Evaluation of miR‑135a/b expression in endometriosis lesions
- Rafaella Petracco
- Ana Cristina de Oliveira Dias
- Hugh S. Taylor
- Álvaro Petracco
- Mariângela Badalotti
- João da Rosa Michelon
- Daniel Rodrigo Marinowic
- Marta Hentschke
- Pamella Nunes de Azevedo
- Gabriele Zanirati
- Denise Cantarelli Machado
- Published online on: September 2, 2019 https://doi.org/10.3892/br.2019.1237
- Pages: 181-187
Since 1860, when endometriosis was first described, its etiology and pathophysiology have not been fully understood (1). Endometriosis is a progressive estrogen-dependent disease characterized by the presence of endometrial cells (epithelial and stromal) outside the uterus, most frequently occurring in the pelvic organs and the peritoneum. Although its prevalence is not clear, it affects 5-10% of women of reproductive age and up to 50% of infertile women (2-4). Pelvic pain is the most common symptom, associated with dysmenorrhea, dyspareunia, or even chronic pelvic pain, which are frequently associated with infertility.
Delayed diagnosis complicates disease management. For earlier detection and a therapeutic follow-up, the identification of non-invasive biological markers with good specificity for this disease is a promising strategy. In cases of endometriosis, environmental, hormonal, immunological and genetic components are involved (5). In addition, epigenetic factors and adult stem cells also affect disease pathogenesis (6,7). Estrogen receptor gene polymorphisms and various exon-deleted progesterone receptor mRNAs are also involved in endometriosis (8). Previous studies have indicated the presence of aneuploidies in chromosomes 11, 16, and 17 and the loss of heterozygosity in chromosomes 9, 11, and 22 in the eutopic endometrium of patients with endometriosis, suggesting the possibility of a genetic component associated with disease development and progression (9-12). Previous breakthroughs in genetic mapping for endometriosis have been described and this technique is gaining importance due to the potential to show molecular aspects associated with diseases (12,13).
MicroRNAs (miRNAs) are small, single-strand noncoding RNA molecules containing ~22 nucleotides, which are essential for gene regulation and are able to simultaneously control numerous genes (14). miRNAs are associated with different physiological processes, including apoptosis, differentiation and hematopoiesis. Following the identification of the role of miRNAs in cell function, miRNA expression has been associated with a number of diseases, in particular various types of cancer (15). Recent research has indicated that miRNAs and their target mRNAs are differentially expressed in endometriosis and other disorders of the female reproductive system.
Impaired endometrial receptivity, abnormal uterine bleeding, and endometriosis are some of the disorders that may be associated to the alteration of cellular and molecular homeostasis of the endometrium. Indeed, the expression of different miRNAs in the female genital tract, in normal and pathological tissues, suggests an association with the physiopathology of numerous diseases, including endometriosis (16,17).
The difference in miRNA expression between eutopic and ectopic endometrial tissues reflects the differential expression of genes involved in cell adhesion, extracellular matrix remodeling, migration, proliferation, immune system regulation and other events directly associated with the establishment of endometriosis implants (18,19).
miRNA135 (miR-135) has two subtypes: 135A and 135B. Previously, we demonstrated an inverse correlation between miR-135 and homeobox protein Hox-A10 (HOXA10) in the mid-secretory endometrium during the implantation window in patients with endometriosis, which may explain implantation failure and the higher incidence of infertility in these patients. HOXA10 was abnormally regulated in the endometrium of women with endometriosis by both miR-135a and miR-135b, suggesting a mechanism for the differential expression of HOXA10 in disease pathogenesis (20). In addition, miR-135 is upregulated in distinct phases of the menstrual cycles and has cell specificity; alterations observed in the early proliferative phase were demonstrated to be associated with a defective implantation window (20,21). HOXA10 expression is downregulated in endometriosis lesions compared with in the eutopic endometrium. Besides, the association between HOXA10 and miR-135a and miR-135b has been detected in the eutopic endometrium. However, the expression of this specific miRNA has never been evaluated in the ectopic endometrial tissue.
The present study aimed to assess miR-135a and miR-135b expression levels in the ectopic and eutopic endometrium and throughout the menstrual cycle. We hypothesized that miR-135a and miR-135b would be upregulated in the proliferative phase in ectopic and eutopic tissues.
Materials and methods
Patients and tissue collection
A total of 31 patients who underwent surgery for endometriosis diagnosis or treatment were recruited between March 2013 and May 2014. The study was approved by Pontifical Catholic University of Rio Grande do Sul Ethical Committees (approval no. 228.944; Porto Alegre, Brazil). Following collection of written informed consent, excised endometriosis lesions and adjacent endometrial biopsies were obtained from women with both surgical and histological diagnosis of endometriosis. All endometrial samples were obtained using a Pipelle catheter® (Laboratoire CCD), and endometriosis lesions were excised during laparoscopy. Endometrium and endometriosis biopsies were placed in a tube containing RNAlater® (Ambion; Thermo Fisher Scientific, Inc.) and stored at -80˚C until further processing.
The diagnosis of endometriosis was confirmed by histology. All 31 patients included in the study cohort were healthy and did not have any other medical issues, with the exception of endometriosis and/or sterility. The patients included had mild or moderate (stages II-III) endometriosis (revised American Society of Reproductive Medicine, classification system, 1997) (22).
Exclusion criteria included: The use of hormonal medications (or supplements) within three months prior to surgery; pregnancy; cancer; endometrial pathologies including polyps and submucosal/intramural fibroids; and patients who did not know their last menstrual period or refused to participate in the study. Out of the 31 participants, 8 were excluded due to insufficient mRNA levels. Following the first analysis, the samples were divided according to the menstrual cycle as follows: Proliferative, day 1-14 (n=11); and secretory, day 15-28 (n=12), according to Noyes criteria (23).
miRNA analysis using reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from the tissue fragment (~1 cm3) using TRIzol reagent® (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Quantification and quality of RNA extracted were analyzed by a fluorometer platform Qubit® 2.0 (Thermo Fisher Scientific, Inc.) by performing serial dilutions according to the manufacturer's protocol. The total RNA was transcribed to obtain cDNA using a poly (A) RT-PCR method through the NCode miRNA first-strand cDNA synthesis MIRC-50 kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For miRNA expression analysis, a RT-qPCR technique was performed using an Applied Biosystems® 7500 RT-PCR platform (Thermo Fisher Scientific, Inc.). Complementary primers to the sequences of miR-135a (forward, 5'-CCAGGCTTCCAGTACCATTAGG-3'; and reverse, 5'-GTTTCCGAGAGAGGCAGGTG-3') and miR-135b (forward, 5'-GCTTATGGCTTTTCATTCCT-3'; and reverse, 5'-GTGCAGGGTCCGAGGT-3') were used. The experiments were performed on individual plaques for each miRNA using the U6 as an endogenous expression control. The primers used for the U6 small nuclear miRNA were (U6 forward, 5'-CTCGCTTCGGCAGCACA-3'; and reverse, 5'-AACGCTTCACGAATTTGCGT-3').
qPCR was performed using Supermix SYBR-Green and ROX (Quatro G P&D Ltda.). A total of 25 ng of cDNA were used for a 50-µl total reaction, according to the manufacturer's protocol. The thermocycling conditions were initiated by uracil-N-glycosylase activation at 50˚C for 2 min and initial denaturation at 95˚C for 10 min, followed by 40 cycles at 95˚C for 15 sec, 57˚C for 20 sec and 70˚C for 30 sec; the final extension was at 72˚C for 5 min. The threshold cycle (Ct) and melting curves were acquired by using the quantitation and melting curve program of the AB 7500 platform. The miRNA level of each sample was normalized according to U6 expression. Relative miRNA level was presented using the 2-ΔΔCt formula (24).
Data are expressed as the mean ± SD. Statistical analysis was performed using paired Wilcoxon signed-rank test to compare the ectopic and eutopic endometrium samples, and an unpaired Mann-Whitney test was used for the comparison between different menstrual cycle phases. All the analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
Population and samples
The mean age of the 23 enrolled patients was 32 years (standard deviation ±2.93; range, 24-40 years). Of these patients, 16 were nulliparous and 7 reported at least one pregnancy. There were no significant differences between age and parity between the proliferative and secretory groups. A total of 1 patient had the clinical suspicion of endometriosis for 10 years, 2 for 3 years, 3 for 2 years, 4 for 1 year, 3 for 3-9 months, and in 10 patients, the diagnosis was made during surgery. The primary symptoms for surgery were pelvic pain (30.4%), sterility (56.5%) or both (13.0%). Table I summarizes the cycle phase characteristics and endometriosis-associated symptoms of all patients in the study cohort.
Expression of miR-135a and miR-135b in ectopic and eutopic endometrium lesions
The expression of miR-135a and miR-135b was analyzed in ectopic and eutopic endometrial tissues, in the proliferative and secretory phases, from women with endometriosis. The secretory phase was not classified into different stages. The results indicated slight decreases in miR-135a and miR-135b expression levels in the ectopic endometriosis endometrium compared with the eutopic endometrium tissue [3.54-fold decrease (P=0.353) and 4.06-fold decrease (P=0.305), respectively; Fig. 1]. When analyzed according to the phases, the expression levels of miR-135a and miR-135b were decreased in the endometriosis lesions compared with the eutopic endometrium tissue in the proliferative [n=11; 1.7-fold (P=0.25) and 0.25-fold decrease (P=0.32), respectively; Fig. 2A] and secretory phases [n=12; 5.31-fold (P=0.266) and 8.14-fold decrease (P=0.129), respectively; Fig. 2B]. All differences between these groups were statistically non-significant.
Expression of miR-135a and miR-135b in different menstrual cycle phases
The expression levels of miR-135a and miR-135b were increased in the secretory phase compared with the proliferative phase in the endometrium tissue [10.8-fold increased (P=0.057) and 7.6-fold increased (P=0.11; Fig. 3]. In the endometriosis tissue, the expression levels of both miR-135a and miR-135b were significantly increased in the secretory phase compared with the proliferative phase [7.9-fold increased (P=0.036) and 9.7-fold increased (P=0.005; Fig. 3].
To obtain a favorable environment for the intrauterine embryo, adequate hormonal signaling must be performed, both with endometrial and myometrial components. miRNAs are essential for normal uterine development and function.
To the best of our knowledge, the present study is the first to compare miR-135 expression levels in the eutopic endometrium in patients with endometriosis in different menstrual cycle phases. We hypothesized that there would be an increase in miRNA expression in the proliferative phase in both types of analyzed tissues, but the increase was only significantly positive in endometriosis lesions.
Most studies have identified differential miRNA expression by comparing disease-free patients with those with endometriosis, and a number of studies have only analyzed the eutopic endometrium in a certain phase of the menstrual cycle (25-27). However, different endometriosis tissues, including those of endometrioma and peritoneal and ovarian endometriosis, express different profiles of miRNAs, and miRNA expression may or may not vary throughout the menstrual cycle phases (28). Ohlsson Teague et al (16) evaluated miRNA expression in different menstrual cycle phases and did not identify any difference compared with miRNA expression in the proliferative and secretory phases.
The difference between the studies may be explained by the fact that compared with the eutopic endometrium, the ectopic endometrium is probably susceptible to hormonal regulation; therefore, the lesions may suffer alteration regarding estradiol fluctuation. Estrogens and progestogens modulate chemotaxis and apoptosis in human endometrium and endometriosis tissues, and contribute to inflammatory responses, abnormal tissue remodeling, therapeutic refractoriness and disease persistence (29).
Progesterone has been suggested to control HOXA10 expression, resulting in increased HOXA10 levels in the secretory phase of the menstrual cycle, also known as the window of implantation (30). However, Petracco et al (21) demonstrated that HOXA10 expression was regulated by both miR-135a and miR-135b in the endometrium of females with endometriosis. Increased miRNA expression appeared to directly downregulate HOXA10 expression, which is required for implantation in this group of patients.
In the present study, it was observed that, independent of the cycle phase, miRNA expression was always decreased in the ectopic tissue (endometriosis) compared with the endometrium tissue; however, this difference in expression was not statistically significant.
When miR-135a and miR-135b expression levels were compared separately in the eutopic endometrium and endometriosis lesions, between the secretory and proliferative phases, the expression levels were identified to be decreased in the proliferative phase. However, statistical significance was only observed in the analysis of endometriosis lesions. Therefore, the highest expression levels of miR-135a and miR-135b were identified in the eutopic endometrium tissue during the secretory phase. The addition of control samples from healthy individuals may improve the results of the present study, primarily to compare the relative expression levels of miR-135a and miR-135b in eutopic and ectopic tissues with basal levels in healthy tissues.
Naqvi et al (31) indicated that even distal endometrial lesions selectively and significantly altered the expression of genes, including HOXA10 and progesterone receptors. The authors demonstrated that there is a uterine effect even when endometriosis is remote from the pelvis. Besides the decreased expression of HOXA10, the diminished progesterone receptor systemically induced by the remote disease may be associated with the increased levels of miR-135a observed in the secretory phase, when increased levels of progesterone are expected. The analysis of the association between miR-135 tissue levels and HOXA10 gene expression in eutopic and ectopic tissues may provide more conclusive results regarding the understating of endometriosis.
Santamaria et al (32) suggested that cells migrate from endometriosis lesions to the eutopic endometrium and disturb gene expression involved in embryo implantation and infertility. The ectopic cells migrate to the uterus and follow a different mechanism that alters uterine receptivity. The genes abnormally expressed in the endometrium of patients with endometriosis affect miRNAs and alter the effect of miR-135. In addition, Santamaria et al (33) in an extensive review regarding the roles of miRNAs in gynecological diseases revealed that 6 miRNAs have the potential to be used as new diagnostic tools. Certain miRNAs, if considered as a panel, exhibited a sensitivity of 96.6% and a specificity of 76.6% in blood endometriosis diagnostic tests (34). Therefore, these results additionally support the hypothesis that circulating miRNAs may be used as valuable non-invasive biomarkers for diagnosis and classifying different stages of endometriosis.
Cho et al (34) analyzed miR-135 expression in the serum of patients with endometriosis and controls according to the menstrual cycle. They demonstrated that during the secretory phase, miR-135a expression was significantly decreased in patients with endometriosis compared with the controls (2.9-fold decrease; P=0.041). The discordance between serum and tissue levels of miR-135a expression may be due to the different expression of each miRNA and its tissue specificity, as miRNA expression patterns depend on the environment and specific tissue type. miR-135 is expressed in the serum of patients with endometriosis and this is a promising result for the future of endometriosis research.
The present study demonstrated that miR-135 is fully expressed in the ectopic and eutopic endometrium of patients with endometriosis. We hypothesize that the use of these data as diagnostic and therapeutic tools may lead to an improved understanding and management of endometriosis.
The roles of a number of miRNAs in the pathogenesis of endometriosis have been established. However, their exact contributions to the development and maintenance of endometriosis are not fully understood. Subsequent investigations are mandatory for the development of diagnostic tools and therapeutic approaches that use miRNA manipulation technology to drive mRNA expression.
There are a number of pieces of evidence demonstrating that the physiopathology of endometriosis has a genetic component, and several studies have associated this disease with different miRNA levels and alterations in the endometriosis tissue (35,36). As the present study detected differences in the levels of miR-135 expression during the menstrual cycle, it may be assumed that they serve a determinant role in the homeostasis of the reproductive tissue. Therefore, the profile of miR-135 expression may affect endometrial functioning and impair embryo implantation.
Little is known about the regulation of miRNA expression, but it has been demonstrated that complex mechanisms are involved. miRNAs are expressed temporally depending on the stage of cellular development and at varied levels between different tissues (37). Therefore, miRNA expression is considered to be extremely specific and is highly regulated.
Although more studies using large sample sizes and healthy tissue as control groups are necessary to elucidate the whole function of miR-135 in endometriosis, this miRNA is becoming an important target for the understanding of the pathogenesis of endometriosis and is a potential biomarker to guide diagnostic tools and therapeutic interventions. While other biomarkers for endometriosis are being studied, including matrix metalloproteinase (MMP)-2 and MMP-9, cancer antigen 125 remains the most recommended marker for suspicion of endometriosis and follow-up. The effects of miRNAs on the expression of genes involved in endometriosis pathophysiology in ectopic and eutopic tissues may be analyzed to elucidate the role of post-transcriptional factors in the genesis of this disease. There is an urgent requirement for a test based on non-invasive molecular biomarkers to identify the symptoms of endometriosis during the early stages of disease development.
The present study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior, Brazil (CAPES– Finance Code 001).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
RP, AP, MB, JdRM and MH were responsible for biopsy sample collection and clinical analyses. ACdOD, HT and DRM performed the molecular analyses, including miRNA extraction and RT-qPCR. PNdA and GZ performed the analysis and interpretation of data. DCM made substantial contributions to conception and design of the study, and the acquisition, analysis and interpretation of data. All authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
The study was approved by Pontifical Catholic University of Rio Grande do Sul Ethical Committees (approval no. 228.944). Signed informed consents were obtained from the patients or guardians.
Patient consent for publication
The authors declare that they have no competing interests.
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