A summary of the data

4.5.2.1 Upregulation of Ebv-miR-BART2-5p

In my study I have shown that the Ebv-miR-BART2-5 is uniquely upregulated in pelvic endometriosis in the proliferative or follicular phase of the cycle this 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.

FIGURE 4-24 EBV negative stain

– control slide EBV positive stain

– control slide EBV negative stain in normal

Tissue microarrays on which in situ hybridisation for EBV was performed

This finding is in keeping with previous studies discounting the presence of active viruses within the tissues but might indicate a viral effect on lymphocytes. This does not discount the importance of the influence of the effects EBV has on the development of disease as implicated by my analysis. The presence of these miRNAs in my case tissue samples further helps to substantiate the role of ebv-BART2-5p as a key player in endometriosis. Potential mechanisms of function may be developed through viral derived endosomes recently shown to play important roles in carcinogenesis, selectively placing EBV miRNAs into the endosome to transfer them to epithelial cells and alter growth characteristics.

Metazoan organisms encode miRNAs which regulate gene expression by binding to the 3’UTR terminal of mRNAs. Herpes viruses are known to induce persistent lifelong infections in their latent stage by evading the host’s immune system⁴⁹⁸. miRNAs have been identified within DNA viruses of the herpes family^499-501. Identified tumorigenic viruses associated with latent infections, evasion of the host immune responses⁵⁰² and epithelial, endothelial or lymphoid malignancies include Cytomegalovirus (CMV)⁵⁰³, Kaposi’s sarcoma associated herpesvirus (KHSV)⁵⁰⁴ and Epstein-Barr Virus (EBV)⁵⁰⁵. Problems with predicting viral miRNA targets with available algorithms exist⁵⁰⁶ as both cellular and viral targets can interact and should be analysed⁵⁰⁷. As viral targets are poorly conserved this is not always possible and their experimental validation has been limited⁴⁹⁹.

FIGURE 4-25

Figure illustrating the various mechanisms involved in development of endometriosis

There are a series of identified mechanisms and pathways which I theorise result in the development of endometriosis (Figure 4-25, Figure 4-10). My findings indicate an EBV viral effect on lymphocytes which potentially plays an important role in development of disease. During latent infection, EBV has been reported to induce certain host miRNAs which play a role in viral oncogenesis such as miR-155 and miR-21⁵⁰⁸ (Figure 4-10). miR-21 controls the oncogene Tropomyosin alpha-1 chain (TPM1). TPM1 has been identified in my study as a potential antibody marker from the serum of patients with disease (Section 6.7.3.8). miR-155 has also been identified in this study as being upregulated in Ovarian endometriosis (P=0.00001720) (Table 9-4) specifically in the follicular/proliferative phase of the cycle (P=0.0004878) (Table 9-5). It is also seen to be upregulated in the normal endometrium of cases (average value of expression from all case samples 6.308624) compared to the normal endometrium from controls (average value of expression from all control samples 3.818766) (see Excel analysis-Thesis excel data (my modified lists of miRNA decideTests_resultsV2).xls).. MiR-21 is also seen to be dysregulated in my study and is expressed in Ovarian endometriosis (P=0.00140219) (Table 9-14). miR-21 is also seen to be dysregulated in this study. It is expressed in Ovarian endometriosis (P=0.00140219) (Table 9-14). miR-21 was also seen in the normal peritoneum of cases (Table 9-12) (average value of expression from all case samples 12.30429) and in the normal peritoneum of controls (average value of expression from all control samples 12.29785). The data from the normal peritoneum does not necessarily refute the presence of this miRNA in peritoneum of women developing disease. It is recognised that small sample numbers might provide a limitation to their true expressed levels. In addition, the location of a peritoneal biopsy well away from the growth of endometriosis might lessen the incidence of deregulated miRNA in these sampled tissues.

EBV was the first virus to be associated with a human tumour⁵⁰⁹ (Figure 4-27) and is seen in lymphomas and epithelial malignancies such as undifferentiated nasopharyngeal carcinoma. In vitro studies show the pivotal role of EBV in oncogenesis. It is capable of transforming over 50% of resting B-cells into continuously proliferating cells with latent virus within a few days⁵¹⁰. Infection of epithelial cells with

EBV is not completely understood. There is the possibility of virus as well as exosomes transferring genetic material. This results in epithelium demonstrating the default program of infection^511,512. Unlike B-cells, epithelial cells are unable to become memory cells and the LMP proteins cannot “turn off”. Thus even in immunocompetent individuals infection can persist. The same theory could be true of endometriosis. Publications show that the presence of healthy, functional NK cells is inversely proportional to the presence of EBV virus and its effects⁵¹³. There are a number of predicted human target genes for EBV BART2-5p which have particular roles enabling ectopic tissue to engraft and survive. The BCLAF1 gene linked to apoptosis, the CDH1/E-Cadherin linked to cell adhesion and the MASPIN linked to p53 signalling are all implicated. Over expression of the BCLAF1 induces apoptosis and T cell proliferation. The expression of BART2-5p provides a survival advantage to ectopic endometrium having reduced apoptosis and avoiding immune surveillance by the t-cells. This study demonstrates the reduction of BCLAF-1 on tissue microarray analysis (TMA). Comparing levels between endometriotic tissue and normal endometrium, there is a statistically significant downregulation of BCLAF-1 in endometriosis compared to normal endometrium from cases (P<0.0001) (Figure 4-16, Table 4-5, Table 4-6).

CDH1/E-Cadherin is a master regulator of the G0/G1 stage of the cell cycle and its role is to reduce cell adhesion, division and cycling. Its suppression in ectopic endometrial tissue predicts an increase in cell adhesion (observed in endometriosis) and removes the suppression on the G0/G1 cell cycle stage moving cells into the S phase. This study demonstrates the downregulation of E-Cadherin in endometriosis on tissue microarray (TMA) analysis. There is a statistically significant downregulation of E-Cadherin in endometriosis compared to normal endometrium from controls (P<0.0001) and normal endometrium from cases (P=0.0017) (Figure 4-12, Table 4-1, Table 4-2). The reduction in E-Cadherin enables the tissues to metastasise and infiltrate causing disease progression and creating an invasive pattern often seen in endometriosis. Maspin is expressed in normal endometrial cells and its suppression by BART2-5p leads to an increase in ability for the endometrium to migrate and survive. An activating p53 binding site was identified and showed Maspin to be the downstream effector gene. My study confirms the reduction of Maspin on tissue microarray (TMA) analysis. Comparing levels between endometriotic tissue and normal endometrium, there is a statistically significant downregulation of MASPIN in endometriosis compared to normal endometrium from controls (P<0.0001) and normal endometrium from cases (P=0.2760) (Figure 4-14, Table 4-3, Table 4-4).

4.5.2.2 Downregulation of hsa-miR-564

In my study, hsa-miR564 was the miRNA found to be downregulated exclusively in the endometriosis tissue samples. Overall, most cancers show aberrant miRNA expression and an overall level of downregulation of relevant miRNAs when compared to normal tissues. This miRNA has been found to be downregulated in leukocytes of patients with chronic myeloid leukaemia⁵¹⁴ and in papillary thyroid carcinoma with lymph node metastasis⁵¹⁵. It has been postulated that the downregulation of this miRNA is mediated by the activity of BCR-ABL tyrosine kinase gene and its protein⁵¹⁴ (Figure 4-26). The ABL oncogenes play a role with Cyclin D1 and K-RAS to induce cellular transformation both in vitro and in vivo models⁵¹⁴. The predicted targets of miR-564(Target Scan predictive analysis tool) include E2F transcription factor 3 (E2F3) and Akt2 gene. The E2F family of genes play a role in the cell cycle, DNA replication and the upregulation of E2F3, an oncogene with strong proliferative potential. It is associated

with poor prognosis in hepatocellular carcinomas⁵¹⁶ and in tumour studies it has been proposed as a marker of poor patient prognosis. In studies involving lung cancer patients, the targeting of E2F3 by a tumour suppressive miRNA (mir-449a) induces remission and improved survival of patients⁵¹⁷. E2F is also a target of transforming proteins and gene products of DNA tumour viruses such as adenovirus E1A, papillomavirus E7, simian virus 40 and poliomavirus large T antigens⁵¹⁸. The Akt2 gene codes for the enzyme RAC-beta serine/threonine-protein kinase and has been described as an oncogene in ovarian carcinoma⁵¹⁹. It has been associated with poor prognostic outcomes and increased tumour aggressiveness when upregulated⁵¹⁹. Ak2 has also been found upregulated in EBV virus immortalised B cells⁵¹⁹.

FIGURE 4-26

Hand drawn diagram showing the effects of HSA-miR-564