Integration analysis of microRNA and mRNA paired expression profiling identifies deregulated microRNA-transcription factor-gene regulatory networks in ovarian endometriosis

Endometriosis is characterized by the presence of endometrial tissue (endometrial glandular and stromal) abnormally outside the uterine cavity [1]. In women of reproductive age, there is a 6–10% incidence rate, which may even reach 35–50% in women who are enduring pelvic pain and/or infertility [2, 3]. Despite its high prevalence, the etiology and pathophysiology of endometriosis are still not fully understood. It is widely accepted that endometriosis is a multifactorial, polygenic disorder [4]. A large amount of molecular aberrations exist between the ectopic endometrium (EC) (endometriotic lesions) and the eutopic endometrium (EU), which could potentially explain the mechanism of the abnormal growth of EU outside the uterus.

microRNAs (miRNAs) are endogenous, single-stranded, 18–22 nucleotide RNAs that mainly inhibit gene expression at the post-transcriptional level [5]. They negatively regulate the translation of target messenger RNAs (mRNAs) via the sequence specific recognition of the “seed sequence” and repress translation and/or degrade the target mRNAs according to the degree of complementary nucleotides [6]. Transcription factors (TFs) are a group of proteins that regulate gene expression by binding to specific DNA sequences at the transcriptional level [7]. Depending on its function, the transcription of an adjacent gene is either activated or repressed [8]. As two vital gene regulatory molecules, miRNAs and TFs can share common target genes and exert effects on each other [9]. As such, the miRNA expression levels can be regulated by TFs, and the mRNAs encoding TFs can be inhibited by miRNAs [10]. In addition, miRNAs and TFs can form a feed-back loop (FBL) or feed-forward loop (FFL) in cooperation to fine tune gene expression [11]. The combinatorial regulation of TFs and miRNAs is suggested to play vital roles in various biological processes and disease pathogenesis [12‐15].

Accumulating evidence suggests that aberrant miRNA and TF expression may be involved in the pathogenesis and development of endometriosis [16‐25]. Although differences in miRNA expression profiling between EC and EU from women with endometriosis have previously been reported, almost no previous studies have focused on the integrative analysis of miRNA-mRNA interactions and solved the complex problem of the identification of miRNA-TF-gene regulatory networks. With the aim of further understanding the abnormal molecule changes in the occurrence and development of endometriosis, we used high-throughput sequencing techniques to detect miRNA and mRNA expression profiling in paired EC and EU. Joint analysis of the miRNA-TF-gene regulatory network can help us survey the key genomic factors and understand the mechanism of endometriosis at the molecular level.

Several microarray studies were conducted to obtain DEMs between EC and EU from women with endometriosis [18, 46, 47], but overlapping data are limited, which suggests that the molecular changes caused by endometriosis might be more complex than we assumed. Additionally, miRNA target prediction methods based on sequence complementarity have been used to annotate the functions of DEMs in these studies, which may contain a high false positive rate and false negative rate [48]. Therefore, the functions of most of the identified DE miRNAs in endometriosis remain unclear due to a lack of definite target genes and confounding downstream validations. Network analysis allows structured grouping of miRNAs, TFs and target genes; thus, network construction is essential for identifying critical molecular changes and understanding their functional roles. To our knowledge, this is the first study to establish concomitant miRNA-TF-gene binary interactions and regulatory networks between the EC and the EU from patients with endometriosis. At first, our study revealed 107 DEMs and 6112 DEGs by sequencing. The large differential expression profiling was refined by PCC to 22 functionally DE miRNAs, 12 TFs and 430 target genes. The network included some well-studied candidate miRNAs and genes (e.g., miR-183-5p, FOXO1, and VEGFA) but also identified many novel objects (e.g., the miR-449 family and miR-106a-363 cluster). Here, we discuss these key regulators in the network, with the hope of providing valuable information for understanding the molecular mechanism of endometriosis.

The highly conserved miR-449 family encoded by the CDC20B gene consists of three members: miR-449a, miR-449b, and miR-449c. Accumulating evidence suggests that the miR-449 family plays an important role in cell proliferation, migration and invasion and might serve as a tumor suppressor in miRNAs [49]. Consistently, miR-449 members were absent or down-regulated in various human malignancies, such as liver cancer [50], breast cancers [51], lung cancer [52], and ovarian clear cell carcinoma [53]. In the current study, we reported the first significant down-regulation of miR-449a, miR-449b-5p, and miR-449c-5p in ectopic endometrial tissue. Considering that endometriosis shares many characteristics with malignant diseases, such as invasion and immigration, it is very likely that the lower expression of miR-449 plays an important role in the pathogenesis of endometriosis. miR-34b and miR-34c are classified into the same cluster and share related functions with miR-449 due to their structural similarities [54, 55]. Much attention has been focused on miR-34 therapeutics for cancer [56, 57]. In addition, miR-34 mimics have been used in several preclinical studies and a phase I clinical trial, and they have shown potential as anticancer therapeutics [58]. Our study identified the down-regulation of miR-34b-3p, which is consistent with the results from Teague et al. [16]. Moreover, we also identified two down-regulated miRNAs, namely, miR-34b-5p and miR-34c-5p, which have been reported by Burney et al. in the eutopic endometrium compared with normal endometrium [17]. Their results suggested that down-regulation of miR-34b and miR-34c was involved in the initiation and maintenance of endometriosis. Given the therapeutic role of miR-34 in cancer and its structural similarity to miR-449 [54], mimics from the miR-449 and miR-34b/c cluster could also be potential miRNA replacement therapies in endometriosis. We therefore suggest that further studies focus on both the miR-34 and miR-449 families by analyzing their function and target genes in endometriosis.

The miR-200 family is another important set of miRNAs that was down-regulated in the present study and has been reported to be reduced in many solid tumors [59]. The miR-200 family contains two groups, miR-200a/b/429 and miR-200c/141, that regulate a cohort of target genes involved in epithelial-mesenchymal transition, metastasis and angiogenesis [60], which are important events in the pathogenesis of endometriosis [61]. Previous studies revealed deregulated miR-200 family members in endometriotic lesions compared with eutopic endometrium [16, 18] and in eutopic endometrium compared with normal endometrium [19]. Circulating miR-200a and miR-141 have also been proposed as candidate biomarkers for endometriosis [62]. The present study is not the first to identify lower levels of miR-141-3p, miR-200a-3p, miR-200b-3p and miR-200c-3p in endometriosis. However, our report confirmed the previous results and identified a series of corresponding genes of these miRNAs, which may be involved in many physical or pathological processes in endometriosis. For example, JAZF1, which is regulated by miR-200b/c, is a proto-oncogene known to be affected by chromosomal translocation in endometrial stromal tumors [63]. In the study by Yotova et al., JAZF1 was hyper-methylated and down-regulated in stromal cells from ovarian ectopic endometria compared with normal endometria [25]. As another example, TP73, which is regulated by miR-200b, is part of the TP53 family and plays a role in the cell cycle, apoptosis and infertility [64], and no previous research has revealed its functions in endometriosis.

miR-363, miR-20b, and miR-106a are transcribed from the miR-106a-363 cluster [65]. All of these miRNAs were down-regulated in ectopic lesions according to our results. Intriguingly, these miRNAs have dual functions either as tumor suppressors or as oncogenic miRNAs. A high level of miR-363-3p suppresses the proliferation of human hepatocellular carcinoma cells [66] and decreases the metastasis of neuroblastoma cells [67], whereas the knock-down of miR-363-3p was reported to suppress carcinogenesis in gastric cancer cells [68]. Li et al. demonstrated that miR-20b acts as an oncogene by contributing to breast tumorigenesis [69], but Hong et al. found that miR-20b was markedly down-regulated and served as a tumor suppressor miRNA in papillary thyroid carcinoma [70]. Li et al. identified increased miR-106a as playing an oncogenic role in pancreatic cancer [71]. In contrast, Zhi et al. reported that miR-106a could inhibit proliferation and promote apoptosis in astrocytoma cells [72]. As no study has previously reported the functions of miR-363-3p, miR-20b and miR-106a in endometriosis, we infer that the decreased expression of these three miRNAs might have tumor suppressive-like functions in the initiation, progression, and metastasis of endometriotic lesions. Additionally, many corresponding target genes, such as ADAMTS family members, which are also known tumor suppressor genes, are down-regulated, consistent with the present study, which confirmed our inference indirectly.

miR-182-5p, which belongs to the miR-183/96/182 cluster, contains the highest node degree in the identified DEMs and shows a 5.3-fold down-regulation in EC versus EU. This finding is consistent with the results obtained from the microarray screening and qRT-PCR validation by Filigheddu et al. [18] Similar to miR-182-5p, miR-183-5p expression in ectopic endometrium was 5.7-fold down-regulated in our study and was confirmed by previously published reports [47, 73]. Additionally, functional analysis indicated that the down-regulation of miR-183 can inhibit apoptosis and enhance the invasive ability of endometrial stromal cells [47]. These findings indicate that aberrant miR-182/183 expression is part of the epigenetic mechanism of pathogenesis and the development of endometriosis.

Based on the integrative regulatory network, a series of TFs also attracted our attention, including FOX family members, GATA family members, and E2F family members, as well as CEBPA, SOX9 and HNF4A. FOX (fork-head box) proteins are a family of transcription factors involved in many pathologic and physiological processes, such as cell growth, proliferation, and longevity [74]. The FOX subfamilies, such as FOXO, FOXC and FOXP, play different roles in different types of diseases [75]. Here, we identified four important FOX subfamily members, FOXO1, FOXC1, FOXL1 and FOXJ1, which cooperatively regulate the integrative networks in endometriosis. FOXO1 serves as an anti-oncogene in various malignancies through diverse mechanisms, such as promoting apoptosis and facilitating DNA repair [76] and was decreased by 3.5-fold in endometriotic tissue in the present study. Previous data showed the same trend with lower FOXO1 levels expressed in endometriosis, which might contribute to an overactive PI3K/AKT pathway, but the underlying mechanisms are not well understood [77]. Additionally, two FBLs, miR-182-5p/FOXO1/NUAK1 and miR-182-5p/FOXO1/SCN9A, were observed and their functions are expected to be explored in the future. In recent years, an elevated expression of FOXC1 has been detected in different tumors, such as esophageal cancer [78], hepatocellular carcinoma [79] and osteosarcoma [80]. The current views on its function and molecular mechanisms reveal that FOXC1 as an oncogene plays multiple roles in cancer progression and metastasis by affecting different targets [78‐81]. The present study evaluated FOXC1 expression by high-throughput sequencing and qRT-PCR, and both results indicated that FOXC1 increased approximately sevenfold in EC compared with EU. Furthermore, FOXC1 was identified as a direct target of miR-133b and shares the same targets including EYA4 and LHFP.

GATA1, GATA2 and GATA6 belong to the GATA family, which mainly plays roles in morphogenesis and organogenesis [82]. Our study observed that GATA1 could increase the miR-202-3p expression level and act on 40 targets in the endometriotic lesion. Although the cause of the high expression of GATA1 is not yet clear, Hawkins et al. reported that miR-202-3p was up-regulated in EC compared to normal endometria [19]. Moreover, we suggest that GATA2 and GATA6, as target TFs of the miR-200 family, might be involved in the pathogenesis of endometriosis together with the miR-200 family.

E2F1 is the most important TF in the E2F family, which is known to integrate cell cycle progression with DNA repair, replication, and multiple checkpoints [83]. Interestingly, the function of E2F1 in tumors is a paradox; both positive and negative effects of E2F1 on tumorigenesis have been observed [84]. The present study showed down-regulation of the E2F1 target miR-449 family members, which suggests that their combination possibly initiates endometriosis. E2F2, another important family member of E2F, is also involved in regulating the cell cycle and is associated with cancer growth [85]. A previous study reported that E2F2 can promote the development of liver cancer [86] and can also inhibit the proliferation of prostate cancer cell lines [87]. In our joint analysis, we observed that the down-regulated E2F2 is consistent with a lower expression of miR-196b-5p. Before our research, Abe et al. found that miR-196b-5p was down-regulated in ectopic endometria compared with normal endometria by microarray. Their subsequent functional experiments revealed that miR-196b can inhibit proliferation and induce apoptosis in endometriotic stromal cells [21], which led us to hypothesize that E2F2 might also play important roles in endometriosis by targeting miR-196b.

CEBPA is an important TF controlling hematopoietic differentiation and homeostasis, gene mutation and aberrant expression, which may contribute to hematological system diseases [88]. We observed that highly expressed CEBPA in EC targets 49 corresponding genes and that like FOXO1, was targeted by miR-182-5p. In a recent study, Wang et al. reported that miR-182 can repress CEBPA in hepatocellular carcinoma and is involved in the AKT signaling pathway [89]. This negative correlation between miR-182 and CEBPA is consistent with our result; however, their specific mechanism and involvement in the downstream signaling pathway in endometriosis need to be further confirmed.

Notably, the integrative data revealed that HNF4A targets 12 miRNAs and thus might be a miRNA “hub” in endometriosis. HNF4A is a critical factor in liver function and digestive diseases [90]. The mechanisms of HNF4A and its target miRNAs have not been fully elucidated yet; it is known that HNF4A can directly up-regulate some specific miRNAs to stabilize the hepatocyte phenotype [91]. This unexpected finding requires further validation to identify the underlying mechanism mediated by HNF4A in endometriosis.

GO and KEGG enrichment analysis delineated 42 functional terms and 27 important signaling pathways, most of which are consistent with the current knowledge on endometriosis. For example, the most significant term and pathway are plasma membrane (GO: 0005886) and the PI3K-Akt signaling pathway, which enriched 145 and 17 DEGs, respectively. Ectopic lesions are exposed to a unique peritoneal microenvironment that is characterized by elevated levels of hormones, inflammation, oxidative stress and iron [92]. These abnormal molecules can influence cell survival and regulate cellular functions through binding to cell membrane receptors, and then lead to the subsequent cascading activation of kinase signaling pathways [93]. Therefore, the plasma membrane term and PI3K-Akt signaling pathway are clearly relevant for endometriosis. The PI3K-Akt pathway, which integrates a variety of extracellular signals and regulates various cellular functions, including cell growth, differentiation, transformation and survival, is being investigated as a therapeutic target in other diseases and thus may also represent a target for endometriosis treatment [94, 95]. In addition, our enrichment analysis revealed some novel dysregulated signaling pathways, such as the Rap1 signaling pathway and the retrograde endocannabinoid signaling pathway, and their functions should be further investigated.

Despite our novel results, the limitations of this study should also be mentioned. First, the number of sequenced samples was relatively inadequate, and only non-hormonal treated patients with moderate to severe stage disease within the secretory phase were enrolled. Endometriosis is a complex disease that is influenced by multiple factors, such as the menstrual cycle, CA125 levels and various subtypes. These differences might impact the expression profile. Although normalized samples are helpful to determine the key pathogenic molecules, our study was most likely underpowered in discovering DEMs and DEGs with subtle expression differences due to the limited sample size. Second, the present results are preliminary and descriptive. Integrative analysis of paired miRNA and mRNA profiling data cannot entirely exclude false positive results. In addition, only some of the identified miRNAs and TFs were validated by qRT-PCR. Therefore, other experiments, such as cross-linking immunoprecipitation or functional experimental validation, are needed to holistically validate the interactions identified by our method. Furthermore, the positive findings that were not included in the network might contain valuable information and are worthy of further discussion. Third, various computational methods have been devised to measure the miRNA-mRNA regulatory relationships in recent years, such as correlation analysis, regression models, Bayesian network learning and causal inference techniques [96]. However, which one is the best method remains an open question in practical use. Here, we used the typical Pearson correlation coefficient to analyze the relationship between DEMs and DEGs, which might lead to some differences from other methods and an inevitable loss of information.

In summary, we provide the first characterization of miRNA-TF-gene co-expression network in paired miRNA and mRNA expression profiling of endometriosis. By establishing the regulatory network in miRNA and mRNA expression profiling, our present study confirms and significantly extends the results of prior studies, thereby defining some crucial miRNAs, TFs and genes involved in the pathogenesis of endometriosis. The identified interactions could also have implications toward targeted therapeutic strategies for endometriosis. Further in-depth functional studies are encouraged to confirm our results.