Aquaporin Expression In Endometriosis And Role On Endometrial Cell Migration

BackgroundEndometriosis is a common disease of women in reproductive period, it shows up ward trend in recent years; this is a benign disease in pathological morphology of performance. However, it has similar characteristics with malignant tumors:planting, invasive and distant metastasis, today it has been one of the major in gynecological disease and a serious threat to women's health. The source of ectopic endometrium has not yet been clarified, at present the main theories are:cultivation of endometrial theory, lymphatic and venous spread theory, body cavity metaplasia theory, induction theory, genetics, immune regulation theory and other factors (including environmental factors, angiogenesis, apoptosis reduction, etc.). Cultivation of endometrial theory was accepted by most scholars, but how the eutopic endometrium grows into the uterine cavity area outside the present is not clear.Endometriosis is a benign disease with invasion capability, invasion are often accompanied by Trilogy:migration, adhesion and angiogenesis. In endometriosis, there are much studies about the eutopic endometrium and ectopic endometrium on the adhesion and angiogenesis. There is no endometriosis migration research. The occurrence process of endometriosis is a continuous progress, how to prevent recurrence of the disease has become an international problem, which makes it particularly critical blocking the disease occurs from the beginning of the planting ectopic endometrium. Thus, reveal the molecular mechanism of the growing process of endometrial cells in movement and control the occurrence of endometriosis is of great significance.As we all know, cell migration is an important physiological process of the body, which is regulated by ion channels, involved in actin dynamics and morphology of the rapid changes in cell and adhesion sites to re-distribution. Cells exist in a complete set of strict mechanisms to regulate cell migration. Cell migration can be decomposed into three steps:Cells in the forefront of extending pseudopodia, actin network contraction, and guide the direction of cell migration; actin dispolymerization with pseudopodia movement, the formation of new actin pseudopod with the election of a new substrate-binding adhesion; tail separation of the cell body adhesion and cell to move forward; tail actin contraction, cell body to move forward. Cell migration occurs because cells in vivo osmotic pressure changes. Changes in osmotic pressure caused by cells in vivo require the participation of a large number of water molecules. At present there is less research on the cell migration and the occurrence of endometriosisFor a long time, it is widely believed that water molecules inside and outside cells in simple ways, through the proliferation of lipid membrane bilayer. In 1988, Agre, etc. occasionally found a new 28ku transmembrane protein in the red blood cell membrane when identified the human Rh blood group antigen.1991-1992, Agre, etc. have completed the cloning and functional identification of proteins and confirmed that the protein has a clear water transport function. Subsequently, it was found a similar specificity in many animals, plants and microorganisms in the transport of water channels, known collectively as the water channel protein (Aquaporin, AQP). Studies have shown that AQP family has a wide range of functions:to promote the rapid passive transport of water molecules; regulate cell osmotic pressure inside and outside of the balance; promote cell migration; regulation of fat metabolism; neural signal transduction. In the female reproductive organs, including the uterus, ovaries, fallopian tubes parts follicle ovulation, menstruation, as well as the formation of the malignant behavior of cancer or the occurrence and development with benign gynecological diseases are related to fluid flow, and intrauterine or follicular cavity volume changes. AQP is a selective water channel water molecules through the pore, broad participation in reproductive organs of the physiological and pathological mechanisms occurring. AQP is a group of selectively transport water molecules across cell membrane of the small and complete glycoprotein, molecular weight of 25-34kD, some AQP subunit can also transport glycerol. AQP's first grade structure across the membrane six times a single peptide chain and its amino and carboxyl terminal are located in the cell, including three cell outer ring and two cell inner ring, the highest homology is located in ring asparagine-feeding acid-alanine (Asn-Pro-Ala, NPA) unit, NPA is a characteristic shared by members of the AQP family structures. AQP's tertiary structure was an hourglass pattern, allowing water molecules (H2O) free passage, but does not allow the water quality sub (H3O) through. AQPs are widely distributed in body tissue, particularly with the absorption of fluid secretion on the epithelial cells and endothelial cells in the content of their subsequent participation in water secretion, absorption and cellular water balance inside and outside. It has now been found in mammals,13 types of aquaporins (AQP0～AQP12), according to the infiltration of specific AQP, they will be divided into two categories, general and specific amino acid sequence match. Class 1 are permeable only to water, including AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, AQP11, AQP12; the first two categories, including AQP3, AQP7, AQP8, AQP9, and AQP10, except transfer to water, but also for other small solutes are permeable, especially glycerol. Eutopic endometrium in women's cells have been found to have AQP expression, our hospital's pre-study found that AQP1,2,8 expressed in the eutopic endometrium, AQP5 expressed in epithelial ovarian cancer cells; but the study of AQP in the ectopic endometrial cells has yet to see the relevant reports.In our hospital, other's early studies have shown that AQP1,2,5,8 expressed in the normal endometrium of healthy women. There is no aquaporin study in endometriosis. This study first detected in patients with endometriosis in eutopic endometrium and ectopic endometrial, explore AQP1,2,5,8 expression in endometriosis, observe AQP1,2,5,8 localization of endometrium; The results showed that, AQP1 located in microvascular in eutopic and ectopic endometrial; AQP2,5,8 located in endometrial glandular cells in eutopic and ectopic endometrium. AQP1,2,4,5,8 selective transport of water molecules of water channel proteins, This hospital has studied AQP1 in normal endometrial capillaries, endometrial hyperplasia and endometrial carcinoma. It has been reported in the literature that AQP2,8 mainly expressed in the secretory cells, at present there is no AQP5 study in secretory cells. This study found that AQP2,5,8 expressed in endometrial glandular epithelial cells, one type of the secretory epithelial cells. To explore AQPs function in the occurrence of endometriosis, design AQP5 as the entry point, apply RNA interference gene silencing AQP5 gene, observe expression of AQP5 in endometrial glandular cells and cell migration changes after RNAi AQP5. Seeks to clarify relationship between cell migration and endometriosis and searching for a key to block the migration of endometrial molecules, in order to provide experimental basis for exploring the prevention and treatment of endometriosis. PartⅠAQP1,2,5,8 Proteins Expression in Eutopic and Ectopic Endometrial TissuesObjectiveTo detect AQP1,2,5,8 expression level in eutopic and ectopic endometrium in patients with endometriosis, preliminary study the relationship between water channel protein(Aquaporin AQP) and the occurrence of endometriosis.Materials and methodsThe specimens (n=70) were obtained from the Pathological Department of Women's Hospital, School of Medicine, Zhejiang University from October 2004 to June 2007, and comprised 70 eutopic endometria,70 ectopic endometrial form endometriomas. Through the immunohistochemical detection of AQP1,2,5,8 protein expression.Results1. AQP1 was mainly abundantly located in microvascular in the eutopic and ectopic endometrial cells. Optical dentisy analysied there was statistical difference of AQP1 expressions between ectopic and eutopic groups(P<0.01). In samples from ectopic group, the AQP1 staining was different in proliferative and secretory phases(P<0.05), but not in eutopic endometria(P>0.05). The AQP1 expression was significantly higher in ectopic endometria than eutopic endometria in proliferativ phase(P<0.05) but not in secretory phase(P>0.05). In eutopic endometrium group, aquaporins 2,5, and 8 were mainly located in luminal and glandular epithelia. The frequency of positive immunostaining for aquaporins 2,5, and 8 decreased in ectopic compared with eutopic endometrial(p<0.001,p=0.004,p<0.001). Aquaporin 2,5, and 8 were found at a low frequency in the endometria in early proliferative phases but at a higher frequency in late proliferative and secretory phases. There were no significant differences in the menstrual cycle of the proliferative phase and secretory phase in the two groups.ConclusionsOver expression of aquaporin 1,2,5,8 in endometrium involved in occurence of endometriosis. PartⅡRole of RNAi AQP5 on Cell Migration in Endometrial Glandular cellObjectiveTo explore AQP5 gene impact of migration in endometrial glandular cells, clarify migration-related water channel protein gene on the endometrium grown in vitro.Materials and methodsThrough RNA interference (RNAi) technology, silent endometrial cancer glandular cell line in the AQP5 gene, Realtime-PCR test mRNA level after RNAi AQP5. Western blot detection AQP5 protein expression changes of glandular cell line after interfence; endometrial glandular cells migration ability changes after siRNA AQP5 through cell perforation experiment examines(Transwell).Results1. After RNAi AQP5 gene, in interference group, AQP5 mRNA and protein expression levels of endometrial glandular cells reduced appearently, there were statistically significant (P=0.03 and 0.007 respectively), while the negative control group and blank control group, the mRNA and protein expression were no statistical difference in the level of change.2. After AQP5 RNA interference, in interference group endometrial glandular cells migration number decreased significantly compared with the negative control group and blank control group (P<0.05) Conclusions1. RNAi is able to silence AQP5 gene in endometrial glandular cells.2. Down-regulated AQP5 expressions may inhibit the migration of endometrial glandular cells.