| 【Background】In China, Gastric carcinoma is one of the highest mortality in the digestive malignant tumor. Current clinical managements are mostly unsatisfactory due to high capability of migration and invasion. As a complex biological process, metastasis of gastric cancer is often accompanied by some cytokines and signaling pathways changes. With expectation of improving therapeutic efficacy, gene therapy is being studied as a promising modality.Especailly, in advanced gastric cancer, Trastuzumab targeting Her-2 amplication, which occupys with less 20% in gastric cancer, combined chemotherapy improved objective response rate and overall survival. Therefore, identifying and exploring the underlying mechanisms of target molecules associated with tumor migration and invasion are especially important to prolong the gastric cancer patients’ s survival time and impoving quality of life.Semaphorin3A(Sema3A), as a member of the type 3 Sema tumor suppressors, is a nerve growth inhibition of exocrine proteins. Recent studies found that Sema3 A is down-regulated expression in some kinds of cancers. Furthermore, it is closely related to the malignant behavior of tumors. Our preliminary results found Sema3 A expression decreased in gastric cancer patient’s serum, which suggesting that Sema3 A plays animportant role in the development of gastric cancer. In this study, further research will be conducted to explore the role and mechanism of Sema3 A in the gastric cancer progression.【Objective】1.To observe the role of Sema3 A in gastric cancer proliferation and invasion, the expression of Sema3 A in gastric cancer patients’ s serum and tissues and gastric cancer cell lines were detected.2. The MTT, clone formation, apoptosis, wound healing and transwell assays were conducted to observe the role of Sema3 A in proliferation and metastasis in vitro.3.The Western blot and RT-PCR were performed to explore the molecular mechanisms of Sema3 A in the proliferaion and metastasis of gastric cells.【Methods】1.The expression of Sema3 A in gastric cancer serum and tissues and different gastric cancer cell lines were determined by ELISA, RT-PCR and Western blot assay.2.Construction of Sema3 A sense expression vector, we infected the high metastasis potential MKN28-M cells of gastric cancer through the lenti-virus packaging, and then obtained exogenous stable expression Sema3 A gastric cancer cell lines.3.The MTT and clone formation assays were used to observe the Sema3 A on gastric cancer cell proliferation. Observing the effect of Sema3 A on cell apoptosis used Flow cytometry.4.In gastric cancer cells, wound healing assay and transwell assay were used to observe forced overexpression of Sema3 A on the ability of invasion and metastasis in vitro.5.Western Blot assay was used to detect the differential expression of cell apoptosis related protein.6.Western Blot and Real-time PCR assay were performed to reveal the relationship between Sema3 A and the process of EMT.【Results】1.Sema3 A expression was decreased in gastric cancer patients’ s serum and tissues.Then,we analyzed the relationship between the SEMA3 A expression level and variousclinicopathological characteristics. The loss of Sema3 A expression in gastric cancer patients were more likely to occur the lymph node metastasis.Sema3 A in the high metastasis potential gastric cancer cells expression was lower than other gastric cancer cell lines.2.Lenti-virus packaging Sema3 A sense expression vector transfection into gastric cancer cells MKN28-M, effectively increased the Sema3 A gene expression.3.Exogenous forced expression of Sema3 A inhibited gastric cancer cell proliferation,increased apoptosis of gastric cancer cells.4.The overexpression of Sema3 A decreased ability of invasion and migration in gastric cancer cell.5.Overexpression of Sema3 A downregulated Bcl-2, Bcl-Xl and upregulated Bax,Fas-L expression in vitro.E-cadherin, an epithelial marker, while it reduced the protein levels of mesenchymal markers, including Vimentin,N-cadherin, compared with the NC vector-transfected cells,which suggested that the loss or decreased expression of Sema3 A promotes gastric cancer metastasis through induction of EMT.【Conclusion】The expression of Sema3 A is decreased in gastric cancer patients’ s tissues and especalliy in gastric cancer high-metastatic MKN28-M cell lines. Further study made a conclusion that Sema3 A overexpression inhibited cell proliferation and induced apoptosis,decreased the metastasis ability of gastric cancer cell. Furthermore, we found Sema3 A increased cell apoptosis significantly by Bcl-2 family proteins and the death receptor pathway, and inhibited metastasis through EMT. These results revealed that Sema3 A may inhibit the development of gastric cancer and will provide some experimental basis for targeted therapy of gastric cancer. |
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