| BackgroundCervical cancer, ovarian cancer and endometrial carcinoma are the three hackneyed malignancies in female genital system. The incidence rate of ovarain cancer only accounts to 4% of female malignancies, but the death rate of ovarian cancer is highest in female malignancies. Despite improvements in the application of aggressive cytoreductive surgery and combination chemotherapy, the five year survival rate of ovarian cancer is obvious improved, ovarian cancer has the most unfavorable total prognosis due to its insidious onset, concealed primary focus, diagnosis at late stage, dissemination, relapse, and tendency to develop chemotherapy resistance. Through considerable efforts aimed at elucidating the tumorigenesis of ovarian carcinoma, which gained thumping developments in early dianosisits, gene therapy and mechanism of chemotherapy resistance, the molecular mechanism of ovarian cancer is still unclear.After a quarter century of rapid advances, cancer researches reveal cancer to be a disease involving dynamic changes in the genome. The foundation of cancer has been set in the activation of oncogenes with mutations of dominants and the inactivation of tumor suppressor genes with recessive loss of function. Sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, reprogramming of energy metabolism and evading immune destruction are eight acquired biological capabilities during the multistep development of different human tumors.Because these capabilities are shared in the development of most and perhaps all types of human tumors, the oncogenes and tumor suppressor genes which are closely related with these acquired capabilities become the hot spot of mechanism and therapy research of cancer.Metastasis-associated in colon cancer-1 (MACC1) is an undescribed gene ever before which was detected by genomewide expression analytical technique in primary focus and metastasis focus of colon cancer. MACC1 could promote proliferation, invasion and hepatocyte growth factor (HGF)-induced scattering of colon cancer cells in vitro, and promote the growth of colon cancer cell transplanted tumor, which could serve as a biomarker of metastasis and prognosis of colon cancer. MACC1 aslo were demonstrated to be closely related with the metastasis and prognosis of gastric cancer, lung cancer and liver cancer. MACC1 was proved as a regulator of MET in HGF/C-met signaling, HGF was also called scatter factor which could promote the growth, adhesion, migration and invasion of tumor cells, HGF also could stimulate the growth of vascular endothelial cells which played key roles in the growth, invasion and metastasis of many malignant tumor cells, including ovarian cancer. There was still none report on the relationships between MACC1 and ovarian cancer up to now, it is still unclear that whether MACC1 implicate in ovarian cancer, whether MACC1 has clinical significances to ovarian cancer, how about the relationships between MACC1 and the biological behaviors of ovarian cancer cells, how about the mechanism of MACC1 overexpression induced ovarian cancer cell malignant transformation.ObjectiveDetect the expression of MACC1 in different ovarian tissues, analyze the relations between overexpression of MACC1 and the clinical pathological features of ovarian cancer, and evaluate the clinical significances of overexpression of MACC 1 in ovarian cancer. Then inhibit the expression of MACC 1 in ovarian cancer cells by RNA interference, investigate the effects of MACC1 knockdown on the growth, apoptosis, migration and invasion of ovarian cancer cells, approach the relationships between overexpression of MACC 1 and the biological behaviors of ovarian cancer cells. At last, analyze the possible signaling in which MACC1 implicated, discuss the possible mechanism of MACC 1 implicate in the development and progress of ovarian cancer. The present research includes four parts as follow:Part I Expressions and Significants of MACC1, HGF and C-met in Epithelial Ovarian CancerMethods1. The expressions of MACC1 mRNA in 20 specimens normal ovary tissues,19 specimens bengin ovarian tumors and 52 specimens epithelial ovarian cancer (EOC) tissues by RT-PCR.2. The expressions of MACC1, HGF and C-met protein in 20 specimens normal ovary tissues,19 specimens bengin ovarian tumors and 52 specimens ovarian cancer tissues by immunohistochemistry and western blot.3. The relationships between abnormal expressions of MACC1, HGF and C-met and the clinical pathological characters of ovarian cancer were analyzed.4. The correlation of abnormal expression in ovarian cancer among MACC 1, HGF and C-met were discussed.Results1. The expressions of MACC1 mRNA were significantly higher in EOC tissues than those in normal ovary and bengin ovarian tumor tissues (F=142.45, P=0.000); more advanced FIGO stage of EOC, higher expression of MACC1 mRNA (F=51.305,P=0.000).2. The expressions of MACC1, HGF and C-met protein were obviously higher in EOC tissues than those in normal ovary and bengin ovarian tumor tissues (χ2=35.845, P=0.000;χ2=24.532, P=0.000;χ2=28.893, P=0.000); more advanced FIGO stage of EOC, higher expression of MACC1 protein (χ2=9.707, P=0.024).3. The relative expression values of MACC1, HGF and C-met prtein were significantly higher in EOC tissues than those in normal ovary and bengin ovarian tumor tissues (F=465.790, P=0.000; F=326.289,P=0.000; F=280.871, P=0.000).4. The overexpressions of MACC1, HGF and C-met were related with FIGO stage, histological grade and lymph nodes metastasis, there were more advanced FIGO stage, worse histological grade and accompanied lymph nodes metastasis, there were more high overexpressions of MACC1, HGF and C-met in EOC (P< 0.05).5. There were positive correlations between overexpression of MACC1 and overexpressions of HGF and C-met (r=0.350, P=0.011; r=0.429, P=0.002); there was positive correlation between overexpression of HGF and overexpression of C-met (r=0.487, P=0.000).Summaries1. Overexpression of MACC1 was closely related with EOC, which could serve as a biomarker of diagnosis, therapy and prognosis of EOC.2. Overexpressions of MACC1, HGF and C-met might synergistically implicate in the malignant progression of EOC. Part II Construction of MACC1 Gene Specific shRNA Eukaryotic Expression Vector and Selection of OVCAR-3 Cells Stably Transfected with Recombinant VectorMethods1. Designed and synthesized MACC1 specific shRNA oligonucleotide strands.2. Constructed MACC1 gene specific shRNA recombinant vector based on eukaryotic fluorescence expression plasmid psuper-EGFP.3. Transfected OVCAR-3 cells with recombinant shRNA vectors, selected OVCAR-3 cells stably transfected with recombinant shRNA vectors by G418.4. Identified the best inhibited effect of MACC1 in transfected OVCAR-3 cells by RT-PCR and western blot.Results1. MACC1 specific shRNA oligonucleotides were annealed and ligated to be double-strand shRNA successfully.2. Identified by digestion and sequencing, double-strand shRNAs were inserted into psuper-EGFP at right sites.3. Transfected OVCAR-3 cells with recombinant vectors, and harvested monoclonal cell lines of OVCAR-3 cells with stably trasnfected recombinant vectors by G418 successfully.4. Identified by RT-PCR and western blot, OVCAR-3-s3 were the cell line with best inhibited effect of MACC1.Summaries1. Constructed specific shRNA recombinant eukaryotic fluorescence expression plasmid against MACC1 gene successfully.2. Harvested OVCAR-3 cell lines with stable expression recombinant shRNA vectors successfully, which lay a foundation for the further investigation about the relationships between MACC1 and the development and progression of ovarian cancer and the mechanism of MACC1 regulated HGF/C-met signaling.PartⅢEffects of MACC1 Gene Inhibition on the Ovarian Cancer Cell Line OVCAR-3 CellsMethods1. The proliferation of OVCAR-3 cells before and after inhibition of MACC1 was measured by MTT assay and monocell plate colony formation assay.2. The apoptosis of OVCAR-3 cells before and after inhibition of MACC1 was measured by FCM assay and TUNEL assay.3. The migration of OVCAR-3 cells before and after inhibition of MACC1 was measured by cell monolayer scratch assay and transwell assay.4. The invasion of OVCAR-3 cells before and after inhibition of MACC1 was measured by Matrigel assay and xenograft modle assay.Results1. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the proliferation of OVCAR-3-s3 was suppressed obviously, and the rate of colony formation was decreased significantly (F=23.725, P=0.000).2. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the apoptosis rate of OVCAR-3-s3 was increased obviously (F=1679.406, P=0.000), and the numbers of apoptosis body were increased significantly (F=271.84, P=0.000).3. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the scrathes of OVCAR-3-s3 healed more slowly obviously after cultivation 24h without FBS, and the numbers of OVCAR-3-s3 cell on the lower polycarbonate membrane of transwell chamber were decreased significantly (F=44.261,P=0.000).4. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the numbers of OVCAR-3-s3 cell on the lower polycarbonate membrane of transwell chamber covered matirgel were decreased significantly (F=40.296, P=0.000) (F=23.725, P=0.000), and the volumes of xenograft tumors of OVCAR-3-s3 cells after injection 35 days were obviously smaller (F=32.571, P=0.000).Summaries1. Inhibition of MACC1 gene could obviously suppress the proliferation, migration and invasion of OVCAR-3 cells, but obviously induce the apoptosis of OVCAR-3 cells.2. MACC1 could serve as a potential gene target for prevention and therapy of ovarian cancer.Part IV Studies on the Overexpression of MACC1 and the Pathogenesis of Development and Progress of Ovarian CancerMethods1. The morphologic changes of xenograft tumors cells caused by OVCAR-3, OVCAR-3-neo,OVCAR-3-NC and OVCAR-3-s3 cells were observed by HE stain.2. The expressions of MACC1 and C-met protein in xenograft tumors caused by OVCAR-3, OVCAR-3-neo, OVCAR-3-NC and OVCAR-3-s3 cells were observed by immunohistochemistry.3. The expressions of C-met, MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 protein in OVCAR-3, OVCAR-3-neo,OVCAR-3-NC and OVCAR-3-s3 cells were measured by western blot.4. The expressions of cyclinDl, caspase3, caspase9, survivin, MMP2, MMP9 and VEGFA mRNA in OVCAR-3, OVCAR-3-neo, OVCAR-3-NC and OVCAR-3-s3 cells were measured by RT-PCR.Results1. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the morphology of OVCAR-3-s3 cell changed obviously.2. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the expressions of MACC1 and C-met protein in xenograft tumors caused by OVCAR-3-s3 cells were decreased obviously.3. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the expressions of C-met, p-MEK1/2 and p-ERK1/2 protein in OVCAR-3-s3 cells were decreased significantly.4. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the expressions of cyclinD1, survivin, MMP2, MMP9 and VEGFA mRNA in OVCAR-3-s3 cells were decreased significantly, but the expression of caspase3 and caspase9 mRNA were increased obviously.Summaries1. Overexpression of MACC1 might implicate in the malignant transformation of ovarian cancer cells.2. Overexpression of MACC1 might implicate in the invasion and metastasis of ovarian cancer via HGF/C-met signaling and MAPK signaling mediated downstream tumor associated genes. Conclusions1. Overexpression of MACC1 was closely related with EOC, which might implicate in the malignant transformation of ovarian cancer cells.2. Overexpression of MACC1 might implicate in the invasion and metastasis of ovarian cancer via HGF/C-met signaling and MAPK signaling mediated downstream tumor associated genes.3. MACC1 could serve as a potential gene target for prevention and therapy of ovarian cancer. |
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