{"paper_id":"f9a644a7-bed5-4ef0-93c7-c9d495edb44d","body_text":"Endometriosis is a debilitating disease that affects as many as 10 to 15% of all women of reproductive age and as many as 40 to 50% of all women with infertility. The disease occurs in women of reproductive age (i.e., menstruating females) and is characterized by the major complaints of pelvic pain, dysmenorrhea, and infertility. Endometriosis is defined as the presence of both ectopic endometrial stromal and glandular tissue. This disease is thought to develop via reverse menstruation of viable endometrial tissue into the peritoneal cavity.\nHowever, because almost all women of reproductive age exhibit some degree of retrograde menstruation, 1 , 2  it is postulated that some other factors must contribute to the development and progression of the disease. Over the course of the past 30 years, additional factors which may predispose this subset of women to developing endometriosis have been proposed. Alterations in the immune system, eutopic endometrium, and stem cell population have also been demonstrated to be altered in women with endometriosis. 3 – 5  While these alterations may contribute to the development and/or progression of endometriosis, a limiting factor is the difficulty in assessing cause and effect. This holds true for examining the potential role of any factor, including mRNAs, proteins, or microRNAs. Once a factor is identified as “mis-expressed” in endometriotic tissue, studies must be conducted to determine if this mis-expression is a cause for (driver of the disease) or a result of endometriosis (passenger).\n\nMicroRNA (MiRNAs) are a class of small noncoding regulatory RNAs that posttranscriptionally regulate gene expression. 6 , 7  The genes which encode miRNAs reside primarily between genes (intergenic) or within introns (intronic) of genes. MiRNAs also make up the entire introns as well as reside with exons, 8 , 9  but this occurrence is less common compared with intergenic or intronic miRNAs. MiRNA transcription is mediated by polymerase II or III as the initial long primary RNA transcript (pri-miRNA) is capped (MGpppG) and polyadenylated. 10  Pri-miRNA transcript then binds with the RNA-binding protein DGCR8 (DiGeorge syndrome critical region 8) and the pri-miRNA–DCGR8 complex is then cleaved by Drosha (an RNAse III enzyme), yielding a stem-loop precursor miRNA (pre-miRNA). The pre-miRNA is then exported from the nucleus via exportin 5 and RAN-GTP into the cytoplasm. Once in the cytoplasm, a second RNAse III enzyme, DICER, cleaves the pre-miRNA into a transient RNA duplex of approximately 18 to 25 nucleotides. Processing of all miRNAs, except miR-451, 11 , 12  is thought to be DICER dependent.\nOnce processed, the miRNA duplex is cleaved, generating two strands. One strand is degraded while the remaining strand is preferentially loaded onto the RNA-induced silencing complex (RISC). The RISC is composed of DICER, a TAR RNA-binding protein (referred to as TRBP) and one of the four different Argonaute (Argo) proteins. 13  Once within the RISC, miRNAs bind to the 3′-untranslated region (3′-UTR) of the target mRNA transcript. If the binding of the miRNA to the 3′-UTR “seed sequence” exhibits perfect base-pairing, the mRNA transcript is degraded and mRNA translation does not occur. 14  If base pair binding homology between miRNA and 3′-UTR of the mRNA is imperfect, mRNA translation is inhibited. This most likely occurs via an inhibition of translation after initiation 14  or through an RISC interaction with eukaryotic translation initiation factor 6 (EIF6) preventing assembly of 80S ribosomes. 15  More recently, miRNA regulation of transcript stability and protein translation has also been proposed to be mediated at the level of the 5′-UTR 16 – 18  as well as the DNA-coding sequence. 17 , 18\nWhile the majority of the literature supports the notion that miRNAs inhibit translation, there is some evidence that miRNAs can actually enhance translation through alterations in the Argo component of the RISC. 7  Thus, while miRNAs appear to primarily regulate translation in an inhibitory manner, they may also enhance translation in certain biological scenarios.\n\nMiRNAs are postulated to play a role in normal biological processes while their mis-expression has been associated with numerous diseases. 19 – 21  The study of miRNAs and endometriosis began nearly 10 years ago with the generation and comparisons of miRNA expression profiles between eutopic endometrium and endometriotic lesion tissue. One of the first assessments of miRNA expression comparing paired eutopic and ectopic endometrial tissue was conducted in 2009. 22  Fourteen upregulated and eight downregulated miRNAs specific to endometriotic lesion were identified by microarray analysis (► Table 1 ). In silico analysis identified 3,851 mRNA transcripts as putative targets of these 22 miRNAs. Of these predicted targets, 673 were also differentially expressed in ectopic compared with eutopic endometrial tissue, as determined by microarray. Using Ingenuity Pathway Analysis (IPA) software, potential molecular pathways were assessed and determined to involve c-Jun, CREB-binding protein, protein kinase B (AKT), and cyclin D1 (CCND1) signaling, all of which have previously been associated with endometriosis pathogenesis.\nFiligheddu and coworkers 23  conducted a similar profiling experiment and compared miRNA expression between ectopic and eutopic endometrium using tissue obtained during the proliferative stage of the menstrual cycle. Fifty miRNAs were considered differentially expressed between the two tissue types (► Table 1 ). Again, the mis-expression of these miRNAs was postulated to allow for dysregulation of several pathways previously proposed to contribute to the pathophysiology of endometriosis, including cytokines, enzymes, growth factors, receptors, and transcription regulators. In both of these studies, 22 , 23  six common miRNAs ( miR-145, miR-143, miR-99a, miR-126, miR-100, and miR-29c ) were reported as upregulated. However, there were conflicting reports on whether or not their expression was influenced by the stage of the menstrual cycle. 22 , 23\nThe first transcriptome-miRNA analysis of endometriotic endometriomas (endometriotic cysts of the ovary) was conducted by Hawkins and colleagues in 2011. 24  MiRNAs expressed at the highest level in endometriomas and the corresponding abundance of transcript in non-endometriosis control endometrium was determined (► Table 1 ). Many of the miRNAs previously reported 22 , 23  were again identified by Hawkins and colleagues. 24  This 2011 study 24  was the only one of the first three studies at the time to analyze the potential function of a differentially expressed miRNA.  miR-29c , which exhibited the highest expression differential between tissue types, was evaluated using primary human endometrial stromal cells in vitro. 24  Putative extracellular matrix (ECM) protein gene targets of  miR-29c  (COL7A1, UPK1B, and TFAP2C) were downregulated in cells overexpressing  miR-29c  and the direct effect on the 3′-UTR of the genes was confirmed. Thus, mis-expression of  miR-29c  in endometriomas appears to functionally contribute to the aberrant expression of ECM proteins associated with the disease at least in isolated endometrial stromal cell culture.\nMore recently, Long et al 25  also investigated the expression of the  miR-29  family in the endometrium samples from women without endometriosis, as well as in paired ectopic and eutopic endometrium samples. As opposed to earlier studies, 22 – 24  these authors observed a decrease in lesion expression of  miR-29c . The investigators further examined the role of  miR-29c  in endometrial cell proliferation, invasion, and apoptosis in vitro.  miR-29c  suppressed endometrial cell proliferation and invasion, promoting cell apoptosis. The authors concluded that  miR-29c  exhibits inhibitory action on endometrial cell proliferation and invasion by inhibiting the expression of c-Jun. It should be emphasized that while these investigators demonstrated a decrease in  miR-29c  expression in endometriotic lesion tissue which would suggest enhanced cell proliferation and invasion due to low  miR-29c  expression, all of the earlier reports demonstrated an increase in  miR-29c  expression. 22 – 24\nSince these initial reports, additional studies have focused on differentially expressed miRNA between paired endometriotic lesion and eutopic endometrium. More recently, Shi and colleagues 26  profiled differentially expressed miRNAs in the normal, eutopic, and ectopic endometrium using miRNA microarray screening analysis (► Table 1 ). Of those differentially expressed,  miR-183 – 5p  was found to be downregulated in the ectopic and eutopic tissues from women with endometriosis compared with eutopic endometrium from women free of disease. Functional analysis indicated that  miR-183 – 5p  may contribute to endometrial stromal cell apoptosis (but had no influence on cell proliferation) and exhibits a negative regulatory impact on the invasive ability of these cells. Stromal cell  miR-183  expression was also examined in response to ovarian steroids (17β-estradiol and progesterone) and inflammatory cytokines (tumor necrosis factor-α and interleukin-6). Administration of all these reagents decreased  miR-183  expression. It was concluded from this study that repressed levels of  miR-183  may modulate the growth and invasive potential of endometriotic endometrial stromal cells contributing to the development and progression of endometriosis.\nYang and colleagues 27  used miRNA microarrays to identify differentially expressed miRNAs between endometriotic lesion tissues from women with stage III/IV disease compared with eutopic endometrium from women without endometriosis. Lesion tissue expressed significantly higher levels of expression of seven miRNAs and significantly lower levels of expression of ten miRNAs (► Table 1 ). Of these miRNAs, two of the upregulated miRNAs ( miR-106 – 5p and miR-16 – 5p ) and six of the downregulated miRNAs ( miR-126, miR-146a-5p, miR-15a-5p, miR-19b-1 – 5p, miR-200b, and miR-200c ) were predicted to target key regulators of angiogenesis. Expression of VEGFA was significantly upregulated, whereas EGFR2, PTEN, and CXCR4 were markedly down-regulated in lesion tissue compared with endometrium from women without endometriosis. The authors concluded that the differentially expressed miRNAs could modulate VEGFA, EGFR2, PTEN, and/or CXCR4 expression and contribute to the pathogenesis of endometriosis.\n\nFrom the initial generation of miRNA profiles, later investigations began to focus on specific miRNAs and putative targets. The potential miRNA targets may be relevant to the pathophysiology of endometriosis and may aid in determining whether these miRNAs function as drivers of the disease. Ramón and colleagues 28  assessed the expression of miRNAs proposed to play a role in angiogenesis. In this study, the investigators correlated the expression of  miR-15b ,  miR-16 ,  miR-17 – 5p ,  miR-20a ,  miR-21 ,  miR-125a ,  miR-221 , and  miR-222  with that of specific angiogenesis factors, vascular endothelial growth factor-A (VEGF-A), and thrombospondin-I (TSP-I). When analyzing paired specimens, ovarian endometriomas exhibited significantly lower levels of proangiogenic VEGF-A mRNA and protein and higher levels of  miR-125a  and  miR-222  compared with corresponding eutopic endometrium. In contrast, levels of the angiogenesis inhibitor (TSP-I) were significantly higher in endometriomas and this was associated with reduced levels of  miR-17 – 5p . Significant inverse correlations were noted between  miR-222  and VEGF-A protein expression and  miR-17 – 5p  and TSP-I protein levels. These correlations suggest that the mis-expression of these miRNAs may at least in part contribute to the observed altered expression of the angiogenic factors and thus contribute to the pathogenesis of the disease.\nOne of the miRNAs evaluated by Ramón and colleagues 28  was also examined by Zhao and coworkers. 29 \n miR-20a  expression was assessed in a total of 40 patients with ovarian endometriosis and 20 controls with benign ovarian tumors. qRT-PCR assessment revealed significantly increased  miR-20a  expression in ovarian endometriosis as compared with control ovarian cyst tissue. This increase in  miR-20a  expression is in contrast to the decrease detected by Ramón and colleagues 28  as well as earlier assessments 22 , 23  which reported lower  miR-20a  expression in lesion tissue. However, the reason for these discrepancies was not discussed. Zhao and colleagues determined the pathways potentially targeted by  miR-20a , and the cell cycle pathway was identified as one of the most relevant. Assessment of predicted  miR-20a  target gene, netrin-4 (NTN4), revealed a significant decrease in patients with ovarian endometriosis. From this study, it was concluded that the increased expression of  miR-20a  may play an important role in the pathogenesis of ovarian endometriosis by suppressing NTN4 expression.\nIn addition to the earlier-mentioned miRNAs, several miRNAs have been further examined in endometriotic lesion tissue and/or cells.  miR-199a  is downregulated in several types of cancers, 30 , 31  but had yet to be reported in endometriotic tissue. Considering that the pathophysiology of endometriosis shares similar characteristics with cancer, Dai and coworkers 32  evaluated  miR-199a  expression in matched ovarian endometriomas and eutopic endometrium as well as endometrium from women free of endometriosis. Compared with eutopic endometrium from women without endometriosis,  miR-199a  expression was lower in both the eutopic endometrium from women with endometriosis and ovarian endometriomas. Forced expression of  miR-199a  in endometrial stromal cells resulted in dampened cell adhesion, invasion, and migration. These cellular phenotypes were associated with suppression of the IKK/NFκB pathway and reduced interleukin-8 expression, suggesting a possible functional role for the mis-expressed levels of  miR-199a  in the pathogenesis of endometriosis.\nMore recently, the above group investigated the role of  miR-199a  in the angiogenic potential of endometrial stromal cells under hypoxic conditions. 33  Forced overexpression of  miR-199a  significantly attenuated its angiogenic potential under hypoxia and was also associated with a reduction in VEGF-A expression in these cells. Reporter gene assays targeting the 3′-UTR of VEGF-A and hypoxia-inducible factor (HIF)-1α were incorporated to demonstrate that  miR-199a  downregulation was mediated at the level of the 3′ – UTR of these target genes. These findings may suggest that  miR-199a  may attenuate the angiogenic potential of stromal cells under hypoxia partly through HIF-1α/VEGF-A pathway suppression, thereby contributing to the pathogenesis of endometriosis.\nmiR-126  is also a proposed regulator of angiogenesis as well as growth, adhesion, and invasion. 34 , 35  Earlier reports 22 , 23  demonstrated increased expression of this miRNA in lesion tissue. Liu and colleagues, 36  in contrast, reported that  miR-126  expression was significantly reduced in ovarian endometriomas and eutopic endometrium from women with endometriosis compared with eutopic endometrium from women free from the disease. Associated with this reduction in  miR-126  was a significant increase in v-crk sarcoma virus CT10 oncogene homolog (CRK) mRNA and protein as well as a highly significant association between endometrioma and eutopic endometrial  miR-126  expression. More severe forms of endometriosis were associated with lower levels of  miR-126  expression in these tissues. Unfortunately, the ability of  miR-126  to modulate cellular events conducive to endometriosis pathogenesis, such as cellular proliferation, adhesion, and/or invasion, was not evaluated in this study. It should also be noted that a more recent array analysis 27  observed decreased levels of ectopic endometrial tissue expression of  miR-126  which is in agreement with the study by Liu and coworkers, 36  but in contrast to earlier array analysis reports. 22 , 23\nThere is considerable evidence that steroidogenic factor-1 (SF-1) expression is increased in stromal cells from endometriotic tissue and this may be due to multiple mechanisms. 37 – 39 \n miR-23a/b  expression is reduced in endometriotic and eutopic endometrium from women with endometriosis. As  miR-23a/b  is proposed to target  SF-1 , the potential mechanistic link between  miR-23a/b  and SF-1 expression in the pathogenesis of endometriosis was examined. 40  Reduced levels of  miR-23a/b  expression was confirmed in ectopic endometriotic tissue and eutopic endometrium from women with endometriosis compared with eutopic endometrium from women free of endometriosis and this reduction was associated with elevated transcript levels of  NR5A1  (SF-1),  STAR , and  CYP19A1 . To confirm that  miR-23a/b  directly regulated  NR5A1  ( SF-1 ) expression, luciferase reporter assays were conducted. Despite the fact that transfection with  miR-23a/b  precursor suppressed  NR5A1  ( SF-1 ) transcript,  miR-23a/b  did not bind to the 3′-UTR for  NR5A1  ( SF-1 ), suggesting that  miR-23a/b  mediation of SF-1 expression is via an indirect mechanism.\nmiR-145  expression has been shown to be mis-expressed in endometriotic tissue compared with eutopic endometrium. 22 , 23 , 41 , 42  Coupled with the fact that  miR-145  putatively targets factors involved in cellular events 41  conducive to endometriosis survival/progression, Adammek and colleagues 43  examined the expression and potential function of  miR-145  in the pathogenesis of endometriosis. Epithelial endometriotic 12Z cells were transfected with  miR-145  which resulted in reduced cell proliferation and invasion, while  miR-145  reduced cellular proliferation in transfected primary ectopic and eutopic endometrial stromal cells from endometriosis patients. In addition, it was discovered that  miR-145  induced posttranscriptional downregulation of targets FASCIN-1, PAI-1, and JAM-A at the transcript and protein level in 12Z cells. Direct binding of  miR-145  to the 3′ – UTR of  JAM-A  was confirmed by luciferase reporter construct assays. In contrast, only FASCIN-1 was shown to be modulated by  miR-145  in primary ectopic and eutopic endometrial stromal cells from endometriosis patients. Cytoskeleton proteins ACTG2, TAGLN, and MYL9 were shown to be differentially regulated by  miR-145  with overexpression of  miR-145  leading to decreased  ACTG2  transcript expression but increased  TAGLN  transcript expression, while  MYL9  expression was unaffected. Similarly,  miR-145  overexpression in 12Z and stromal cells from both eutopic and ectopic endometrial cells from women with endometriosis resulted in decreased transcript expression of pluripotency and stemness-related markers. Modulation of protein expression or verification of direct binding via 3′-UTR reporter assays was not performed to further validate these targets. Although initial results on  miR-145  expression in different endometriosis samples are conflicting, 22 , 23 , 42  these data suggest that  miR-145  appears to inhibit endometriotic cell proliferation and invasion as well as regulation of stem cell properties. As discussed earlier, miRNAs are proposed to regulate steroid action within the endometrium to control the cellular events necessary for “normal” endometrial function. Mis-expression of miRNAs is in turn thought to contribute to the pathogenesis of diseases of endometrial tissue origin such as endometriosis. Within the field of endometriosis research, several miRNAs have emerged as potential players in modulating the events conducive to the establishment, survival, and progression of the ectopic implant. Of these, functional data exist for only a few. To truly understand which miRNAs are important in the pathogenesis of endometriosis and how they contribute to the disease, greater effort must be put forth to standardize what constitutes true “control” groups and what constitutes “endometriotic tissue.” Reported and unpublished observations strongly suggest that not only does miRNA expression vary based on the “type” of implant (ovarian endometrioma vs. peritoneal implant) among study subjects but that this expression also varies by type of implant within study subject.\nBuilding upon their earlier observations that  miR-183  is decreased in endometriotic lesion tissue, 23 , 26  Chen and colleagues incorporated microarray analysis to identify  miR-183  targets that may be relevant to cellular functions associated with endometriosis pathophysiology. 44  Twenty-seven differentially expressed genes were identified of which four of those downregulated genes ( ITGB1, AMIGO2, VAV3, and PSEN2 ) were further assessed based on potential function and relevancy to endometriosis. Assessment of protein expression by Western blots demonstrated that integrin β1 (ITGB1), but not AMIGO2, was affected by  miR-183  over-expression. Expression of detectable levels of neither VAV3 nor PSEN2 proteins was observed. Luciferase reporter assay verified that  miR-183  targeted ITGB1 and that ITGB1 was overexpressed in the endometrium of endometriosis patients. Forced expression of ITGB1 could also rescue the repressive effects of  miR-183  on endometrial stromal cell invasiveness. From these studies, the authors concluded that  miR-183  may be involved in the development of endometriosis by regulating stromal cell ITGB1 expression and function.\nBuilding upon the initial report by Ohlsson Teague and colleagues which demonstrated decreased levels of  miR-142 – 3p  in endometriotic lesion tissue, 22  Kästingschäfer and coworkers 45  investigated the functional effect of  miR-142 – 3p  expression in endometrial stroma cells in vitro. Increased expression of  miR-142 – 3p  via transient transfection resulted in a significant reduction in cell viability as well as reduced expression of steroid sulfatase, interleukin-6-coreceptor gp130, and interleukin-6-mediated activation of the STAT3 pathway. These data were interpreted to imply that  miR-142 – 3p  may modulate cell viability and that this may involve both steroid hormone- and cytokine-mediated signaling events. While these results would be in agreement with reduced levels of  miR-142 – 3p 22  contributing to the survival of endometriotic lesion tissue, the reader should be aware that Yang and colleagues 27  observed increased levels of this miRNA in endometriotic lesion tissue.\nmiR-451  is one of the few miRNAs associated with endometriosis 24  which has been examined not only in human tissue/cells but also in animal models for the disease. Our laboratory reported the initial study examining both  miR451  expression and function in human tissue and cells. 46 \n miR-451  is elevated in endometriotic lesion tissue compared with paired eutopic endometrium from women with the disease. 24 \n miR-451  expression exhibited an inverse association with transcript and protein expression of putative target, macrophage migration inhibitory factor (MIF). Using luciferase reporter constructs for MIF, we demonstrated that  miR-451  specifically bound to the 3′ – UTR to regulate its expression. Furthermore, forced expression of  miR-451  induced a significant downregulation of both MIF mRNA and protein in a human endometrial cell line which was associated with a significant reduction in cell survival.\nAn additional study by Joshi and colleagues 47  identified  miR-451  mis-expression in women with endometriosis as well as in baboons with induced disease. Induction of endometriosis in baboons results in altered expression of several miRNAs including  miR-451 . Reduction in eutopic endometrial  miR-451  expression was associated with increased expression of  miR-451  target,  YWHAZ , and this was observed in both baboon and human tissue. Regulation of  YWHAZ  by  miR-451  was confirmed using 3′-UTR reporter assays in vitro. Lastly, overexpression of  miR-451  in an immortalized human endometriotic epithelial cell line (12Z) led to the decreased expression of its target YWHAZ which was correlated with decreased cell proliferation. Collectively, both our study 46  and that of Joshi and colleagues 47  demonstrated that  miR-451  is capable of modulating cell proliferation and this is associated with alterations in the expression of targets MIF and YWHAZ.\nOne of the major functional endpoints which have been examined with respect to miRNA function in endometrial/endometriotic cells has been angiogenesis.  miR-15a-5p  was reported to be suppressed in endometriotic lesion tissue and proposed to mediate angiogenic events associated with the disease. 27  In a follow-up to their initial study, 27  Liu and colleagues further examined  miR-15a-5p  expression in endometriotic lesion tissue as well as assessed its functional role. 48  Compared with control samples,  miR-15a-5p  was downregulated in patients with endometriosis, supporting their initial array analysis observation. 27  Forced expression of  miR-15a-5p  suppressed cell proliferation, migration, and invasion in endometriotic ectopic stem cells by targeting the 3′-UTR region of VEGFA. Thus, the suppressed levels of  miR-15a-5p  in endometriotic tissue coupled with the suppressive effects of this miRNA on cell proliferation in vitro may suggests that reduced  miR-15a-5p  levels may be conducive to lesion proliferation and survival.\n\nIn summary, early analysis of differentially expressed miRNAs between endometriotic tissue and eutopic endometrium has identified dozens of differentially expressed miRNAs. Subsequent studies have begun to dissect the role of these miRNAs to distinguish between drivers, those miRNAs which modulate key events conducive to the pathophysiology of endometriosis, and passengers, those miRNAs whose expression may change as a result of the disease pathogenesis. Driver miRNAs have been shown to modulate key components of biological pathways highly relevant to the pathophysiology of endometriosis including cellular proliferation, invasion and angiogenesis. While there is agreement among some studies with respect to which miRNAs are differentially expressed and the level of their expression compared with control tissue, there is also disagreement among these levels of expression. These discrepancies may be rooted in differences in the study groups, such as the types of samples that were included in their control groups as well as what “types” of endometriotic lesions were evaluated in their study groups. It is evident that for progress to be made in understanding the role of miRNAs in the pathogenesis of endometriosis, greater emphasis needs to be placed upon standardization of study designs and populations. By doing so, it will not only lead to more consistent experimental outcomes but also enhance experimental rigor and reproducibility. Nonetheless, the data that exist to date strongly suggest that miRNAs are mis-expressed in endometriotic lesion tissue where they are proposed to contribute to the pathogenesis of the disease. Future studies are expected to more clearly define the roles of these mis-expressed miRNAs in endometriosis pathophysiology by identifying the pathways and their components that are regulated by these miRNAs.","source_license":"CC0","license_restricted":false}