Roles of microRNAs in Regulating Apoptosis in the Pathogenesis of Endometriosis

Endometriosis is a gynecologic disorder characterized by the presence of endometrial tissues outside the uterine cavity affecting reproductive-aged women. Previous studies have shown that microRNAs and their target mRNAs are expressed differently in endometriosis, suggesting that this molecule may play a role in the development and persistence of endometriotic lesions. microRNA (miRNA), a small non-coding RNA fragment, regulates cellular functions such as cell proliferation, differentiation, and apoptosis by the post-transcriptional modulation of gene expression. In this review, we focused on the dysregulated miRNAs in women with endometriosis and their roles in the regulation of apoptosis. The dysregulated miRNAs and their target genes in this pathophysiology were highlighted. Circulating miRNAs as potential biomarkers for the diagnosis of endometriosis have also been identified. As shown by various studies, miRNAs were reported to be a potent regulator of gene expression in endometriosis; thus, identifying the dysregulated miRNAs and their target genes could help discover new therapeutic targets for treating this disease. The goal of this review is to draw attention to the functions that miRNAs play in the pathophysiology of endometriosis, particularly those that govern cell death.


Introduction
Endometriosis is a benign disorder affecting 5-10% of women of reproductive age. It is characterized by the presence of endometrial glands and stroma on ectopic locations such as the pelvic cavity, ovaries, and rectovaginal septum [1,2]. It is an estrogen-dependent chronic inflammatory condition associated with pelvic pain, dyspareunia, dysmenorrhea, and infertility [3,4]. The incidence of the disease is as high as 35-50% in the general population, with symptoms of pain and infertility. Still, the disease remains underdiagnosed, and it takes a long time for a diagnosis to be made [5]. The prevalence of endometriotic disorders peaks between 25 and 35 years of age [6,7]. However, about 62% of endometriosis cases were detected among adolescents, confirmed by laparoscopy with the manifestation of chronic pelvic pain, which occurred cyclically or acyclic, as reported by Laufer et al. [8]. Meanwhile, cancer antigen-125 (CA-125) is one of the most investigated blood biomarkers, with an adequate evaluation of the test accuracy. Although this method is minimally invasive, cost-effective, and readily available compared to surgery, the effectiveness of these biomarkers remains to be assessed, since there is still insufficient evidence to determine their efficiency. Moreover, these biomarkers do not appear to be as effective as diagnostic surgery for application in clinical settings [9]. For women with endometriosis-associated pain, medical or surgical treatment can be adopted with oral contraceptives to be the first-line treatment [10]. Pain-dependent progestin therapy is better suited for its variable efficacy, as reviewed by Abdul Karim et al. [11]. Oral progestins such as norethisterone acetate ation in the gene expression for cell adhesion [38] and invasion [39] resulted in functional changes in the eutopic tissues and contributed to the ectopic implantations. The regulation of the genes is influenced by a small, non-coding RNA, microRNA (miRNA). Multiple human reproductive tract diseases such as preeclampsia [40], endometrial adenocarcinoma [41], uterine leiomyomata [42,43], ovarian cancer [44], and endometriosis [21,[45][46][47], have documented the irregular expression of miRNA. Dysregulated miRNAs may contribute to the mechanism underlying the pathogenesis of endometriosis [46,48].
Many studies reported miRNA expression in endometriosis, but only a small group are well-characterized. Thus, there is a lack of understanding of the endometriosis pathophysiology pathway and the involvement of miRNAs in pathogenesis. In this review, we summarize the findings that evaluated the altered miRNA expression in the ectopic and eutopic endometrial tissue of women with endometriosis and the healthy controls and the potential of circulating miRNAs as biomarkers of the disease. In addition, this review also focuses on apoptosis as the pathophysiology pathway that impacted endometriosis.

microRNAs
microRNAs (miRNAs) are naturally occurring, small, non-coding RNA fragments that regulate various cellular processes, including cell proliferation, differentiation, and apoptosis [49]. A single miRNA molecule may affect different mRNA targets traversing several cellular pathways [50]. From miRNA sequences identified in 271 organisms, there are 38,589 hairpin precursors and 48,860 mature miRNAs (miRBase.org, updated March 2018, Release 22) [51,52]. These miRNAs act by suppressing gene expression posttranscriptionally by base pairing to the 3 untranslated region (3 UTR) of the target mRNAs [53]. microRNA is believed to act as a gene repressor by targeting mRNA cleavage and translational repression [54]. Therefore, if a miRNA is upregulated, its functional targets should be downregulated due to the increased miRNA binding to its 3 UTRs. Alternatively, if a miRNA is downregulated, it will upregulate its functional target mRNA.
The biogenesis mechanism of miRNA is shown in Figure 1. The majority of miRNAs are embedded in intronic regions of protein-encoding genes [55,56] and are transcribed in the nucleus as the primary miRNA (pri-miRNA) by RNA polymerase II [57]. Further maturation processing of pri-miRNA utilizes endonucleases, Drosha [58], and cofactor DCGR8 into 70nt hairpin pre-miRNAs [59]. The pre-miRNAs were exported into the cytoplasm through exportin-5 [60] and Dicer cleaved the pre-miRNAs into a mature miRNA-miRNA duplex [61,62], and the helicase unwound them into single-stranded transcripts. The mature single-stranded miRNAs will guide the RNA-induced silencing effector complex (RISC) to complementary mRNA sequences [46], which results in the degradation of target mRNA or the inhibition of translation. Several studies indicate that miRNAs can be transported into systemic circulation in exosomes or microvesicles, which can be incorporated into distant cells [63]. microRNAs seem to be highly stable in circulation, and their plasma profiles change markedly depending on diseases, thus having potential as disease biomarkers [64].

Dysregulated miRNAs in Endometriosis
The endometrial profiling studies of miRNAs involved in the pathogenesis of endometriosis usually compare the expression levels between all tissues. The most common method is to compare the eutopic endometrium, endometrial lining inside the uterus, and ectopic endometrium from the same patient. Still, some studies compare the eutopic endometrium of the patient with endometriosis and the healthy controls [24]. Several studies have shown that many miRNAs are involved in endometriosis, and their expression is altered in eutopic endometrium and ectopic endometrium of the same women with endometriosis [21,46]. Therefore, significant miRNAs are expected to inhibit gene expression, as proinflammatory and immune responses, angiogenesis, cell cycle progression, and adhesion molecules are expressed differently in eutopic and ectopic endometrial tissue of women with endometriosis.
Using TARGETScan and PICTAVERT, Filigheddu et al., (2010) predicted the target mRNAs of the differentially expressed miRNAs, resulting in more than 3000 predicted targets. The functions of each predicted target were then mapped, and the molecular pathway was identified using Ingenuity Pathways Analysis (IPA 7.5) [21]. The molecular networks corresponding to biological functions associated with endometriosis (cell proliferation, cell cycle, cell motility, and reproductive system disorders) represented in IPA results were highly significant [21,24].
The molecular network converging on estrogen receptor 1 (ESR1) includes DNA methyltransferases (DNMT3A and DNMT3B) that are validated targets of miR-29b; miR-29c; and miR-29b, miR-29c, and miR-148a, respectively. These DNA methyltransferases were expressed differently in endometriosis, and there may be abnormal methylation of HOXA10 and the progesterone receptor PR-B that affects gene expression [21]. In addition, a validated target of the miR-200 family known as Smad Interacting Protein 1 (SIP1), a factor involved in epithelial-to-mesenchymal transition (EMT) and tumor metastasis [72]. In endometriotic lesions, an observed downregulation of the miR-200 family may play a role in the development of ectopic tissue.
In the Hawkins et al., (2011) study, the expression of miR-29c was upregulated, and the overexpression led to a blunted decidualization response [65]. miR-29c affects the different extracellular matrix gene expressions in the endometrial stromal cells. It has been suggested that miR-29c is a progesterone-responsive miRNA, as it is upregulated in the mid-secretory endometrium [73]. miR-34c-5p was significantly downregulated in the early secretory endometrium of women with endometriosis, and miR-193a, miR-29c, miR-203, and miR-200c were downregulated in the late proliferative endometrium [65]. Shen et al., (2013) demonstrated an increase in Steroidogenic Factor 1 (SF-1) mRNA expression in eutopic and ectopic endometrium, and this was associated with reduced levels of miR23a and miR23b. These miRNAs may function as negative feedback regulators to mediate the posttranscriptional regulation of estrogen signaling pathways, resulting in reduced SF-1 mRNA expression in the corresponding tissues [68].

microRNAs Expression in Eutopic Endometrial Tissue
Studies that demonstrated the altered miRNAs expression in the eutopic endometrial tissue from women with endometriosis are listed in Table 2. Six miRNAs were found to be downregulated (miR-9, miR-9*, miR-34b*, miR-34c-5p, and miR-34c-3p, and miRPlus_42 780) when eutopic endometrium from women with endometriosis and without endometriosis was compared [45]. miR-9 was the most significantly dysregulated miRNA in the microarray dataset, and qRT-PCR showed a significant reduction in women with endometriosis. One of the miR-9-predicted targets is BCL2, a gene encoding an antiapoptosis protein and were found to have higher expression in endometriosis patients. Three members of the miR-34 family were downregulated in the endometriosis patients. The downregulation of miR-34c and miR-34b in women with endometriosis may regulate progesterone resistance and enhance proliferation and ectopic tissue survival [45]. In addition, Corney et al., (2007) concluded that miR-34b and miR-34c act as mediators of the p53-dependent suppression of proliferation [74].  [79]. The overexpression of miR-196a in the eutopic endometrium inhibited the expression of the progesterone receptor and progesterone receptor B. Additionally, miR-196a upregulates MEK/ERK signaling by downregulating PGR expression in the eutopic endometrium of women with minimal or mild endometriosis [79]. The upregulation of miR-21 was found in patients with severe endometriosis after comparing miRNA expression in the eutopic endometrium from mild endometriosis and severe endometriosis [75]. Therefore, the predicted targets for miR-21 are the tumor suppressor genes PTEN, PDCD4, E2F1, and TGFBRII [80].
We observed limited concordance in the dysregulated miRNA expression identified by these studies. This could be due to the variation of clinical studies, the different locations of tissue biopsy and the experimental design, which may contribute to no or little overlap between miRNAs found in the studies. There is no overlap of miRNAs between the studies that compared the expression of miRNAs in eutopic endometrium of women with endometriosis to the controls.

Circulating miRNAs as Diagnostic Biomarkers in Endometriosis
The gold standard for diagnosing endometriosis is laparoscopic visualization of endometriotic lesions with histological confirmation of the lesion [81]. However, most women who suffer from endometriosis experience a diagnostic delay of 6-12 years, because this delay is usually associated with the disease's severity [82]. There are still challenges in proposing a reliable, noninvasive diagnostic biomarker for endometriosis, so serum/blood samples of miRNAs will reduce the surgical risk and may allow for early treatment of the disease. In addition, there is the potential for serum miRNAs to be used as biomarkers of disease progression, since there is evidence of dysregulated miRNAs in the blood of women with endometriosis [49]. Six studies in this review examined the circulating miRNAs using high-throughput assays of either serum [83][84][85] or blood/plasma samples [86][87][88] from women with or without endometriosis (Table 3). These studies showed a slight overlap of miRNAs expression across studies.  Two studies confirmed the let-7 family was dysregulated in the serum of women with endometriosis [83,88]. Let-7 promotes cell differentiation and may act as a tumor suppressor. Meanwhile, let-7b is associated with cyclin D1, an estrogen-regulated gene involved in cell proliferation [83]. Another study showed the downregulation of let-7, which targets the KRAS gene. The loss of this miRNA may initiate the progression of ectopic lesions. It may represent a link between inflammation and tumor-like growth in endometriosis [88]. Moreover, twenty-seven miRNAs were downregulated in a study comparing plasma samples from women with and without endometriosis [86]. miR-17-5p and miR-20 downregulation cause upregulated mRNA targets BCL2 and cell cycle repressor cyclin-dependent kinase inhibitor 1A, which enhances cell survival and represses cell proliferation [86]. The downregulation of miR-17-5p/20a is also associated with the upregulation of hypoxia-inducible transcription factors (HIF-1a) [89] and vascular endothelial growth factor (VEGF-A) [90], which contributes to neo-angiogenesis.
Serum miR-199a-5p [84] and miR-370-3p [85] were downregulated in women with endometriosis, affecting cell proliferation. miR-370-3p targets SF-1, a gene regulator involved in estrogen biosynthesis; thus, the overexpression of SF-1 will increase local estrogen production [85]. The expression levels of miRNA may differ according to the time of the day when the blood sample is collected [91]. Thus, there is a challenge in the consistency of the studies investigating the expression levels of miRNAs. The heterogeneity of the patient population, the timing of the sample collection, and the menstrual cycle may be confounding factors in the inconsistency of these studies; thus, standardization in sampling design and bioinformatic assessments of miRNAs assays are required to ensure an improvement in identifying diagnostic markers involved in miRNAs for endometriosis.

Apoptosis
Apoptosis is a form of programmed cell death that eliminates cells from the tissue without evoking an inflammatory response [92]. The three phases of the programmed cell death cascade are signal activation triggering apoptosis, regulation and execution, and cellular structural modifications [92]. During the menstrual cycle, apoptosis is essential in regulating cellular homeostasis through the elimination of cells from the functional layer of the uterine endometrium, and it is detected in the glandular epithelium of the late secretory and menstruating phase [93,94]. Bcl-2 family and Fas/FasL are known to be involved in apoptosis in normal endometrium, with Bcl-2 exhibiting an inhibitory effect on apoptosis during the proliferative phase while Fas showed peak expression during the secretory phase in the menstrual cycle and initiate the caspase cascade by activating caspase-8 [95,96]. The activation will trigger the death pathway and cause apoptosis in the endometrium. Other genes in Bcl family are Bax and Bak which have a proapoptotic function which peaks during the secretory phase [97].
There is evidence of a reduced level of spontaneous apoptosis in the ectopic endometrium compared with the eutopic endometrium of women with endometriosis [93].
Furthermore, for the first time, Gebel et al., (1998) displayed a significant reduction of apoptosis in the eutopic endometrium of women with endometriosis compared to healthy controls [93]. Thus, it is hypothesized that the development of endometriotic lesions may be due to the reduced sensitivity of endometrial tissue to apoptosis.

microRNAs Regulate Apoptosis in Endometriosis
Ten studies from 2016 to 2022 on the dysregulated miRNAs that modulate apoptosis in endometriosis were found and listed in Table 4. These studies used different cell lines to represent endometriotic cell lines to study the effect of overexpression or knockdown of microRNAs on the target genes expressed. The miRNAs were downregulated, while the target genes were upregulated; these modifications may contribute to the antiapoptotic behavior of the endometrial stromal cells, thus causing the progression of endometriosis.
miR-2861, known to induce apoptosis, was found by Yu et al., (2019) to be significantly downregulated in ectopic endometrial tissues when compared to the eutopic endometrial tissue of healthy controls. miR-2861 markedly inhibited ectopic endometrial cell proliferation and induced apoptosis, implying its inhibitory effects on endometriosis [71]. This study confirmed that STAT3 and MMP2 were the targets of miR-2861 in cell proliferation and apoptosis [71]. For women with endometriosis, Yoo et al., (2016) found that the activation of STAT3 may be necessary for the development of the inflammatory phenotype of eutopic endometrium [98], and the aberrant activation of STAT3 signaling could contribute to the pathogenesis of endometriosis [99]. Thus, the downregulated miR-2861 may contribute to the upregulation of STAT3 and MMP2, thereby promoting cell proliferation and inhibiting apoptosis of the ectopic endometrial cells. STAT3 was found to be targeted by both miR-2861 [71] and miR-424-5p, and their downregulation could inhibit apoptosis in endometriosis. Downregulation of miR-33 in ectopic endometriosis increases VEGF and matrix metalloprotein (MPP) expression and results in endometrial cell apoptosis [108]. MMP has been implicated in the conversion of TNF-α and FasL to active soluble forms, suggesting that these molecules can activate or release the factors involved in the apoptotic process [109]. In endometriotic lesions, the expression levels of MMP-2, MMP-9, and Mt1-MMP are significantly higher than a normal eutopic endometrium [110], implying that endometriotic tissue has a greater capacity for invasion. Thus, the higher expression of MMP9 and MMP2 from the previous studies may contribute to the inhibition of apoptosis of endometrial stromal cells in endometriosis. In the study of Gao et al., (2019), the eutopic endometrium of endometriosis patients and cell lines had considerably lower expression levels of miR-451 than the controls. The expression of miR-451 and the ASRM stage of endometriosis were not shown to be significantly correlated [102]. Cell growth is inhibited, and apoptosis is induced when miR-451 is overexpressed in endometriosis cell lines. Bioinformatic research of the GSE7846 dataset revealed the possible target genes for miR-451 to be YWHAZ, OSR1, TTN, and CDKN2D. Joshi et al., (2015) discovered the relationship between the expression of YWHAZ and miR-451 by encouraging the growth of ectopic cells in baboons with endometriosis [111]. In the study by Hirakawa et al., (2016), it was discovered that the hypermethylation of the miR-503 gene caused overexpression of this miRNA, which inhibited cell proliferation, VEGF-A production, extracellular matrix (ECM) contraction, and angiogenic activity of ECSCs, as well as induced apoptosis activity and G0/G1 cycle arrest of these cells. Since cyclin D1 was discovered to have a negative correlation with the expression of miR-503 and to inhibit cell proliferation, it was established that Cyclin D1, Bcl-2, and VEGF-A are direct targets of miR-503 in ECSC [101]. As an antiapoptotic factor with an elevated expression in ECSC, Bcl-2 may be a target for miR-503, which causes endometriosis cells to die by downregulating Bcl-2 expression. According to Ma et al., (2019), the overexpression of miR-142-3p inhibits the proliferation and migration of CRL-7566 cells and causes a decrease in miR-142-3p expression in ectopic endometrial tissues [104]. KLF9 is miR-142-target 3p's gene, and this miRNA causes apoptosis via encouraging KLF9-mediated autophagy. By directly controlling the production of vascular endothelial growth factor A (VEGFA), KLF9 also encourages angiogenesis. The overexpression of miR-142-3p inhibits cell proliferation, as evidenced by a decrease in the proportion of cells in the S phase and an increase in the proportion of cells in the G1 phase in CRL-7566 and ECSC. MiR-142-3p controls endometrial cell invasion and migration, and it lowers VEGFA expression, which prevents vascularization. MiR-142-3p targets KLF9, and the overexpression of this gene in CRL-7566 cells promotes the expression of Bax and p62 while suppressing the expression of Bcl-2 and LC3B.
As evidenced by Zhang et al., (2019), there is an increase in the expression of lentivirus miR-205-5p-transfected EC109 and EC520 endometriotic stromal cells relative to the negative controls, and miR-205-5p plays a suppressive role in endometriosis in vitro. Increased miR-205-5p expression decreases endometriotic stromal cells' capacity for cell invasion and migration while increasing apoptosis [105]. Through the AKT/ERK signaling pathway, the ANGPT2 levels were significantly decreased in overexpressed miR-205-5p EC109 and EC520 cells [103]. This study also concentrated on the relationship between miR-205-5p downregulation and ANGPT-2 upregulation and the severity of endometriosis progression. Through a multivariate analysis, they discovered a significant correlation, demonstrating their promising potential as endometriosis biomarkers. The overexpression of Sirutin 1 (SIRT1) and Bcl-xL (antiapoptotic marker) in the ectopic and eutopic endometrial tissues of women with endometriosis compared with normal endometrial tissues may suggest their association with the progression of endometriosis, as studied by Rezk et al., (2021) [106]. This study found there is a reduced expression of miR-34a and Fox0-1 in the ectopic and eutopic endometrial samples. This implies that miR-34a may play a role in regulating the expression of SIRT1 by the p53 pathway and subsequently regulating FoxO-1 expression in endometriosis tissue by decreasing apoptosis in the endometrium. Downregulated miRNAs that link to cell apoptosis in endometriosis are illustrated in Figure 2.

Abnormal microRNA Expression and Failed Decidualization and Their Association with Apoptosis in Endometriosis
Infertility or failed embryo implantation is one of the most prominent symptoms of endometriosis. According to Hu et al., (2018), endometriosis patients' serum samples had significantly lower expression levels of miR-370-3p compared to the controls. StAR, CYP19A1, and SF 1 downstream gene expression were shown to be regulated by miR-370-3p. According to the study, endometriotic cells that have miR-370-3p overexpression undergo apoptosis and have decreased cell growth. The endometrium's ability to decidualize, which results in infertility, is inhibited by SF-1, which also encourages aberrant uterine gland morphogenesis, changes the endometrial immune homeostasis, and causes a physiological inflammatory response [85].
This review has its strengths. The findings and discussion about the miRNAs and their target mRNAs that regulate cell apoptosis in endometriosis are comprehensive and robust. We also highlighted the circulating miRNAs that may give insight into the diagnosis and management of endometriosis. However, our review also has two limitations. First, we could not find suitable circulating miRNA biomarkers or common miRNAs that link to cell apoptosis in endometriosis. Second, the limited English full articles might contribute to the limitations of our findings.

Conclusions
microRNAs regulate various biological pathways involved in endometriosis by repressing the translation of the target mRNAs. They play a role in inflammatory and immune responses, cell adhesion, cell invasion, cell proliferation, angiogenesis, apoptosis, cell differentiation, and hormone synthesis. There are pieces of evidence of miRNAs in regulating the development and progression of endometriosis, which is important for future directions in the treatment of the disease. Many studies show different expression profiles of miRNA in endometrium tissue and endometrial lesions. A microRNA expression profile may therefore play a diagnostic role, as well as a prognostic factor for the efficacy of novel therapies.
In conclusion, the significance of miRNAs in the regulation of the development of endometriosis is well-known currently, but there are still many questions left to be answered. New research techniques and functional studies on the role of miRNA and their target mRNAs will be needed to improve our knowledge of the pathophysiological impact of these miRNAs in endometriosis. This will enable a more effective treatment and potential diagnostic biomarkers to be used as the future therapeutic targets for the disease.