Translational Genomic Approaches in Endometriosis

Sub-project 1 : Translational genomic approaches in endometriosis Endometriosis occurs when shed endometrium from the female reproductive tract grows outside the uterus, which might cause infertility. The pathophysiology in endometriosis—abnormal tissue growth, invasion, and adhesion formation—are similar to those seen in tumorous tissues. The aetiology of endometriosis is not well understood. Several factors such as immunological, genetic and environmental factors may involve the susceptibility to develop endometriosis. Each year, about one fourth of above 800 women who accepted IVF have been diagnosed of having endometriosis in the Center of Reproductive Medicine, Taipei Medical University Hospital. The causes and mechanisms of endometriosis remain unclear so far, moreover, the diagnosis and treatment of endometriosis in clinical has lots of problems including: the diagnosis still needs invasiveness laparoscopy and the treatment which based on surgical removal of endometrial tissue and hormone medication, but both methods all have defects such as relapse of disease or drug side effect. In the previous studies that based on cDNA microarray, we have discovered several genes including: integrins/ cell adhesion molecules, metalloproteinases, cathepsins, estrogen receptors, VEGFs, aromatase, EGFR, MAPK and cytokines (IL-6, SDF-1, CXCR4 ). Therefore, in this project we will combine function genomics and clinical research to study the gene expression pattern, mechanism and signal transduction of endometriosis, and verify genes that would alter the cell growth and invasiveness of endometrial cells. Furthermore, next-generation sequencer (SOLiD 3plus, ABI) would be used to analyze the genomic pattern of patients who have family history of endometriosis. There are four specific aims for this sub-project: 1. To analysis the gene expression pattern and signal pathways related to endometrial cell growth and invasiveness. 2. To find the possible gene mutation related to endometriosis by analyzing the genome of patience that has family history of endometriosis using high-throughput next generation sequencer. 3. By integrate our findings from above and results from other subprojects including endometriosis-related microRNA and protenomics analysis, we hope to find out possible pathological mechanisms and bio-markers for diagnosis and prognosis. 4. By studying the genes and signal transduction which would interfere the cell growth and invasiveness of endometrial cells. We will use the possible targets to select some potential leading compounds in our established herbal drug bank. We will also precede animal model for potential targets. This project will not only add the information for our understating on the pathogenesis of endometriosis, but also provide the new strategies for diagnosis and treatment in endometriosis. Sub-project 2 : The Role of microRNAs and Regulating Mechanisms in Endometriosis Endometriosis is a common gynecologic disorder. Although it has been proposed that endometriosis is a hormonal disease, an autoimmune disease, a genetic disease, our understanding of its etiopathogenesis is still inadequate. In the very recent years, evidence has emerged that endometriosis may be an epigenetic diseases in which microRNA may involved. microRNAs are short single-stranded RNAs that regulate gene expression at the post-transcriptional level. The microRNAs acting as agents of the RNA interference pathway have been implicated in the regulation of a variety of cellular processes, and even the pathogenesis process of human diseases including endometriosis. The differential expression of microRNAs in endometriosis, and the putative molecular pathways constituted by their targets, suggests that microRNAs may play an important role in endometriotic lesion development. The miRNA expression profile is quite different in paired ectopic and eutopic endometrial samples from women in endometriosis, thus, some miRNA-regulated molecular pathways that are likely to contribute to the pathogenesis of endometriosis has been proposed. Unfortunately, most of the evidences about endometriosis and microRNA are from micro array or bioinformatics analysis. We all face the same problem which is too many indeterminate targets will be proposed, but only few of them have been proved after followed by biological experiment. The critical roles played by the microRNAs and its target genes in endometriosis can’t be conformed simply from array data. For these reasons, to set up an effective screening method for microRNA/target scan is important. The objective of this sub-project is to find out accurate microRNAs and its targets which involved in endometriosis by effective bio-informatics analyzing method. By integrate different data bases and our specific finding; we will establish a screening system for microRNA and its targets. This project will not only establish a bioinformatics analysis platform, but also provide the information of microRNAs and its target genes on the pathogenesis of endometriosis. More specifically we intent in the project: (1) To establish an efficient, accurate analyzing platform for microRNA/target gene study. (2) Identification of endometriosis related miRNAs and target genes. (3) To investigate the host genes and the down-stream gene target of miRNAs (4) Evaluation of MiRNA expression profile in clinical metastasis project 3 : Role of Inflammatory Cytokines and Endometrium Stem Cells in Endometriosis and Clinical Implication Endometriosis is the growth of endometrial tissues eutopically and/or ectopically. This disease affects nearly 10 % of reproductive aged women with symptoms of persistent pelvic pain, abnormal uterine bleeding, dyspareunia, dysmenorrheal, and infertility. Stem cells in endometrial basalis layer have been hypothesized to mediate endometrium regeneration as well as the initiation of endometriosis. However, mechanism involved in endometriosis transformation processes still remains largely unknown. In clinic observation, chronic inflammation is tightly associated with endometriosis. In this aspect, we then hypothesized that inflammatory cytokines may result in transformation of endometrium stem cells and initiate the processes of endometriosis. To test this hypothesis, we have established a serum-free culture system to generate mouse epithelial-like endometrium stem cells (mEESCs). These mEESCs showed in alkaline phosphatase activity, expressed stem cell markers (such as Oct-4, SSEA-1, CD49f, CD63, and CD34), and IGF-1/IGF-1R (stemness regulatory signaling). Interestingly, by real-time PCR analysis, the mEESCs showed in increasing of stemness (such as Oct-4, Sox2, c-Myc, Klf-4, and IGF-1) under treatment of inflammatory culture condition. To further correlate the pluripotent gene expression (Oct-4 and Nanog) with endometriosis progression in human, tissues of hyperplasia (N= 37), endometriosis (N= 43 ), and adenomyosis (N= 8 ) were collected and the clinical CA125 score was examined. In our results, endometriosis showed in higher expression of Oct-4 and Nanog as well as tight correlation with CA125. In this proposal, we hypothesized that inflammatory cytokines might play an important role in initiation and development of endometriosis by transformation of endometrium epithelial stem cells. To test our hypothesis in humans, four specific aims will be addressed: 1. To generate human endometrium stem cells using a serum-free culture system 2. To examine the effects of inflammatory cytokines on transformation of human endometrium stem cells into endometriosis 3. To examine the epigenetic regulation, e.g. miRNAs or methylation, of the mEESCs under inflammation treatment 4. To built up a human endometriosis animal model for clinical drug selection Sub-project 4 : Molecular Identification of Cell Adhesion Molecules(CAM) Ligand-modulated Endometriotic Cell Growth Endometriosis is one of the most frequent diseases in gynecology. It is a benign disease in which endometrium-like tissue is found outside the uterus. The pathogenesis of endometriosis is unknown, but invasive and metastatic mechanisms have been implicated in the development of the disease. Analogous to tumor metastasis, it is likely that cell adhesion molecules are central for the invasion and metastasis of endometriotic cells. In affected individuals, shed endometrium is thought to attach and spread within the pelvis through specific cell adhesion receptors. Cell adhesion molecules (CAM) are associated with the development of metastatic behavior in endometriosis. Cell adhesion and detachment play an important part in tumor invasion and metastasis, and a variety of cell adhesion molecules have been found on endometriotic cells. Recently, enhanced expression of adhesion molecules, such as αvβ3 and CD44, was found in the endometriosis. Osteopontin（OPN）is a component of the extracellular matrix (ECM) that interacts with cell surface receptors, including integrins, to mediate cell adhesion, migration, differentiation, survival, and immune function. It was reported to be a key molecule for tumor progression and metastasis. OPN is a general ligand of αvβ3 and CD44. Enhanced OPN expression was found in the endometriotic tissue and in rat endometriotic model. From the preliminary findings, the differential CAM and ECM-related gene expressions were detected in the ectopic endometrium. A significant increase of OPN was found in the ectopic endometrium. Cell morphology, proliferation, migration and invasion were studied. Under immunofluorescent microscopy, we found that OPN transfected cells promoted lamepodia and stress fiber formation. Additionally, reduced E-cadherin and increased N-cadherin expression were found in the ectopic endometrium and associated to epithelial to meschymal transition. We also found that steroid could augment OPN expression and secretion, and induced cell migration. In this study, we propose that CAM-ECM molecules promote the endometriotic cell migration, invasion, and ectopic growth and contribute to endometriotic epithelial to meschymal transition process. In the first year, we plan to survey the specific CAM-ECM molecules and validate the molecular regulation of OPN or specific CAM-ECM molecules-induced endometriotic cell migration and invasion. In the second year, we plan to address whether OPN or specific CAM-ECM molecules induce endometriotic epithelium-mesenchymal transition. Finally, we will study whether the candidate microRNA and inflammatory cytokine could modulate OPN gene expression and identify the molecular pathway of steroid-induced OPN expression and cell migration. The results will unravel the causal role of CAM-ECM molecules in the regulation of endometriotic cell growth, which may promote the development of CAM-ECM-directed therapies for patients with endometriosis. Sub-project 5 : Epidemiological study on environmental and genetic risk factors of Endometriosis in Taiwan Objectives Objectives of this study are to establish a biorepository under single and efficiency management, to investigate differences in characteristics of patients with endometriosis in Taiwan and other countries, to explore the genetic and environmental factors of this disease, and to clarify the probably epidemiological model in endometriosis. Finally, to suggest a world common treatment and prevented guideline. Background and importance: The fertility problems were critical public issues in Taiwan. The patients searched for clinical reproduction technology were increasing. Endometriosis, defined by the presence of viable endometrial tissue outside the uterine cavity, is a common condition in women of reproductive age, and may affect fertility. Our understanding of the association between the early stage of endometriosis and infertility remains incomplete. Endometriosis is a complex trait, in which genetic and environmental factors act together to produce the phenotype. It has been reported that dioxin and nonylphenol have adverse reproductive effects and may attribute to human infertility. And, research into candidate genes has largely been based on biological and clinical hypotheses. Thus, it is chosen for this study. This two-year project aims to find the characteristics of these genetic and environmental factors for endometriosis in Taiwan. Results of this study could provide the researcher with new etiological avenues to explore. Designs: The combined investigation of environmental and genetic risk factors in complex traits will refocus attention on the case-control study of endometriosis. Use of newly diagnosed over prevalent cases is preferable, as the latter may alter risk estimates and complicate the interpretation of findings. Potential confounding will be addressed both in studies of environmental and genetic factors. The first year, metal elements and other contaminants were detected. The second year, the polymorphism of candidate genes will be identified The Third year, blood dioxin levels and nonylphenol levels will be monitored in all subjects and integrated analysis with environmental and other factors will be made. Sub-project 6 : Building a Biorepository and Information Infrastructure for Reproductive Medicine and Screening of Biomarkers for Endometriosis We have previously used proteomic approach to identify several serum proteins that may associate with the development of endometriosis. Based on the previous study, in 2008, the first US patent in a title of Diagnosis Method of Endometriosis by Detecting Biochemical Markers and Usage of These Biochemical Markers, W-C Yang, C-R Tzeng, and H-J Wang , US7399598, 2008/2.19~2025/10/14 was approved. Subsequently, the ROC patent was also approved (I 305837, 2009/2/1 – 2025/11/24. Republic of China). In addition, in 2009, the 3 inventors, W-C Yang, C-R Tzeng, and H-J Wang received Golden Award from 2009 National Invention and Creation Award from economic department. The PI of the grant, Dr. W-C Yang also received 2009 Crazy Idea-Biotechnology & Medical Application Competition Award from CPC. However, it may require more efficient data management system to collect and manage the data from genomic and proteomic studies. In this project, we will collaborate with the Center for Translational Medicine in Taipei Medical University to establish a tissue bank for biorepository of reproductive medicine and set up a data management system to integrate the clinical data including electric medical record and medical and molecular image and the genomic and proteomic data together. It will facilitate the researches for endometriosis as well as for the researches of reproductive medicine in TMU. The goal of this research project is to develop the first diagnostic kit for endometriosis and find new strategy for the treatment of endometriosis.