The Study Of Expression And Mechanism Of Smad4 In HO-8910 And HO-8910PM

Objective : Malignant tumor is one of the most devastating diseases threatening human health nowadays. Metastasis of malignant tumor leading to failure of multi-visceral organs is the main cause of tumor related death. Now preventing metastasis is one of the key ways to treat tumors. With the increasing research of multi-stages, multi-factors and multi-steps theory which was regulated by metastasis gene ,people have a more further cognition on tumor metastasis.To discovery and confirm the metastasis related gene becomes hot spot in the research area of tumor metastasis mechanism . Because these genes not only enable us to understand tumor metastasis mechanism .And more importantly these genes and its products may become the anti-tumor metastasis taget or the index to observing prognosis.The transforming growth factorβ(TGF-β) superfamily is composed of many multifunctional cytokines including TGF-βs, activins, inhibins, and bone morphogenic proteins. These proteins regulate a variety of biological responses such as proliferation, differentiation, apoptosis, and development . Members of this family signal through cell surface transmembrane serine /threonine protein kinases known as type I and type II receptors. TGF-βbinds to a heteromeric complex of type I and type II receptors. The constitutively active type II receptor phosphorylates and activates the type I receptor which phosphorylates Smad2 and Smad3 (receptor-associated Smads). Receptor-associated Smads bind the common mediator Smad4 and are translocated to the nucleus, where they associate with DNA-binding cofactors to modulategene transcription. Smad4 is the central molecule in the signal transduction. Now, the investigation in TGF-β/Smad signal transduction mainly concentrate on gastroenteric tumor such as pancreatic carcinoma but few reports on orary cancer. Ovary cancer is one of three malignant tumors in feminine genitalia. Until now lacking the effective early diagnosis method. Patients always have been in the metaphase or terminal. The ovary cancer is of the following characte -ristics: cancer cells fall off from the primary focus, enter ascites, plant on the surface of caul, chorion and mesentery widely.The investigation indicates that over-expression of Smad4 can suppress the proliferation , synthesis extracellular matrix and induce apoptosis.Recent years,it conform that the expression level of Smad4 is generous related with the development of malignant tumor, biological behaviour and prognosis.Many scholars consider that the inactivation of DPC4 take place in the later stage of tumor development belonging to later stage molecule event and relating with tumor invasion and metastasis.In order to discuss the mechanism of tumor metastasis, we utilize high and low metastasis cell lines of human ovary cancer HO-8910 and HO-8910PM as the subjects to exploit the molecular mechanism.We detect the different expression of smad4 in the two cell line. And then,we construct a model which can get over expression of smad4 by gene transfection, detect the expression of PAI-1,VEGF in downstream of TGF-β/Smad signal transduction and another E-cad gene which related with cancer metatasis.We use Transwell chamber and cell scarification assay to detect the capability of the invasion and metatasis in order to investigate the relationship in smad4 gene and tumor metatasis as well as moleculer mechanism.Methods: 1.detect the different expression of smad4 in cell lines of human ovary cancer HO-8910 and HO-8910PM :①detect the cellular localization of smad4 by immunochemical method.②detect the different expression of smad4 in cell lines of human ovary cancer HO-8910 and HO-8910PM by Western blot. 2.construct smad4 eukaryotic expression vector, transient transfection smad4 gene by liposome,24 hours later, detect the expression of Smad4,PAI-1,VEGF,E-cadherin. 3.transwell chamber to detect the different capability of cell invasion. 4. cell scarification assayto detect the capability of cell migration. 5. adhesion assay to detect the different capability of adhesion with extracellular matrix. 6. gelatinase zymography assay to detect the activity and expression of MMP2 and MMP9.Results: 1.Immunocytochemical method showed that Smad4 is expressed in the cytoplasm and nucleus, highly expressed in HO-8910PM and lowly expressed in HO-8910. 2.Western blot method showed that Smad4 highly expressed in HO-8910PM and lowly expressed in HO-8910. 3.transient transfection smad4 gene can down regulate the expression of PAI-1 and up regulate the expression of E-caherin,but it can not influence the expression of VEGF. 4. Transwell chamber invasion assay showed that cell number invading through Matrigel filter was significantly decreased in the PM/Smad4 cell compared with the negtive group(P<0.05). 5.The result from cell scarification test indicated that PM/Smad4 cell migrate less quickly than negative untransfected group. 6.The result from cell adhesion test indicated that PM/Smad4 cell adhere more than negative untransfected group (P<0.05). 7. the activity and expression of MMP2 and MMP9 have not obviously difference in HO-8910,HO-8910PM,PM/Smad4 cell except the precursor of MMP2.Conclusion: 1.Smad4 highly expressed in HO-8910PM and lowly expressed in HO-8910 indicated that it may related with tumor metastasis. 2. Smad4 gene can suppress the capability of metatasis in vitro not through MMP2 and MMP9. 3. Smad4 gene can suppress the capability of migration of HO-8910PM in vitro. 4. Smad4 gene can increase the capability of adhesion of HO-8910PM with extracellular matrix. 5.Smad4 gene can suppress tumor metastasis by regulate the expression of PAI-1 and E-cadherin.