Expression And Signifiance Of Aquaporin-5 And Aquaporin-9 In Epithelial Ovarian Tumors

Background and ObjectiveOvarian cancer is one of the gynecologic malignant tumors, whose mortality rates are almost highest. And its incidence rise significantly in recent years. Because ovarian cancer spot locates in the deep pelvic bottom absenting early symptoms and signs. The most patients don't go to receive a medical examination until they can touch a package of block in the abdomen or feel windy because of the massive ascites,Most of them have already been the later stage, had the shift in the abdominal cavity even distant place.5-year survival rate of the patients of the later period was at only 35%. If the ovarian cancer can be found, diagnosed and treated early, the 5-year survival rate in early-stage ovarian cancer can reach to 90% without postoperative chemotherapy and radiation, improving the prognosis of the ovarian cancer. One of the biological characteristics of malignant tumor is to soak and shift, which promote the development of ovarian cancer. Explore the mechanism of invasion and metastasis of ovarian cancer and looking for can predict the transfer and biological indicators to guide clinical treatment of ovarian cancer have a high value in clinical application. The aquaporins are small, very hydrophobic, intrinsic membrane proteins. It exists widely in nature from mammals, amphibians, plants, yeast, bacteria to various lower organisms. The aquaporins can transport other small molecules material except of water. The aquaporins are related to the course of physiology and pathology of water, exciting mainly in epithelial and endothelial cells of organs and organizations, which infect the water metabolism in mammals. In recent years, It is find that aquaporins take part in the progress of tumors, and various forms of tumor with excessive expression The aquaporins support water and nutrients to the rapid growth of tumors by transporting water and other small molecules such as glycerine selectively and efficiently. It is find that aquaporins stimulate the growth of vascular endothelium cells of the tumor, contributing to the tumor angiogenesis, promoting the growth, invasion and metastasis of tumors. It is considered as a new cancer-causing genes and metastasis-enhancing factor.Ovarian cancer mortality,5-year survival rate, poor prognosis and factors related to the gynecologic oncology researchers had been the focus of attention. There are a lot of reports about aquaporins in many sorts of tumors, but the report in ovarian cancer is rare. This study adopts immunohistochemistry and in situ hybridization methods to detect the expression levels of AQP5 and AQP9 protein and mRNA in epithelial ovarian tumors, observe the kinds of proteins and the relationship between epithelial ovarian cancer in the clinical and pathological parameters and the correlation between them. Making a primary discussion on function of aquaporins in progress of tumors to explore a new molecular biological marker related to measure and treatment of epithelial ovarian cancer.Materials and Methods1 Experimental samples:we collected 90 tissue cases of the Operations fixed in formalin and embedded with paraffin in department of gynecology and obstetrics in the third affiliated hospital of Zhengzhou University between Jan 2007 and Dec 2009. Serial paraffin sections were made from every tissue at 6 um, each section is done in duplicate for SP staining and in situ hybridizations (ISH) staining.90 tissue cases (normal:10 cases, benignant:15 cases, borderline tumor: 15 cases and malignant:50 cases) have been confirmed by pathological. In Ovarian cancer:Ⅰstage was in 8 cases,Ⅱstage was in 10 cases, III stage was in 23 cases, IV stage was in9 cases (FIGO2000); there are ascites in 34 cases, no in 16 cases,; histological grade wasⅠin 13cases,Ⅱin 18 andⅢin 19 (WHO2000); 28 were serous cystacarcinomas, 15 were mucinous cystacarcinomas,7 were endometrioid carcinomas; 29 cases had lymphatic metastasis,21 cases had no lymphatic metastasis. Their ages varied from 18 years to 80 years with a peak age of (47.12±6.59) years, the patients with tumor have received no any chemotherapy, radiotherapy and immunization therapy before operation.2 Experimental reagents:rabbit anti-AQP5 polyclonal antibody(Beijing Biosyn thesis Biotechnology CO.LTD), rabbit anti-AQP9 polyclonal antibody, immunohistochemistry S -P kits and DAB developer(Beijing Zhongshan Corp), AQP5> AQP9 cRNA probe(Beijing Sunbiotech CO.LTD), In situ hybridization kit(Wuhan Boston Corp) and so on.3 Experimental methods:Using immunohistochemical SP and in situ hybridization methods detected the protein and mRNA of AQP5 and AQP9 expression in 10 cases of normal tissues,15 cases benignant tumors,15 cases of borderline tumors and 50 cases of carcinomas, and analysis of them expression and various clinical pathological data of the various inter-relationships.4 Statistical analyses:The data were analyzed by statistics software SPSS 13.0 package. Multi-samples rate comparison used contingency table chi-square test, rank-sum test and two samples rate used fourfold table chi-square test. Spearman rank correlation analysis was used to analyze the relevance. The test standard was a=0.05.P<0.05 statistically significant.Results1 There are expression of AQP5 and AQP9 protein in parenchymal cell cytoplasm and Membrane of normal ovarian tissues and Ovarian Epithelial Tumors, shallow brown to yellow brown particle. Protein positive rate and staining increased with the tumor histological grade. the positive expression rate of AQP5 and AQP9 protein respectively, normal 20.00%(2/10) and 20.00%(2/10), benign 33.30%(5/15) and 40.00%(6/15), borderline 73.30%(11/15)and 6O.63%(9/15), malignant 84.00% (42/50) and 86.00%(43/50). There were significant differences in the relative contents of AQP5 and AQP9 protein among cancer tissues and normal tissues, benign tumors, (P<0.05) and so AQP5 protein between borderline and normal tissues (P <0.05), There was no statistically significant difference in other groups (P>0.05). There are expression of AQP5 and AQP9 mRNA in parenchymal cell cytoplasm of ovarian borderline tumors and carcinoma, blue particle. There is no expression of AQP5 and AQP9 mRNA in normal tissues and benignant tumors. the positive expression rate of AQP5 and AQP9 mRNA respectively, borderline 46.70%(7/15) and 46.70%(7/15), malignant 66.00%(33/50) and 68.00%(34/50). positive expression rate of AQP5 and AQP9 mRNA in borderline tumors and carcinomas are obvious higher than that of normal tissues and benignant tumors (P<0.05), There was no statistically significant difference in other groups (P>0.05)2 The expression of AQP5 and AQP9 mRNA and protein In poorly differentiated cancers was obviously higher than that in highly differentiated cancers, there was statistically significant difference in them (P<0.05); In ascite cases the expression was obviously higher than that in no ascite cases, there was statistically significant difference in them (P<0.05); there was no associated with pathological types (P >0.05). In stageⅢandⅣthe expression of AQP9 mRNA and protein was significantly higher than that in stageⅠandⅡ, there was statistically significant difference in them (P<0.05); In lymphatic metastasis cases the expression was obviously higher than that in no lymphatic metastasis cases, there was statistically significant difference in them (P<0.05); In stageⅢandⅣthe expression of AQP5 mRNA and protein was higher than that in stageⅠandⅡ, but there was no significant difference in them(P<0.05); In lymphatic metastasis cases the expression was higher than that in no lymphatic metastasis cases, there was no significant difference in them (P<0.05)3 The expression of AQP5 and AQP9 protein in ovarian cancer tissues were positively correlated. There were 48 cases whose AQP5 protein expression was positive,40 of them AQP9 protein expression were positive, There were 8 cases whose AQP5 protein expression was negative,5 of them AQP9 protein expression were negative (r=0.61 P=0.000); The expression of AQP5 and AQP9 mRNA in ovarian cancer tissues were also positively correlated (r=0.594 P=0.000)Conclusion1. The expression of AQP5 and AQP9 mRNA and protein increased with the increased tumor malignancy, and it is colsely correlated with ascites formation, clinical stages, histopathological grade and lymphnode metastasis. These results suggest that mRNA and protein of AQP5 and AQP9 may be correlated with the occurrence and development of epithelial ovarian carcinoma.2. The expression of AQP5 and AQP9 protein and mRNA in ovarian cancer tissues was positively correlated. This result suggests that both of them have a synergistic effect in development of epithelial ovarian carcinoma.