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The Molecular Mechanism Of HGF And TSP-1 Regulation Development In Ovarian Cancer

Posted on:2011-09-03Degree:DoctorType:Dissertation
Country:ChinaCandidate:W WeiFull Text:PDF
GTID:1114360305950171Subject:Obstetrics and gynecology
Abstract/Summary:PDF Full Text Request
Ovarian cancer is one of the common malignancy in female reproductive system tumors, and its incidence rate ranks third in gynecologic cancer; However, due to ovarian cancer patients with ascites or pelvic mass for treatment, the majority of the patients are clinical late stage already. Its mortality rate is the highest gynecological malignancies, and 5-year survival rate is still hovering at about 30%. There is currently a lack of the understanding of the exact molecular mechanism of occurrence and development in ovarian cancer. Therefore, to explore the pathogenesis of ovarian cancer and research on the related molecules involved in the occurrence and development of ovarian cancer can lay the theoretical foundation and provide experimental evidence for diagnosis and targeted treatment.Tumor invasion and metastasis is an extremely complex process involved in multi-factors. Research shows that among in the many impact factors, growth factor-mediated intracellular signal transduction pathways closely related with tumor progression, which has been a hot spot of the cancer research field in recent years. A number of factors play regulatory roles, among which hepatocyte growth factor (HGF) is currently the most widely known biological activity of growth factors. Studies have shown that HGF play an important biological functions in promoting cell division, proliferation, differentiation, movement, morphology and angiogenesis, etc. HGF play a regulatory role through multiple pathways in the cell. Mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase pathway are the two most important of growth factor signal transduction pathway, which regulate many cellular processes, such as growth, proliferation, differentiation and malignant transformation, and with close relation with the progress and prognosis of malignant tumors. HGF act on the receptor c-Met, stimulate the signals into the cells, start the series of cascade reactions, and lead to a variety of substrate phosphorylation, which led to a series of intracellular signal transduction and certain the expression of the downstream molecules and biological effects. However, the molecular regulation mechanism of HGF is not clear. To explore the role and signal transduction pathway of HGF in ovarian cancer invasion, there is of great significance to clarify the molecular mechanisms and targeted therapy for the occurrence and development of ovarian cancer.Thrombospondin 1 (TSP-1) is the one glycoprotein with a variety of biological functions, and play an important role in regulating tumor cell growth, adhesion, apoptosis and other biological behaviors and tumor angiogenesis. In the current, the research of TSP-1 in ovarian cancer is still confined to the respect of abnormal expression, and the effect of TSP-1 on biological function of ovarian cancer cells has not been reported. Studies have found that TSP-1 expression in cells may be subject to the regulation of a variety of mechanisms, for example, growth factors, oncogenes and tumor suppressor genes. At present the HGF and TSP-1 expression in ovarian cancer and the possible association existing between each other in mechanism have not been reported.This study was designed to explore the molecular mechanism of ovarian cancer, and provide new specific target for the study of the treatment for ovarian cancer. Based on the important roles of HGF and TSP-1 in the regulation of cell apoptosis, cycle and invasion, this study is divided into three parts:the first part investigated the relation between the expression of HGF and TSP-1 in ovarian cancer tissues; the second part detected the HGF regulation of TSP-1 expression in ovarian cancer SKOV-3 cells, and TSP-1 was constructed eukaryotic expression vector to transfect into SKOV-3 cells, to further study the role of TSP-1 in the HGF-regulated biological behaviors in ovarian cancer cells, for a preliminary study of the HGF-regulated mechanisms; the third part apply the specific blocking agent of MAPK/ERK and PI3K/AKT signaling pathways, and studied the relevance of HGF and its mediated downstream signaling pathways, to provide new strategies for further understanding of the mechanism of HGF function in ovarian cancer progression.PARTⅠTHE EXPRESSION OF HGF AND TSP-1 IN OVARIAN CANCERObjective:(1) To detect the expression levels of HGF and TSP-1 in ovarian cancer and benign ovarian tumor tissue, and the relationship with the clinical pathological factors.(2) To study the correlation between HGF and TSP-1 expression at the protein level in ovarian cancer tissue, to a better understanding the relationship of HGF and TSP-1.Methods:(1) Real-time quantitative PCR was applied to detect HGF and TSP-1 gene mRNA expression in ovarian cancer and benign ovarian tumor tissue.(2) Immunohistochemistry was applied to detect the location and expression of HGF and TSP-1 protein in ovarian cancer and benign ovarian tumor tissue.Results:(1) The expression of HGF and TSP-1 mRNA in ovarian cancer and benign ovarian tumor tissues:the level and positive expression rate of HGF mRNA in ovarian cancer tissue was significantly higher than benign organizations (P<0.05), which was 7.43±0.90 compared with that in the benign ovarian tumors. HGF-positive rates were 89.47% and 35%, respectively. The level and positive expression rate of TSP-1 mRNA in ovarian cancer tissue was lower than benign organizations (P<0.05), whose positive rates were 75% and 36.84%, respectively. The TSP-1 relative expression level in ovarian cancer was only 0.34±0.03.(2) The expression of HGF protein in ovarian cancer and benign ovarian tumor tissues: in ovarian benign tumor tissues, HGF protein was almost no color in epithelial cells, and HGF positive expression rate was 22.73%. In ovarian cancer, HGF showed high expression mainly in cytoplasm of tumor cells of varying degrees, and the positive expression rate was 74.29%, significantly higher than benign organizations (P<0.05). The positive expression of HGF protein in ovarian cancer was related with FIGO stage, and lymph node metastasis(P<0.05), but not with patient's age, pathological grading.(3) The expression of TSP-1 protein in ovarian cancer and benign ovarian tumor tissues:in ovarian benign tumor tissues, TSP-1 protein showed strong positive expression in epithelial cells, and positive expression rate was 77.27%. In ovarian cancer, TSP-1 protein expressed weakly positive mainly in the cytoplasm of tumor cells and a small amount of mesenchymal, and positive expression rate was 40%, significantly lower than the benign organization (P<0.05). The absence expression of TSP-1 protein in ovarian cancer was significantly related with FIGO stage and histological grade(P<0.05), but not with age and lymph node metastasis.(4) The correlation between HGF and TSP-1 protein expression in ovarian cancer tissue:among the 52 cases of HGF expression, TSP-1-positive expression was only 14 cases; among the 18 patients with negative HGF expression, TSP-1-positive expression was 14 cases. The intensity of these positive expression in ovarian cancer tissues were significant negatively associated with each other (rs =-0.575) (P<0.01).Conclusions:In ovarian cancer, there exist HGF over-expression and TSP-1 down-regulation, which are related.PARTⅡTHE REGULATION OF HGF ON TSP-1 EXPRESSION AND BIOLOGICAL BEHAVIORS IN OVARIAN CANCER CELLSObjective:(1) To detect the regulation of exogenous HGF on the expression of TSP-1 in ovarian cancer cell line SKOV-3.(2) To explore the role of TSP-1 in HGF regulation of the biological behavior in ovarian cancer cells, for a preliminary exploration on the possible mechanism of HGF regulation of proliferation, apoptosis and invasion pathways.Methods:(1) Real-time quantitative PCR and Western blot were applied to detect the effects of different concentrations of HGF on the TSP-1 mRNA and protein expression levels in SKOV-3 cells at different time.(2) Empty vector pEGFP-N1 and the recombinant plasmid pEGFP-N1-TSP-1 were transfected into SKOV-3 cells, observed the expression and localization of TSP-1 in SKOV-3 cells under the inverted fluorescence microscope, and detect TSP-1 protein expression by Western Blot.(3) After transfection, cells were divided into the following six groups for the following detection of biological behavior:SKOV-3 group; HGF+SKOV-3 group; SKOV-3/pEGFP group; HGF+SKOV-3/pEGFP group; SKOV-3/TSP-1 group; HGF+SKOV-3/TSP-1 group.(4) Detection the effect of TSP-1 on HGF-regulated proliferation activity in SKOV-3 cells by MTT assay.(5) Detection the effect of TSP-1 on HGF-regulated cell cycle distribution and cyclin D1 protein expression in SKOV-3 cells by flow cytometry.(6) Detection the effect of TSP-1 on HGF-regulated apoptosis rates in SKOV-3 cells by flow cytometry.(7) Detection the effect of TSP-1 on HGF-regulated invasiveness and MMP-9 levels in SKOV-3 cells by transwell experiments and ELISA assay.Results: (1) HGF reduced the expression level of TSP-1 in SKOV-3 cells:With the increased concentration of HGF and extension of the stimulation time, TSP-1 mRNA expression levels decreased gradually (P<0.05). HGF regulation of TSP-1 mRNA expression were dose-dependent and time-dependent. Western blot results showed that TSP-1 protein level changes are the same with mRNA level.(2) The recombinant plasmid pEGFP-N1-TSP-1 was transfected transiently into SKOV-3 cells:in SKOV-3/pEGFP cells, green fluorescence was uniformly dispersed throughout the intracellular distribution; while in SKOV-3/TSP-1 cells, green fluorescence was visible only in the cytoplasm under inverted fluorescence microscope. TSP-1 protein expression was significantly increased in SKOV-3/TSP-1 cells by Western blot (P<0.05).(3) TSP-1 transfection significantly reduced the effect of HGF on promoting proliferation in SKOV-3 cells:cell growth curve showed that cells in each group of adding HGF, cell proliferation significantly accelerated (P<0.05). Compared with HGF+SKOV-3/pEGFP group, the proliferation of HGF+SKOV-3/TSP-1 cells was significantly reduced (P<0.05).(4) TSP-1 transfection significantly altered the HGF-regulated cell cycle distribution and cyclin D1 protein expression in SKOV-3 cell:after HGF stimulation, the cell cycle distribution have apparent change, and S-phase fraction of each group were significantly increased (P<0.05). Cyclin D1 expression also increased significantly (P<0.05). Compared HGF+SKOV-3/TSP-1 group with HGF+SKOV-3/pEGFP group, S phase cells percentage and cyclin D1 expression were significantly lower (P<0.05).(5) TSP-1 transfection had no effect on HGF regulation of apoptosis rate in SKOV-3 cells:HGF treatment significantly reduced the number of apoptosis cells (P<0.05), but we compare HGF+SKOV-3/TSP-1 group and HGF+SKOV-3/pEGFP group and found that the apoptotic cells of two groups has no significant difference (P>0.05).(6) TSP-1 transfection significantly reduced the effect of HGF on the invasiveness and the supernatant MMP-9 content in SKOV-3 cells:HGF treated increased cell number to pass through membrane in each group (P<0.05), and induce MMP-9 secretion. In comparison penetrate cells and MMP-9 secretion in HGF+SKOV-3/TSP-1 group and HGF+SKOV-3/pEGFP group, we can see that the invasive ability and MMP-9 content were significantly lower in cells of HGF+SKOV-3/TSP-1 group (P<0.05).Conclusions:HGF down-regulation the expression of TSP-1 may contribute to proliferation and invasion in ovarian cancer cells, and its mechanism may be related to the increase of cyclin D1 and MMP-9 expression.PARTⅢTHE ROLE OF MAPK/ERK SIGNALING PATHWAY IN HGF-REGULATED BIOLOGICAL BEHAVIORS IN OVARIAN CANCER CELLSObjective:To investigate the role of MAPK/ERK and PI3K/AKT pathway in HGF regulation of TSP-1 expression and the induced biological behaviors.Methods:(1) Western blot was applied to detect the effect of exogenous HGF and signaling pathway inhibitors on ERK1/2,AKT phosphorylation and TSP-1 protein expression in SKOV-3 cells.(2) Detection the effect of MAPK/ERK signaling pathway on proliferation activity in SKOV-3 cells by MTT assay. Group are as follows:control group; HGF group; HGF+U0126 group.(3) Detection the effect of MAPK/ERK signaling pathway on cell cycle distribution and cyclin D1 protein expression in SKOV-3 cells by flow cytometry.(4) Detection the effect of MAPK/ERK signaling pathway on cell apoptosis rates in SKOV-3 cells by flow cytometry. (5) Detection the effect of MAPK/ERK signaling pathway on the invasiveness and MMP-9 content in SKOV-3 cells by transwell experiment and ELISA assay.Results:(1) HGF induced MAPK/ERK and PI3K/AKT signaling pathway activation in SKOV-3 cells:HGF rapidly induced p-ERK1/2 and p-AKT expression levels in SKOV-3 cells (P<0.05). P-AKT and p-ERK1/2 expression were similar, which appear after the HGF stimulation for 5min and achieve maximum effect at 15 min.(2) U0126 block HGF down-regulation on TSP-1 expression:after pre-treatment with U0126 or LY294002, HGF can not induce ERK1/2 and AKT phosphorylation; after pre-treatment with U0126, HGF can not downregulate TSP-1 protein expression (P<0.05); while after pre-treatment with LY294002, HGF can still downregulate TSP-1 protein expression.(3) Blocking MAPK/ERK signaling pathway significantly reduced proliferation activity of SKOV-3 cells:U0126 pretreatment significantly reduced the HGF-induced cell proliferation activity. Compared with HGF group, the difference was statistically significant (P<0.05).(4) Blocking MAPK/ERK signaling pathway significantly altered cell cycle distribution and cyclin D1 protein expression of SKOV-3 cells:U0126 significantly reduced the HGF-induced S-phase cells fraction, and there was a significant difference compared to HGF group (P<0.05); the expression levels of cyclin D1 was also significantly decreased.(5) Blocking MAPK/ERK signaling pathway had no effect on apoptosis rate of SKOV-3 cells:compared U0126 group with HGF group, the percentage of apoptotic cells between the two groups has no significant difference (P> 0.05).(6) Blocking MAPK/ERK signaling pathway reduces the invasiveness and MMP-9 content of SKOV-3 cells:U0126 significantly reduced the HGF-induced invasive cells, and there is significant difference compared with HGF group (P<0.05). ELISA results showed that MMP-9 level was in line with the number of invasive cells. MMP-9 secretion in the supernatant of HGF group and the U0126 group cells, the difference was significant (P<0.05). Conclusions:HGF activate the MAPK/ERK pathway and regulate TSP-1 expression level in ovarian cancer cells.
Keywords/Search Tags:ovarian cancer, HGF, TSP-1, SKOV-3, proliferation, cell cycle, invasion, MAPK/ERK
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