Uterine fibroids: a look at the problem

Introduction

The relevance of this problem is due to the fact that in the structure of gynecological diseases in women of fertile age in most countries of the world, uterine leiomyoma (UL) occupies a leading place. Data on the incidence of the disease in patients of different age groups indicate its increase in accordance with age: from 20 to 30 years, the prevalence of UL is 4%, from 30 to 40 years — 11–18%, and from 40 to 60 years — 33–40% [1].

According to Russian and foreign literature sources, in 25–50%, UL is accompanied by symptoms, the type and severity of which correlate with the localization, size, and number of myomatous nodes, the presence or absence of degenerative disorders in them [2]. In patients of reproductive age, the presence of leiomyoma is often accompanied by abnormal uterine bleeding (AUB), less often intermenstrual bleeding [2]. AUB occurs in about 40–50% of women with fibroids at reproductive age. So, according to the results of the study by Fonseca-Moutinho et al. [3], if there are complaints about AUB during ultrasound diagnostics, 73.3% of women have myomatous nodes [3].

Surgical treatment of UL remains leading. The only method of surgical treatment leading to a complete cure is an operation in the volume of total hysterectomy; at the same time, myomectomy is considered the most urgent operation in women of reproductive age interested in the realization of reproductive function. For example, in the USA, the removal of the uterus is in second place after cesarean section among surgical interventions in women. About 600 thousand hysterectomies are performed in America per year, 200 thousand of them for UL [4]. In Russia, the removal of the uterus for leiomyoma accounts for 40–42% of the total number of hysterectomies [5–6]. It was noted that 50–70% of patients suffering from UL at reproductive age underwent organ-preserving surgical treatment [5]. However, due to the insufficient effectiveness of the methods used, the high frequency of relapses of the disease and side effects against the background of drug treatment, there is a need to develop new directions in UL therapy.

Nowadays, there is no clear opinion about the causes of the occurrence and recurrence of UL, but due to advances in molecular medicine, significant progress has been made in understanding the hormonal and molecular genetic mechanisms of initiation, formation, and growth of the myomatous node. Further study of the pathogenesis of the disease is necessary to improve existing therapies and discover new ones.

Etiology of uterine leiomyoma

The etiology of UL is unclear, definitely multifactorial; UL results from the proliferation of a single clone of smooth muscle cells. The myometrium and UL contain multipotent somatic stem cells, which are a reservoir that supports growth and self-renewal. Stem cells from UL carry genetic mutations, whereas myometrial tissue does not, which suggests that a mutation as the first “blow” is necessary for the formation of UL from a myometrial stem cell.

Some authors believe that UL develops in response to cyclical fluctuations of steroid hormones. Ovarian estrogen and estrogen induced locally by aromatase activity are key regulators in UL cells. Estrogen maintains progesterone receptor levels, and progesterone, interacting with the receptors, promotes UL growth. Progesterone is necessary for the maintenance and growth of UL and, according to research, it turns out to be an even more important regulator than estrogen. Estrogen and progesterone and their nuclear receptors (ER and PR, respectively) stimulate the formation and growth of UL.

Other studies have shown that estrogen and progesterone activate signaling pathways such as WNT/β-catenin, which leads to stimulation of various transcription factors and downstream signaling, which ultimately leads to the clonal proliferation of UL. Progesterone and progesterone receptors affect UL proliferation by regulating the expression of growth factors such as epidermal growth factor (EGF) and signaling pathways. Progesterone receptors can bind directly to the anti-apoptotic promoter BCL-2, preventing apoptosis in UL cells. The use of antiprogestins or selective progesterone receptor modulators in clinical trials is another convincing proof of the effect of progesterone on UL growth [7].

In the study by Tskhai et al., [8] it was shown that the most significant parameters influencing the probability of UL recurrence were the parity of labor (the lower the parity of labor, the higher the probability of UL recurrence), the presence of multiple nodes removed during myomectomy, the presence of intense expression of the marker of angiogenesis VEGF, and the presence of weak expression of the apoptosis inhibitor marker BCL-2. Considering the fact that the pathogenesis of uterine fibroids is based on a violation of the mechanisms of apoptosis, proliferation, and angiogenesis, the course of the disease and the likelihood of recurrence directly depend on the ratio of these markers. As for the other parameters, such as age, heredity, and rapid growth of nodes, these parameters showed unstable and variable indicators, on the basis of which it is impossible to trace a certain pattern. In general, the use of this method in predicting the disease is promising for further study of various patterns using various additional factors [9].

Provoking factors and molecular mechanisms of tumor transformation of UL smooth muscle cells are being actively studied. It is known that various stressful effects play an important role in this, especially hypoxia and contractions of the uterine muscles during menstruation, pregnancy, and childbirth. The presence of stem cells in the myometrial tissue has been discussed for a long time, due to the ability of the uterus to expand rapidly and increase in volume during pregnancy. For the first time, stem cells in the uterus were detected by flow cytometry. It was also found that these cells had a low concentration of progesterone and estrogen receptors [10]. Therefore, markers such as Stro-1/CD44 or CD34/CD49(2) were found in stem cells [11].

Recent studies have shown the importance of the expression of RANKL (Receptor activator of nuclear factor kappa-B ligand) genes, which is regulated by progesterone, for the activation of fibroid proliferation and development [12]. Other authors have established the role of bone marrow stem cells that repair the endometrium [13], participate in the healing of the myometrium [14], are necessary during pregnancy [15], and contribute to the development of endometriosis [16]. Probably, bone marrow cells have a similar role in the development of myometrium and leiomyoma, as well as in the development of endometrium and endometriosis. Preventing stem cell engraftment may limit the growth of leiomyoma. Moridi et al. [17] showed in their study that the myometrium synthesized and produced the chemoattractant CXCL12, and the leiomyoma produced it even more, which most likely led to an even greater growth of fibroids due to the engraftment of stem cells. The authors suggest blocking CXCL12 as a possible treatment option for leiomyoma [17].

Role of stem cells

The UL stem cells interact with mature myometrial cells through paracrine pathways, which leads to the proliferation and accumulation of extracellular matrix (ECM). Uterine fibroblasts produce a large number of ECM proteins, such as fibronectin, collagen type I and III, vimentin and versican, which leads to a dense UL structure. It is reported that this dysregulation is induced by environmental stimuli and intracellular signaling pathways. Several intracellular pathways are involved, including Wnt/β-catenin, TGF-β (transforming growth factor-β)/SMAD, MAPK (mitogen-activated protein kinase)/p38, PI3K (phosphotidylinositol-3-kinase/protein kinase B)/AKT/mTOR (target of rapamycin in mammals) and JAK (janus-kinase)/STAT (signal converter and transcription activator). Previous studies suggest that targeting Wnt/β-catenin signaling may be a promising therapeutic approach to UL due to their aberrant activation compared to myometrial cells. Thus, understanding the complex relationship between Wnt/β-catenin signaling and MM development is crucial for targeted therapy [18]. So, Liu et al. [19] analyzed the levels of RANKL micro-RNA in 86 patients with UL. RANKL micro-RNAs were higher in leiomyoma tissues compared to normal myometrium. RANKL increases the expression of proliferation genes (cyclin D1) and stem cell factors (KLF4 and NANOG), but does not affect the expression of differentiation markers (ESR1, PGR, and ACTA2). These findings suggest that RANKL leads to UL growth due to selective induction of proliferation in the stem cell population [19].

Role of genetic mutations

UL pathogenesis remains poorly understood, which hinders progress in identifying risk factors, prognosis, and the development of non-invasive treatment methods. Some cellular mechanisms are involved in the formation and growth of fibroids. Abnormal stem cells of the myometrium and fibroids demonstrate an increased response to the effects of estrogens and progesterone, stimulating processes such as cell proliferation, inhibition of apoptosis, and formation of ECM. New genetic methods have provided new opportunities in understanding UL pathogenesis. Four main subtypes of genetic mutations are known: mutations of the mediator of the transcription MED12, fumarate hydratase (FH), high mobility translocation of the AT-hook 2 group (HMGA2) and deletion of the collagen gene, also include the COL4A5-COL4A6 genes.

Genetic mutations play an important role in UL development; approximately 70% of patients with UL have specific mutations in MED12. Therefore, MED12 encodes a subunit of the mediator complex, regulates the initiation and prolongation of transcription by attaching regulatory elements in the promoters of the RNA polymerase initiation complex. So, MED12 mutations in UL mainly occur in exons of the 1 and 2 MED12 gene, and most of them are deletion, insertion, and missense mutations. The role of these mutations has not been fully studied, but evidence suggests that MED12 mutations are involved in the regulation of the Wnt/β-catenin signaling pathway. The same group showed that inhibition of the β-catenin/MED12 interaction suppressed the activation of β-catenin in response to Wnt signaling. It has been shown that MED12 is necessary for the reference transmission of Wnt signals, and MED12 restricts β-catenin-dependent growth during mouse embryonic development, thus it can be postulated that mutations of the MED12 gene lead to the absence or defects of the MED12 subunit, which can lead to growth dependent on the β-catenin pathway. Indeed, ULs with MED12 mutations are associated with an increase in the expression of the Wnt, Wnt4 ligand in MM cells when compared with UL without the MED12 mutation [20]. Other authors reported that UL with aberrations in HMGA2 (high mobility group A) showed a significant increase in the regulation of PLAG1 (pleiomorphic adenoma gene 1), suggesting that HMGA2 promotes oncogenesis through activation of PLAG1 [7]. Translocations at the 12q14–15 site in leiomyomas increase the expression of the HMGA2 gene. The altered expression of this protein, which is involved in transcription regulation, is associated with the growth of fibroids, including indirectly through stimulation of angionegesis, ERa-induced cell proliferation [21]. It is currently known that HMGA2 is a target of the let-7 family of micro-RNAs. The expression of miRNA-29b in UL tissue was lower than in myometrium; the recovery of micro-RNA-29b inhibits excessive accumulation of ECM and UL growth. Blocking of micro-RNA-29b in myometrial transplants was insufficient to cause tumor growth. It is important to note that the study confirmed previous observations that estrogens and progesterone can suppress the regulation of miRNA-29b and, as a consequence, increase collagen expression [22]. Paul et al. [23] included three types of tissues in the study: normal myometrium, leiomyoma tissue, and tissue of normal myometrium obtained from the uterus with leiomyoma. A total of 1,169 genes were differentially expressed when comparing normal myometrial tissue and healthy myometrial tissue obtained from a uterus with leiomyoma, including fibroblast growth factor receptor 1 (FGFR1), cyclin D1 (CCND1), protocadherin 11 X-linked (PCDH11X), vitamin D receptor(VDR), N-Myc down-regulated gene 2 protein (NDRG2), and inositol polyphosphate-4-phosphatose type II B (INPP4B). The regulation of these genes was disrupted in leiomyoma tissues, which suggests their role in the pathogenesis of UL. The FGF signaling pathway leads to the activation of mitogen-activated protein kinase (MAPK) and an increase in the ECM. It is known that the FGFR1 gene is involved in the pathogenesis of leiomyoma. Also, the increased expression of this gene is associated with increased expression of CCND1, which is an important regulator of the cell cycle and proliferation. The CCND1 gene was the only one found in all three tissue samples. Therefore, microRNA-93 was also found to block cell growth and stimulate apoptosis in UL. On the contrary, the SERPINE1 gene is found in the normal myometrium associated with fibroids, and there is also a decrease in its expression in MM tissues. Thus, there is an assumption that CCND1 is involved in angiogenesis and UL growth [23].

Epigenetic regulation role: signaling pathways

Signaling pathways that are involved in the growth and proliferation of uterine fibroids can be used as therapeutic targets. Changes in signaling pathways are critical for the development of uterine fibroids. Signaling pathways are various growth factors; SMAD-protein; phosphatidylinositol-3-kinase (PI3K); mammalian rapamycin target (mTOR) – PI3K/AKT/mTOR. Genome reprogramming is observed when the microenvironment changes under the influence of inflammatory factors. For example, cytokines can cause symptoms associated with uterine fibroids, but the current research focuses on their role in UL growth. Various cytokines are involved in the pathogenesis of UL. TNF-α and TGF-β are among the main factors determining clinical manifestations. TGF-β regulates the expression of antifibrotic micro-RNAs. From the conducted studies, it has become known that miRNA-21 enhances the synthesis of excess ECM by Smad7 protein. Meanwhile, the increased expression of micro-RNA-26a causes the deposition of collagen also through proteins of the Smad group.

Other authors believe that karyotypic changes are of secondary importance in the development of fibroids, regardless of whether they are primary or occur during the clonal development of UL. It is assumed that the induction of genetic or epigenetic damage is caused by previous genetic stimuli, conditions, or damage, that is, acquired genetic changes can be regarded as secondary, which is a potentiating factor [1]. The molecular classification of UL is based on mutations of “leading genes” (MED12, FH, HMGA2). However, some ULs do not fit into any category or fit into several at once. This demonstrates the need for further study of genetic subtypes and the creation of a more accurate classification.

Along with the already established genetic stratification into four main subtypes, epigenetic dysfunction is an important mechanism in the violation of gene regulation in the pathogenesis of UL [23]. The main mechanisms of epigenetic regulation include DNA methylation, modification of histones, micro-RNAs, and long non-coding RNAs (lncRNAs). microRNAs are small (<22 nucleotides), non-coding RNA molecules that regulate post-transcriptional gene expression by inhibiting them. The predicted target genes of these microRNAs modulate processes that may play an important role in the pathophysiology of uterine fibroids, including cell proliferation, apoptosis, and ECM synthesis. Therefore, UL growth may be associated with epigenetic abnormalities caused by adverse environmental effects. It has been shown that a number of tumor suppressor genes are abnormally hypermethylated in UL when compared with normal myometrium, collagen-forming and regulating genes, and a subset of estrogen receptor (ER) genes. Twenty-two genes, including COL4A1, COL6A3, GSTM5, NUAK1, and DAPK1, have a specific sequence of response to ER. Others have noted that a decrease in HDAC6 expression leads to a low expression of ERa in UL cells, resulting in suppression of proliferation. Thus, the authors suggest that histone acetylation may modulate estrogen receptors. It is known that 46 microRNAs are expressed differently in comparison with normal myometrium. Among these, 19 have increased expression in UL, and 27 have suppressed expression. The expression of miRNA 21, 34a, 125b, 139, and 323 was confirmed using real-time PCR diagnostics. The same group of researchers noted that the expression of members of the miRNA-29 family (29a, 29b and 29c) was suppressed in UL compared to normal myometrium in vivo. They claim that the differential expression of microRNAs contributes to the excessive synthesis of the ECM, which is observed in UL [7].

Epigenetic regulation role: microRNA

The expression of miR-29c in the leiomyoma is significantly reduced, but the expression of TGF-β3 is increased compared to normal myometrium. miR-29c levels are suppressed in myomas compared to myometrium and are controlled by ovarian steroids, transcription factors SP1 (protein specificity 1), NF-kB (kappa light chain nuclear factor-enhancer of activated B cells) and epigenetic regulation. Taken together, these studies indicate a very important role of the miR-29 family in the pathogenesis of fibroids. The analysis showed that the expression of miR-29c was reduced, while the expression of TGF-β3 at both mRNA and protein levels was increased in leiomyomas compared to normal myometrium. The regulatory interaction between miR-29c and TGF-β3 provides a direct communication mechanism that can explain the growth of leiomyoma due to the accumulation of ECM and is an ideal therapeutic target for interrupting the growth of fibroids [24].

It has recently been demonstrated that the signaling pathway regulating apoptosis and cell proliferation, TAZ and YAP, play a key role in tissue fibrosis and activation of myofibroblasts. This pathway controls not only the transition from epithelium to mesenchyma but also the transition of epithelium/fibroblast to myofibroblast, which is characterized by the expression of α-SMA and active ECM synthesis [25].

Results and discussion

Therefore, the research is ongoing to study the prediction of microRNA functional targets by integrative microRNA modeling in order to experimentally identify genes that are suppressed by 25 microRNAs. This RNA-seq dataset is combined with publicly available micro-RNA target binding data to systematically identify micro-RNA targeting features that are characteristic of both micro-RNA binding and target suppression. By integrating these common features into the machine learning framework, an improved computational model for genome-wide prediction of the target of micro-RNA is formed. Computational analysis of goals requires high-quality data not only to identify relevant target functions, but also for proper evaluation and the ability to combine these functions to build final forecasting models. It should be noted that CLIP (crosslinking and immunoprecipitation — sequencing of cross-binding and immunoprecipitation) is able to identify transcript targets associated with the functional complex of micro-RNA-induced silencing (RISC). In addition to CLIP-seq, another popular strategy for target analysis is the identification of downregulated transcripts resulting from overexpression of micro-RNA. Targets identified in this way are more likely to be functionally relevant, which implies a significant suppression of expression. However, there are also concerns about the micro-RNA overexpression strategy, as it is often difficult to distinguish direct microRNA targets from indirect targets (that is, genes that are indirectly suppressed due to the suppression of direct targets). Another problem is that some targets identified by overexpression of miRNA in cell culture may be physiologically insignificant. In addition, the analysis of overexpression of micro-RNA is also severely limited due to the lack of high-quality profiling data of the entire transcriptome. In particular, most existing datasets are small-scale, focusing only on a few micro-RNAs in any single study, and therefore are not ideal for training a general goal prediction model. Although data from several small studies can be combined, significant heterogeneity between different experiments presents a serious problem for accurate modeling purposes. Despite the aforementioned problems, microarray data from micro-RNA overexpression studies have proven valuable for target analysis and have been used to train several widely used target prediction models [26].

Conclusion

Further work on the identification of microRNAs that are involved in the pathogenesis of UL may inspire the creation of new pathogenetic treatments. Such treatment is especially relevant for those groups of patients of reproductive age for whom surgical treatment may be ineffective. Targeted treatment can also prevent the recurrence of UL, hence the need for repeated surgical intervention. Thus, further study of the relationship between the processes of proliferation and apoptosis in various variants of proliferative diseases of the uterus, along with their regulating mechanisms, seems appropriate and useful for understanding the subtle links of the pathogenesis of isolated and combined proliferative processes in the uterus, which will allow personalizing pathogenetically based therapeutic tactics.