Cloning And Expression Of MMP-26 And Its Effects On Biological Behavior Of Glioma U251 Cells

Tumor invasion and metastasis are the most distinctive characteristics of malignant neoplasms. Metastasis or invasion is a multi-stage complicated process accompanied by extracelluar matrix degradation and changes in cell surface receptor expression. Matrix metalloproteinases (MMPs) are among the most important proteolytic enzymes and play important roles in tumor development. Their functions in tumor invasion and metastasis include degrading extracelluar matrix, promoting tumor angiogenesis, promoting tumor growth and proliferation, regulating cell adherence and spreading.MMP-26 is a novel and the most simple structure member of MMPs family. At present, the research on MMP-26 is still on the initial stage. The physiological and pathological function of MMP-26 is still unclear. Processed research show that MMP-26 contributes to the injury restoration of the body and tumor development. It could be a potential marker of the malignant of tumor progression.In this study, in order to elucidate the function of MMP-26 that contributed to the biological phenotype and behavior of glioma U251 cells, we established the MMP-26 overexpressed tumor cell model using gene transfection. After then, We hope to investigate the the role of MMP-26 in tumor development.The results are as follows:1. Expression of MMP-26 in different tumor cell lineWe detected the MMP-26 expression in different tumor cell line using immunocytochemistry and RT-PCR. MMP-26 had positive expression in A549 and PC-3M but negative in U251,PC-3 and MCF-7.2. Transfection, Selection and Identification of MMP-26 gene in U251 Cells.The vector that carrying MMP-26 gene was introduced into U251 cells using liposome-mediated transfection. G418 was used to select the positive cell clones. The positive cells which expressed MMP-26 were identified by immunocytochemistry, RT-PCR and flow cytometry.3. The effect of MMP-26 on the biological behavior of U251 cells.(1) The effect of MMP-26 on U251 cell growth and proliferation. We obtained growth curves and performed cell cycle analysis. Growth curves showed that cell transfected with MMP-26 grew faster than the untransfected cells and cells transfected cells with the vector alone. Cell cycle analysis showed that the number of transfected cells which stayed in G2-M phase was greater than the number of untransfected cells.(2) Effects of MMP-26 on adherence, spreading and migration of U251 cells. The adherence and spreading assay showed that the MMP-26 transfected cells attached to extracellar matrix more quickly and showed a lager size, a polygonal shape on the Matrigel and Fibronectin coated wells. The migration assay showed that the MMP-26 transfected cells migrate faster than the control group cell. The Boyden chamber assay showed that MMP-26 could significantly enhance the invasion ability through Matrigel of U251 cells.4. Effects of MMP-26 on tumor biological behavior using nude mice heterogeneous transplantation model(1) Effects of MMP-26 on tumor growth.The MMP-26 transfected cells and other two control groups cells were injected subcutaneously into nude mice. There was no difference in tumor formation time among these groups. But U251-MMP-26 group showed bigger tumor volume and heavier tumor weight than than that of two control groups. HE staining showed that there were more cells of mitotic figure in MMP-26 transfected cell group.(2) Effects of MMP-26 on tumor local invasion.The HE staining showed that the lobulated tumor grew infiltratively and it had pseudocapsule. There were more sub lobes and necrosis in the U251-MMP-26 group tumors. Tumor cells in the U251-MMP-26 group penetrated into blood vessels to form tumor embolus. They also invaded connective tissue and muscle tissue around the tumor. Some tumor cells broke through the prtitoneum and then infiltrate most of organs in the peritoneal cavity. RT-PCR analysis showed that there was significant difference expression of MMP-2,MMP-7 and MMP-14 among these groups.5. Effects of MMP-26 on angiogenesis.In this stuy, we elucidated the effects fo MMP-26 on angiogenesis by performing migration assay of ECV304 on Matrigel, tube formation assay, MVD assay and tumor cell induced angiogenesis assay. U251-MMP-26 cells migrated significantly faster on Matrigel than the control cells. Tube formation assay showed that U251-MMP-26 condition medium could significantly increase the tube formation ability of ECV304 on Matrigel. The vessels induced by transfected MMP-26 cells in nude mice were abundant in number, wider in diameter, longer in total length than by nontransfected U251 cells or by cells that had been transfected by the vector alone. They also formed a vascular network. This demonstrated that MMP-26 could induce tumor angiogenesis efficiently.In conclusion, we successfully cloned and expressed the proMMP-26 gene in glioma U251cells, and confirmed that MMP-26 could promote U251 cell growth, proliferation and migration on ECM. We also demonstrated that MMP-26 plays an important role in promoting the infiltration through basement membrane, local invasion and angiogenesis of tumor. MMP-26 may be one of the signals that regulates the capability of tumor cells to invade and metastasize.