| Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Over three-quarters of the EOC patients have already established regional and distant metastasis when diagnosed for the first time. Despite a well initial response to chemotherapy, most of the patients with advanced-stage cancer eventually develop recurrent disease that is almost incurable. The lack of valuable diagnostic and prognostic biomarkers and therapeutic strategies results in unchanged poor survival of EOC patients for several decades. Therefore, identification of specific molecules that contribute to the development and progression of EOC, as well as prediction of patients’ prognosis, has still remained a big challenge. Through screening methods, we found glia maturation factor-y (GMFG) is overexpressed in EOC tissues and could be considered as a predictor for prognosis of EOC patients. It was shown that GMFG expression was significantly correlated with FIGO stage and chemosensitivity of EOC patients. However, the precise biological function of GMFG is not known till now. The zebrafish is an important vertebrate model organism in scientific research especially for studies of vertebrate development and gene function. As a model biological system, the zebrafish possesses numerous advantages for scientists. Its genome has been fully sequenced, and it has well-understood, easily observable and testable developmental behaviors. Its embryonic development is very rapid, and its embryos are relatively large, robust, and transparent, and able to develop outside their mother. Furthermore, well-characterized mutant strains are readily available. We tried to study the biological function of GMFG by using zebrafish model, in order to reveal the mechanism of ovarian cancer malinant behavior. We found that GMFG mRNA was ubiquitously expressed and highly enriched in the head region and main vessels of the body. Further study showed that GMFG, but not GMFB, is required for ISVs development in zebrafish. GMFG is a regulator, probably via modulating STAT3, VEGF, and MMPs expression, in angiogenesis during zebrafish development. The in vivo study in zebrafish indicated GMFG, which was found overexpressed in ovarian cancer may participate in cellular migratory behavior. So, we further investigated whether GMFG play a role in the migratory and invasive behavior. The data showed GMFG enhenced ovarian cancer migratory and invasive ability. This process may be by regulated by altered actin cytoskeleton reorganization and which might consequently lead to the aggressiveness and metastasis of EOC. Our results suggest that GMFG may be a potential prognostic predictor for EOC patients. Part I The role and mechanism of GMFG on vascular tip cell migration in zebrafishObjective:Angiogenesis is a vital process for proper embryonic development, wound healing, malignant tumor growth and metastasis. Two glia maturation factor genes, glia maturation factor-y (GMFG) and glia maturation factor-β (GMFB), presenting different expression patterns and distinct biological functions are found in vertebrates. But, the role of GMFG and GMFB in vascular development remains largely unknown. In present study, we cloned the genes of GMFG and GMFB and studied the expression pattern. Furthermore, we revealed the role of GMFG in anioggenic sprouting in zebrafish embryogenesis.Methods:Time course whole-mount in situ hybridization, western blot study was used to detect the temporal and spatial expression pattern of GMFG and GMFB. Using gene sepecific Morpholino based on embryo microinjection technology to knockdown the expression of GMFG and GMFB separately. In vitro mRNA transcription was used in the rescue experiment. Applications based on in vivo real-time two-photon microscope imaging technology and transgenic zebrafish model to observe pecific parts of organs, tissue morphogenesis and cell levels of exercise behavior. Fluorescence-activated cell sorting was used to sorting the specific cell type. qRT-PCR was used to detect the expression level variantion of GMFG downstream genes.Results:1. We cloned zebrafish gene GMFG and GMFB for the first time. 2. GMFG and GMFB genes are highly conserved in vertebrates, which indicating they may play a basic role in cellular behavior in vertebrate.3. GMFG and GMFB are differently expressed. GMFG was predominantly expressed in endothelial cells and GMFB was highly enriched in brain.4. Knockdown of GMFG, but not GMFB, severely disrupted angiogenic sprouting of intersegmental vessels (ISVs), but this angiogenic defects were prevented by overexpression of a MO-resistant form of zebrafish GMFG mRNA In vitro cellular assays revealed that GMFG promoted cell migration and invasion.5. The expressions of angiogenic factors VEGF-A, STAT3, MMP2, MMP9, and MMP13were significantly decreased in endothelial cells of GMFG morphants. Conclusions:1. GMFG and GMFB have different expression pattern during zebrafish embryogenesis.2. Highly enriched GMFG in zebrafish vessel tip cell is essential for angiogenic sprouting. Part II The role and mechanism of GMFG on migratory behavior in ovarian cancerObjective:The aim of this study was to characterize the clinical significance of GMFG, a novel ADF/cofilin superfamily protein, and investigate its role in cell migration and invasion in epithelial ovarian cancer (EOC).Methods:The expression of GMFG in EOC tissues and ovarian cancer cell lines was evaluated by immunohistochemistry and immunoblotting respectively. The data were statistically analyzed for the associations of GMFG expression with clinicopathologic parameters and survival. In vitro cell migration and invasion assays were performed to determine the role of GMFG in cell migratory behaviors. The effect of GMFG on reorganization of actin cytoskeleton was investigated by immunostaining.Results:1. GMFG was overexpressed in EOC.2. Up-regulated GMFG expression was closely correlated with advanced FIGO stage and chemoresistance of the disease.3. EOC patients with higher GMFG expression showed poorer progression-free survival (PFS) and overall survival (OS)4. In vitro cellular assays revealed that GMFG promoted cell migration and invasion.5. GMFG expression altered actin cytoskeleton organization probably by interacting with Arp2/3complex. Conclusions:1. GMFG expression independently predicts poorer prognosis in patients with EOC.2. Ectopic overexpression of GMFG contributes to the malignant biological behavior of ovarian cancer cells. |
PDF Full Text Request