherapy and so on, and might also help to find new prognostic factors.Epithelial-mesenchymal transition (EMT) refers to the process by which cells transit from epithelial phenotype to mesenchymal phenotype. EMT is important for embryonic development, wound healing, and invasion of carcinomas; its prominent hallmarks are loss of expression of epithelial markers such as E-cadherin and induction of mesenchymal markers such as N-cadherin. To explore the molecular mechanism of EMT, also help to find new prognostic factors to gastric cancer.MicroRNAs (miRNAs), a new class of endogenous, noncoding and single-stranded RNAs, were recently discovered in both animals and plants. They trigger trans-lational repression and/or mRNA degradation mostly through complementary binding to the3’-untranslated regions of target mRNAs. Studies have shown that miRNAs can regulate a wide array of biological processes such as cell proliferation, differentiation, and apoptosis. Aberrant expression and metabolism of miRNAs are associated with human diseases including malignancy.Methods In the first part, The levels of miR-200a and EMT-associated hallmarkers ZEB1/ZEB2, E-cadherin, N-caderin and (3-catenin in64samples of gastric carcinoma tissue and10samples of normal gastric mucosa tissue were detected with fluorescence in situ hybridization and Immuno-histochemistry. The correlation showed significant correlations existed between miR-200a expression and those of ZEB1/ZEB2, N-cadherin, E-cadherin and β-catenin.In the second part, The levels of EMT-associated hallmarkers ZEB1/ZEB2, E-cadherin, N-caderin and P-catenin in64samples of gastric carcinoma tissue and10samples of normal gastric mucosa tissue were detected with Immuno-histochemistry. The correlation showed significant correlations existed between miR-200a expression and those of ZEB1/ZEB2, N-cadherin, E-cadherin and β-catenin. Transfection with miR-200a mimics in SGC7901cells, we tested the effects of miR-200a elevation on ZEB1and ZEB2protein expression in SGC7901, using immunofluorescence staining. We investigated the underlying mechanism of miR-200a regulation of epithelial-mesenchymal transition (EMT) in gastric carcinoma cells.Transfection with miR-200a mimics in SGC7901cells, we tested the effects of miR-200a elevation on ZEB1and ZEB2protein expression in SGC7901, using immunofluorescence staining. We investigated the underlying mechanism of miR-200a regulation of epithelial-mesenchymal transition (EMT) in gastric carcinoma cells.In the third part, subcutaneous SGC7901gastric cancer model was established in nude mice. Mice were randomly selected for the control, scrambled miR-treated, and miR-200a-treated groups. A mixture of oligonucleotides containing scrambled miR-200a mimics and Lipofectamine2000was injected into the xenograft tumors, the tumor volumes were measured. The expression of ZEB1/ZEB2, E-cadherin, N-caderin, and β-catenin and so on were measured among three groups.Results In the first part, analysis using FISH showed that levels of miR-200a, miR-200b and miR-200c decreased significantly in GA compared to normal gastric mucosa tissue. MiR-200a, miR-200b and miR-200c expression negatively correlated with WHO GA grades. In the second part, Compared to those in normal gastric mucosa tissue, the expression of ZEB1/ZEB2, N-caderin and β-catenin were elevated, but the E-cadherin expression was down-regulation in the gastric carcinoma.Levels of ZEB1/ZEB2, N-cadherin, P-catenin, Twist and Snail2protein were positively correlated with the WHO GA grades. However, the expression of E-cadherin was negatively correlated with the WHO GA grades.we transfected miR-200a mimics into SGC7901cells, and then used immunofluorescence staining to inhibits EMT by upregulating E-cadherin and downregulating ZEB1/ZEB2, N-cadherin, β-catenin, Twist and Snail2. In the third part, the group with miR-200a mimics formed substantially smaller tumors than did the control and the scramble groups. We determined protein expression of these genes immunohistochemically. N-cadherin,β-catenin, Twist and Snail2were prominently downregulated, and E-cadherin was upregulated in tumor specimens of the miR-200a mimic-treated group.Conclusion1.Downregulated miR-200a expression to decrease E-cadherin, increase N-cadherin, β-catenin and so on and inhibit the Wnt/β-catenin pathway.2. After transfection with miR-200a mimics in SGC7901cells, miR-200a epression were increased by RT real-time PCR. Overexpression of miR-200a expression may increase the level of E-cadherin and suppress Wnt/β-catenin pathway by targeting ZEB1and ZEB2in gastric adenocacinoma.3. The established subcutaneous SGC7901gastric cancer models in nude mice are treated with miR-200a.miR-200a inhibits EMT and tumor growth. Overexpression of miR-200a expression may increase the level of E-cadherin and suppress Wnt/β-catenin pathway by targeting ZEB1and ZEB2in gastric adenocacinoma. |
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