A study of Gene, Protein and miRNA alterations in women with Endometriosis.
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The aim of this thesis was to improve understanding of the underlying genetic and proteomic alterations potentially contributing to endometriosis. Assessment of the genetic mechanisms and pathways controlling angiogenesis, apoptosis and inflammation allowed identification of potential aberrations contributing to disease. Tissue miRNA expression experiments show that, the ebv-mir-BART2-5p is detected in endometriosis. Endometriosis cells contained higher levels of ebv-mir-BART2-5p compared to eutopic endometrium and this finding was confirmed by quantitative PCR. In situ hybridisation for EBV on tissue microarrays did not confirm the presence of active EBV within the endometriotic epithelial cells (Figure 4-24) but 5 of the 42 endometriotic samples on TMA-A gave a positive reading for EBV presence in some of the lymphocytes. PCR on the peripheral blood monocytes confirms overall higher levels of EBV DNA in the monocytes of people with endometriosis compared to controls (see Table 9-39 in the Appendix). There were no detected EBV levels in the surgically confirmed control patient samples. The presence of ebv-mir-BART2-5p is a permissive event for the development of endometriosis potentially acting as an initiator for engraftment of endometrial cells to the peritoneum causing the development of endometriosis. It also aids in disease development by suppressing T-cell function and encouraging adhesions and angiogenesis in affected tissues. Alterations in cellular genotype also enable the ectopic endometrial cells to evade NK cells and Lymphocytes, promoting proliferation and metastasis. Effects of genes on various stages of the cell cycle checkpoints and pathways have been explored as contributors to disease development. Downstream proteins from EBV upregulation are also confirmed to be affected. Tissue microarray studies demonstrate the upregulation of Cyclin D1 (Figure 4-22) and downregulation of E-Cadherin, Maspin and BCLAF-1) (Figure 4-11, Figure 4-13 and Figure 4-15). Galectin 3 (a prominent anti-apoptotic and angiogenic member of the lectin family) proteins were also upregulated in endometriosis. Galectin-3 had already been shown to be a good therapeutic target in humans and may provide alternatives to current surgical or hormonally repressive therapy. A set of in vitro experiments have been performed and show effective disease repression with Galectin therapeutics (Galectin 3 inhibitor GCS-100), potentially opening a window for the development of novel therapeutics. Serum was analysed for miRNA and antibody protein expression profiles. Pathways linked to identified biomarkers have been explored aiding in the understanding of disease development at a molecular level. Identified miRNAs are seen to interact with a number of important pathways listed below. There could potentially be effects on cellular mitosis and meiosis, cellular structure, intra and intercellular signalling, vesicular transport including exo and endocytosis and cellular apoptosis. All of these changes can result in endometriotic cells that replicate at accelerated rates, adhere to ectopic sites, enable angiogenesis and growth, evade normal apoptotic mechanisms and evade immune responses. Certain identified pathways act as tumour suppressors that could potentially explain the non-malignant properties in the majority of cases of endometriosis. These include the p38 MAPK1 pathway and the p53 tumour suppressor genes2. Other pathways involved are known to be associated with tumorigenesis and have pro-oncogenic properties, these include the RAS, WNT, TRK receptors and MAPKKKK pathways. It is the fine balance between tumour suppressors and oncogenes that probably control the transition between endometriosis and endometrial carcinomas. Identification of protein and miRNA expression profiles predicting the presence of endometriosis allowed us to work towards developing a panel of non-invasive biomarkers (Patent P063434GB) for earlier identification and treatment of disease and aids in unlocking new methods of molecular targeting as treatment.
AuthorsSchembri Deguara, Christine Anna Maria
- Theses