Endometriosis is a common condition that results from the presence of endometrial glands and stroma outside the uterus. The current prevalence of endometriosis is estimated to be up to 10% 1, and it is found in 40–60% of women with pelvic pain 2 and in 33% of women with infertility 3. Endometriosis is a disease with too many theories; the metaplasia theory suggests that under diverse influences, celomic tissue could be transformed into endometrium 4, and according to this theory, ectopic endometrium develops in situ from local tissues, including germinal epithelium of the ovary and remnants of the Müllerian and Wolffian ducts. Another theory proposes that the physiological phenomenon of endometrial reflux in the fallopian tubes during menstruation may, under certain conditions, overcome local defense mechanisms, implant, and proliferate 5. However, the occurrence of endometriosis in sites very remote from pelvic organs directed the research toward other theories such as genetic background 6, embryonic rest theory 7, and stem cell dysfunction 8. The stem cells are primitive cells that have the capacity to self-renew and differentiate into one or more mature cell types 9. A growing body of evidence has implicated stem cells as possible endometrial progenitors. In one study, bone marrow-derived stem cells have been identified in the endometrium of women who were bone marrow transplant recipients; these cells appear histologically indistinguishable from endogenous endometrial cells and express markers of glandular and stromal differentiation 10. We proposed in a case–control study the presence of an endometriosis-inducing factor(s) in the blood of women with endometriosis, where mesenchymal stem cells transformed into endometrial-like cells after coculture with the serum of women with different degrees of endometriosis. This differentiation occurred after variable periods of time on the basis of disease severity. Moreover, the differentiated cells expressed tissue markers of endometrial tissues 11. MiRNAs are a class of endogenous small RNAs that negatively regulate gene expression at the post-transcriptional level. Accumulating experimental evidence shows that miRNAs regulate cellular apoptosis, proliferation, differentiation, and migration, all of which are features associated with endometriosis. Dysregulation of miRNA expression leads to various human diseases and MiRNA maturation is regulated at multiple steps by different mechanisms, including miRNA editing, hairpin loop binding, self-regulation, and cross-talk with other signaling pathways 12. We aimed to study whether the regulatory networks in the blood of endometriotic women that allow the transformation of mesenchymal stem cells into endometrial-like cells may involve an miRNA component.
Patients and methods
This was a case–control pilot study that recruited 12 women. Women provided written informed consent and the study was approved by the bioethical committee of the National Research Centre. All women had primary infertility and had undergone diagnostic laparoscopy during their workup investigation for their infertility problem. Three women were free from endometriosis and were in included in the control group, whereas five women were diagnosed with severe endometriosis and another four had mild endometriosis (these women were included in the study group). The severity of endometriosis was graded clinically during the laparoscopy procedure and was based on the revised AFS scoring system 13. Women were between 20 and 35 years of age, and none of them had a history of chronic diseases, for example, diabetes mellitus, auto immune diseases, atherosclerosis, etc. The women had not received any type of hormonal therapy 6 months before the diagnostic laparoscopy procedure.
Blood samples were collected from all the groups in PAXgene Blood RNA Tubes (BD Vacutainer, Franklin Lakes, New Jersey, USA), which contained a reagent that lyses blood cells and immediately stabilizes intracellular RNA to preserve the gene expression profile. Total RNA was isolated from these stabilized blood samples using the PAX genemiRNA isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The quality and yield of the miRNA were analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Foster City, California, USA). Reverse transcription of miRNA samples into first-strand cDNA was carried out using the RT-miRNA First Strand Kit (Qiagen). An SYBR Green-based real-time PCR array (miFindermiRNA PCR Array; Qiagen) was used to assess the expression of well-characterized 88 miRNAs in miRBase (http://www.miRBase.org) using 7500 fast real-time PCR. A set of controls present on this array enables a relative quantification (ΔΔCT method), assessment of reverse transcription, and PCR performance using Sequence Detection System V2.05 software (Applied Bio-systems, Foster City, California, USA). All samples were run in triplicate in three separate experiments.
Statistical analysis was carried out using the SPSS 11.0 software (IBM, New York, USA). Student’s t-test or the one-way analysis of variance test was used to compare continuous variables, with the Tukey–Kramer test for multiple comparisons. Statistically significant differences among the groups were analyzed using the Kruskal–Wallis test with Dunn’s test. Data from the three independent experiments were analyzed and P less than 0.05 was considered statistically significant. The fold regulation of miRNAs represents the fold-change results in a biologically meaningful way. Fold-change values are equal to the fold-change and more than one change indicates a positive regulation or an upregulation. Fold-change values less than one indicate a negative regulation or downregulation, and the fold regulation is the negative inverse of the fold change.
The expression levels of 88 miRNAs according to the miFindermiRNA PCR array platform showed that miRNA-130a was significantly increased in the study group compared with the control group. The fold regulation of miRNA-130a in the study group was 17.03 in the sever endometriosis group and 4.25 in the mild endometriosis group compared with the control group (Fig. 1). We found that 29 miRNAs were significantly downregulated in the mild endometriosis group (Table 1; Fig. 2), whereas only two were downregulated in the severe endometriosis group (Table 2; Fig. 3) compared with the control group. All other miRNAs were within the normal expression level in the study group compared with the control group.
Let-7e expression was significantly reduced in both the severe endometriosis group (−32.7051) and the mild endometriosis group (−4.255), whereas let-7f was significantly reduced only in the severe endometriosis group. The average threshold cycles of let-7a, let-7d, miR-150, miR-322, and miR-222 were greater than the defined cut-off value (default 35) in all groups, making this fold-change erroneous and uninterpretable (Figs 2a and 3a). The patterns of expression of different genes across different patients were grouped into distinct clusters (Fig. 4), in which genes in the same cluster were assumed to be potentially functionally related or to be influenced by a common upstream factor (miR-130a). Such a cluster structure is used to aid the elucidation of regulatory networks of miR-130a.
MicroRNAs are a new class of endogenous small noncoding RNAs that can pair with sites in 3′ untranslated regions in the mRNAs of protein-coding genes to downregulate their expression 14, and are associated with a variety of human diseases, including cancer, heart failure, vascular disease, diabetes, etc. In the last several years, miRNA has been investigated extensively, and it is now recognized that miRNA plays a key role in regulating fundamental cellular processes 12. Blood miRNAs have been considered as promising novel biomarkers 15,16. The results of this study clearly uphold the basic hypothesis that miRNAs are present in blood and represent useful clinical biomarkers. We have shown that the blood levels of miR-130a can serve as a marker for endometriosis. This result is easily understood as miR-130a is shown to be significantly increased to around 4 fold in the mild endometriosis cases and 17-fold in the severe cases. The correlation of miR-130a with the different grades of endometriosis may at least partly a role in the progression of this condition. Endometriosis is characterized by angiogenesis, and new tissue formation and proliferation. MiR-130a is located on chromosome 11 and the expression of this miRNA was found to be rapidly upregulated under high serum culture conditions 17.
Interestingly, miR-130a has a proangiogenic function. It downregulates (at the post-transcriptional level) the antiangiogenic homeobox genes, that is, GAX and HoxA5 and functionally antagonizes its inhibitory effects on endothelial cell proliferation, migration, and tube formation 17, thereby facilitating the angiogenic process.
MiR-15b and miR-16 comprise a downregulated miRNA cluster in endometriosis 18 that was repressed under hypoxic conditions in a human epithelioid carcinoma cell line 19. Our results showed that both of these miRNAs were repressed in the blood of mild cases and were associated with an increased expression of mi-R130a. Repression of miR-15b and miR-16 activities resulted in increased transcription of two of its confirmed targets: the proangiogenic factors vascular endothelial growth factor A (VEGF-A) 19 and cyclooxygenase-2 20. In endometriosis, increased expression of VEGF-A and cyclooxygenase-2 may thus be a response to hypoxic injury, and may be strongly implicated in the growth and survival of endometriotic deposits 21. Cyclooxygenase-2 promotes prostaglandin production, neoangiogenesis, and estradiol-mediated cellular proliferation in endometriotic tissues. Apoptotic resistance is mediated by intracellular proteins such as B-cell CLL/lymphoma 2 (BCL2), leading to enhanced survival of stressed endometrial cells in endometriosis 22. Both miR-15b and miR-16 target BCL2 23, and the reduced expression of these miRNAs in the blood may possibly contribute toward increased activities of this antiapoptotic protein in endometriosis. Therefore, it is possible that increased expression of miR-130a and reduced expression of miR-15b and miR-16 may work synergistically to induce the growth and survival of endometriotic deposits and promote an inflammatory environment by upregulating VEGF-A cyclooxygenase-2 levels.
The proangiogenic properties of miR-130a may affect the transition of an undifferentiated stem cell to a differentiated phenotype, which plays a critical role in the pathogenesis of endometriosis. The origins of the mesenchymal cells participating in tissue repair and pathological processes are poorly understood. Oosterlynck et al. 24 found that the transforming growth factor β is upregulated in the peritoneal fluid of women with endometriosis, and several studies have identified this growth factor as a central component in molecular signaling networks and mediate a switching from an initially ischemic and inflammatory environment that causes tissue damage and necrosis to a ‘healing’ milieu that promotes cellular proliferation and tissue remodeling during endometriotic lesion development 21,25–27. Translation of this factor is repressed by miR-21 28, which is downregulated in the blood of mild cases in association with overexpression of miR-130a, leading to an enhancement in its activity in the transformation of peritoneal stem cell into endometriotic-like cells. Also, miR-21 was found to be upregulated in eutopic endometrium throughout the menstrual cycle in severe versus mild endometriosis 29. Most miRNAs that were downregulated in the blood of mild cases represent a cluster of miRNAs responsible for the regulation of several physiological and pathological conditions in endometriosis 30. These miRNAs are upregulated in the severe group in association with an increase in the expression of miR-130a, which may reflect their role in inducing endometriosis. The Let-7 family is a regulator of cell cycle, proliferation, and apoptosis 31. Let-7e was significantly downregulated in both the severe and the mild groups compared with the control group. This downregulation was exponentially proportionate to the severity of endometriosis, which may be a clue of its regulatory role in the severity of endometriosis.
Thus, miR-130a appears to be a potent regulator of gene expression in endometriosis (Fig. 5), raising the prospect of using blood miRNAs as biomarkers and therapeutic tools in endometriosis. In the future, it would be conceivably beneficial to examine the function of miR-130a in the initiation and progression of endometriosis in a large number of cases.
miR-130a is a potent regulator of gene expression in endometriosis, leading to enhanced transformation of mesenchymal stem cell into endometriotic-like cells. Furthermore, the blood levels of miR-130a may serve as indicators for endometriosis. It also explains the pathophysiology of endometriosis. This may open up an exciting new avenue for targeted anti-angiogenesis therapy for such a devastating disease.
The authors acknowledge the major contributions of Tamer Taha, Hitham Badran, Morad Selim, Mohamed Abd-Elaty, Mazen Abd Elrasheid, and Ehab Nabeil during the planning phase of this study and for kindly providing samples. The authors also would like to thank Mohamed Reda and Zaynab Algammal or their assistance in sample preparation.
Conflicts of interest
There are no conflicts of interest.
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