The Role of miRNAs in the Regulation of Endometrial Cancer Invasiveness and Metastasis—A Systematic Review

Simple Summary Endometrial cancer (EC) is one of the most frequent cancers with increasing annual death rates. Therefore, it is of great clinical importance to understand the mechanisms of endometrial cancer invasiveness and metastasis. MiRNAs are small single-stranded RNAs that regulate gene expression. They were discovered to play a role in all steps of cancer development. This study aimed at conducting a systematic review of the role of miRNAs in endometrial cancer invasiveness and metastasis. The collected data demonstrate that miRNAs regulate EC invasiveness and metastasis by different targets. MiRNAs seem to be potential candidates for diagnostic and prognostic biomarkers, as well as possible therapeutic targets. Abstract Endometrial cancer (EC) is the most common genital cancer in women with increasing death rates. MiRNAs are short non-coding RNAs that regulate gene expression on the post-transcriptional levels. Multiple studies demonstrated a fundamental role of miRNAs in the regulation of carcinogenesis. This systematic review is a comprehensive overview of the role of miRNAs in the regulation of cancer cell invasiveness and metastasis in EC. The literature was searched for studies investigating the role of miRNAs in the regulation of invasiveness and metastasis in EC. We explored PubMed, Embase, and Scopus using the following keywords: miRNA, metastasis, invasiveness, endometrial cancer. Data were collected from 163 articles that described the expression and role of 106 miRNAs in the regulation of EC invasiveness and metastasis out of which 63 were tumor suppressor miRNAs, and 38 were oncomiRNAs. Five miRNAs had a discordant role in different studies. Moreover, we identified 66 miRNAs whose expression in tumor tissue or concentration in serum correlated with at least one clinical parameter. These findings suggest a crucial role of miRNAs in the regulation of EC invasiveness and metastasis and present them as potential prognostic factors for patients with EC.


Introduction
Endometrial cancer (EC) accounts for the most frequent cancers with growing incidence rates [1,2]. The outcome depends on the progression of the disease and applied treatment [3,4]. That makes effective management of EC risk factors, early diagnosis, and effective therapy strategies of EC the clinically important challenges. There are two most common EC staging classifications, TNM and The International Federation of Gynecology and Obstetrics (FIGO) that differentiate endometrial cancer tumors according to the depth of invasion and presence of metastases ( Figure 1) [5]. Furthermore, there is a FIGO grading based on the level of glandular differentiation. A higher grade is associated with a non-glandular, non-squamous growth [6]. MiRNAs are small non-coding single-stranded molecules that regulate all hallmarks of cancer by influencing gene expression post-transcriptionally. The formation of miRNAs begins in the nucleus where polymerase II (Pol II) transcribes pri-miRNA. The pri-miRNA is cropped by the DROSHA complex and exported by exportin 5 to the cytoplasm where mature single-stranded miRNAs arise with the participation of DICER and Argonaute 2 (AGO2) [7,8]. During carcinogenesis, the profile of miRNAs expression undergoes a substantial dysregulation [9,10]. It is a result of multiple changes, including amplification and deletion of miRNA genes or dysregulation of epigenetics [9]. Moreover, miRNAs expression is dysregulated in cancer as an effect of defects in miRNA biogenesis machinery, including DICER and DROSHA [11].
MiRNAs take part in all steps of tumor cell invasiveness and metastasis including migration, local invasion, epithelial-mesenchymal transition (EMT), and systemic circulation [12]. The same miRNAs may play opposite roles in different tumors, promoting tumor growth (oncomiRNAs) or acting as tumor suppressor miRNAs [13]. By targeting 3 untranslated region (UTR) of multiple mRNAs they regulate all hallmarks of cancer defined by Hanahan and Weinberg, including proliferation, invasion, angiogenesis, as well as they influence cancer cells chemoresistance [12,[14][15][16][17].
This systematic review aims to highlight the complex role of miRNAs in regulating endometrial cancer invasion and metastasis. We focus on the aberrant expression of different miRNAs in endometrial cancer tissues and cell lines and their role in the regulation of tumor invasion, metastasis, and patients' outcomes.

Search Strategy
The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) were used to ensure reporting transparency ( Figure 2). Two reviewers (KK and TMG) collected, screened, and performed an independent assessment of the quality of the studies.
Discrepancies were discussed and resolved by consensus. The literature systematic search was undertaken using MEDLINE (PubMed), Embase, and Scopus (9 March 2021) with the terms ('microRNA' OR 'miRNA') AND ('metastasis' OR 'invasiveness') AND ('endometrial cancer' OR 'endometrial carcinoma'). Duplicates were deleted. The references of the found studies were reviewed to find other records.

Inclusion and Exclusion Criteria
The articles were included if they answered the PICO question ( Table 1). The studies with the assessment of miRNA expression levels and the role of miRNAs in endometrial cancer cell invasion, migration, or outcome were allowed. We assessed only original articles written in English with full-text available. Review articles, chapters, conference abstracts, and retracted articles were excluded. Full texts were assessed for eligibility. We excluded articles that did not meet inclusion criteria, articles with no assessment of miRNAs function or expression in the endometrial cancer model, and no validation with RT-qPCR (real-time quantitative PCR) of miRNA expression.   [74,88] ↓-downregulated in endometrial cancer compared to normal cells, ↑-upregulated in endometrial cancer compared to normal cells, n/d-no data, *-inconsistent data of miRNAs expression.
Included studies involved established EC cell lines, including Ishikawa, HEC-1A, HEC-1B, HHUA, AN3CA, ECC-1, RL-95-2, KLE, SPAC-1-L, HEC-50, HOUA-I, and JEC cell line (EC cell lines have been reviewed by Van Nyen et al. [174]). Transient upregulation of a given miRNA with synthetic miRNA or downregulation with complementary anti-miRNA revealed regulation of tumor cell migration and invasiveness in vitro assays by 97 miRNAs. Further, luciferase reporter assays confirmed direct binding of 102 targets by miRNAs regulating invasiveness of EC cells creating a complex regulatory network. Tumor-suppressor miRNAs that are downregulated in EC were identified to bind 77 targets, including some of the well-known oncogenes. OncomiRNAs were identified to promote EC cell migration and invasiveness by binding 25 targets.

Relationship between miRNA Expression and Clinical Parameters
Further, we collected data from articles investigating the association between the expression of invasion-related miRNAs in tumor tissue and clinical parameters, including OS, DFS, PFS, FIGO stage, histological grade, myometrial invasion, and lymph node metastases. We identified 51 articles that correlated upregulated expression of 25 miR-NAs and downregulated expression of 41 miRNAs with at least one clinical parameter (Table 4)    ↓-decreased, ↑-increased, n/d-no data, a-lympho-vascular invasion, *-inconsistent data of miRNAs expression and clinical parameters, OS-overall survival, DFS-disease-free survival, PFS-progression-free survival, FIGO stage-The International Federation of Gynecology and Obstetrics staging classification.

Discussion
Tumor expansion and progression are enabled by coordinated dysregulation of various mechanisms. The process of tumor invasion and metastasis is composed of several steps, including primary tumor growth, migration, local invasion, intravasation, survival in the circulation, extravasation, and pre-metastatic niche formation. All of these steps are regulated by a variety of different miRNAs [12].
In this work, we provide a comprehensive overview of miRNAs that are dysregulated in EC and contribute to tumor progression. We identified 106 dysregulated miRNAs through a systematic literature review. Small RNA-seq analysis revealed that 239 out of 359 detected miRNAs are dysregulated in EC compared to healthy adjacent endometrial tissue [182]. Further, analysis of miRNA-seq data from The Cancer Genome Atlas (TCGA) database expanded the list of dysregulated miRNAs to 531 [179]. Previous systematic reviews identified, respectively, 106 miRNAs [183], 261 miRNAs [184], 310 miRNAs [185] dysregulated in ECs. However, these studies included also many miRNAs detected by high throughput methods, including microarrays and small RNA-seq, without confirmation by the RT-qPCR method that is a gold standard for miRNAs analysis [186]. Moreover, in contrast to previous reviews, we included only miRNAs with a confirmed role in the regulation of EC cell migration, invasiveness, and metastasis in vitro and/or in vivo.

Regulatory Network of Invasion-Associated miRNAs
MiRNAs orchestrate tumor invasion and metastasis by targeting various mRNAs. Complex regulation of multiple signaling pathways, including PI3K/AKT [187], and cellular processes, including EMT and cytoskeleton remodeling [188], by miRNAs enable the control of the invasiveness and metastasis of cancer cells. We classified targets of miRNAs into 15 categories based on their function. Notably, multiple miRNAs target regulators of multiple cellular processes and create a complex network of interactions. Hence, they have been assigned to a given category based on their best-described function in the EC (Figure 4).

Hormone Signaling
Dysregulation of hormone signaling is one of the most important factors in EC development. From them, estrogen seems to be crucial in EC pathogenesis. Estrogen binds to estrogen receptors (ER) or G protein-coupled estrogen receptor 1 (GPER1) to regulate gene transcription and to promote cancer cell pathways [210][211][212]. Estrogen signaling and miRNAs are in complex interaction since estrogen modulates miRNAs expression but also miRNAs target their receptors [213]. In this study, we identified miRNAs that suppress EC progression via binding to estrogen receptors. MiR-22 and miR-206 target ER what inhibits EC cell invasion and migration [121,156,168]. Moreover, miR-195 targets GPER1 and inhibits EC migration, invasion, and EMT in vitro [139]. On the other hand, miR-107-5p and miR-222-3p targeting ERa and miR-205 targeting Estrogen Related Receptor Gamma (ESRRG) act as oncomiRNAs by enhancing migration, invasion in vitro or/and tumor growth in vivo [19,52,68].

Apoptosis
Apoptosis is a process of programmed cell death via the activation of different pathways [215]. Novel therapies targeting apoptosis inhibit B-cell lymphoma-2 (Bcl-2), induced Myeloid Leukemia Cell Differentiation Protein 1 (MCL-1), or target the P53 pathway [216]. In this study, we identified tumor suppressor miRs and oncomiRNAs that target mRNAs of proteins involved in the regulation of apoptosis. Tumor suppressor miR-101 that targets MCL-1, a protein belonging to the Bcl-2 family that inhibits apoptosis, inhibits migration, invasion, and EMT in vitro and tumor growth in vivo, and influences patient survival. Moreover, we recognized invasion-suppressing miR-129 that targets Glycogen synthase kinase-3β (GSK3β), a serine/threonine-protein kinase that inhibits apoptosis via NF-κB activation [217]. MiR-106a targets proapoptotic Bcl-2-like protein 11 (BCL2L11) and acts as oncomiRNA and stimulates migration, invasion in vitro, and tumor growth in vivo [218].

Extracellular Matrix (ECM) Remodeling
Remodeling of ECM is crucial for a cancer cell to initiate an invasion of adjacent tissues. Among multiple factors regulating this process, the most important are Matrix Metalloproteinases (MMPs) and their inhibitors Tissue Inhibitors of Metalloproteinases (TIMPs). MMPs degrade collagen, fibrinogen, fibronectin (FN1), and others and in this way enable tumor cells to invade [12]. In this study, miR-200b targets TIMP-2, miR-200c targets FN1 and both miRNAs act as tumor suppressor miRNAs. On the other hand, miR-103 targets TIMP-3 and stimulates invasion. Moreover, oncomiRNA miR-183 targets MMP-9 and miR-544a targets Reversion Inducing Cysteine Rich Protein with Kazal Motifs (RECK). These results show the complexity of the regulatory processes involved in ECM remodeling.
One target may be regulated by several miRNAs, as well as miRNAs may have a role in the regulation of invasion by different mRNAs [219].

Janus Kinase/Signal Transducers and Activators of Transcription (JAK-STAT) Signaling
JAK-STAT pathway is a known regulator of tumor progression [220]. JAK-STAT pathway is dysregulated in endometrial cancer cells what leads to increased proliferation [221]. In this study, we identified three miRNAs (miR-20a-5p, miR-124, and miR-361) that target STAT3. MiR-20a-5p inhibits invasion and EMT [40]. MiR-124 decreases migration, invasion, and EMT in vitro and is associated with patients' outcomes [50,99]. Conversely, miR-361 inhibits migration and invasion in vitro and tumor growth in vivo, as well as is negatively correlated with histological grade [169].

Angiogenesis
Angiogenesis is one of the hallmarks of cancer according to Hanahan and Weinberg [14] playing a crucial role in the progression of EC. One of the most important proangiogenic factors is the Vascular Endothelial Growth Factor (VEGF) [229]. VEGF is often targeted in novel therapies including anti-VEGF antibody (bevacizumab), VEGF trap (aflibercept), or Tyrosine Kinase Inhibitors (TKI). Therapy with bevacizumab was approved by FDA in different tumors including colorectal cancer and is currently tested in EC [229,230]. An increased number of clinical trials are assessing the efficacy of that novel therapies in monotherapy as well as in a combination with other applied therapies but still lacks a personalized approach [229]. In this study, we found two miRNAs (miR-29a-5p and miR-545-3p) that target VEGF and thus influence EC progression. 4.1.13. Cyclic Adenosine Monophosphate (cAMP) Signaling cAMP pathways are described to inhibit the invasion of cancer [231,232]. cAMP promotes apoptosis and decreases tumor growth [233]. The cAMP-specific phosphodiesterase 7A (PDE7A) hydrolyzes cAMP [97]. MiR-1 and miR-133a target PDE7A which leads to the inhibition of EC cell migration and invasion in vitro.

The Role of miRNAs in EC Diagnosis and Management
There is a great clinical interest in the determination of biomarkers for the diagnosis and management of EC, especially to enable individualized cancer care in the light of genomic classification [234]. Despite the identification of many prognostic biomarkers in EC [235,236], none of them are routinely used for diagnostic or prognostic purposes. Recent systematic reviews and meta-analyses revealed the clinical utility for the use of miRNAs as biomarkers in a variety of cancer, including bladder cancer [237], prostate cancer [238], ovarian cancer [239], and breast cancer [240], which makes miRNAs promising candidate for biomarkers in EC ( Figure 5). Figure 5. miRNAs as diagnostic biomarkers and therapeutics. miRNAs are promising biomarkers that can be analyzed either from tumor tissue (1) or from blood obtained from EC patients (2). miRNAs may support EC diagnosis and indicate primary and adjuvant treatment. Moreover, their expression is an important prognostic factor. miRNA-based therapies that include tumor suppressor miRNAs and anti-oncomiRNAs are currently tested in preclinical and clinical trials (3). EVs-extracellular vesicles, FFPE-formalin-fixed, paraffin-embedded. Figure was created using Biorender.com. Importantly, dysregulation of miRNAs expression can be also detected in plasma or serum ( Figure 5). It was identified that the concentration of 19 miRNAs is increased in EC patients and the concentration of 10 miRNAs is decreased compared to healthy women [184]. Exploring publications we found that the concentration of six miRNAs was decreased and the concentration of 11 miRNAs was higher in EC patients and five from them (miR-27, miR-29b, miR-95, miR-203, and miR-449a) were associated with tumor myometrial invasion or lymph node metastases [60, 77,81,110].
MiRNAs are also promising therapeutics in clinical oncology. Among described in this study miRNAs regulating tumor invasiveness, the effectiveness and safety of miR-34 have been tested in clinical trials. Despite the good efficacy of MRX34 (the liposome-miR-34 complex) in intravenous administration with dexamethasone premedication, the clinical trial was terminated due to severe adverse events including deaths [241,242]. One of the challenges with using miRNAs or their inhibitors in clinical practice is finding the correct route of administration because of their toxicity, limited stability, and low penetrance to target cells. Several delivery strategies have been developed including local delivery, viral delivery, lipid-based and polymer-based vectors [243]. So far, despite numerous preclinical studies and clinical trials none of the miRNA-based therapeutics have complied with expectations [243][244][245].
Some limitations should be acknowledged in this systematic review. First, to minimize the rate of false-positive results, we excluded studies that did not confirm the expression of miRNAs by RT-qPCR. Thus, we excluded studies that performed only global miRNA profiling. It may introduce selection bias and cause exclusion of relevant miRNAs as well as limits the number of comparable results. Second, most of the studies did not distinguish types of EC in the analyzed cohort or focus on EC type I which may introduce bias. Indeed, some studies demonstrate the differences in the miRNA profile in different types of EC [246,247]. Third, the included studies present heterogeneity in sample types (fresh frozen or formalin-fixed, paraffin-embedded (FFPE) tissues), types of controls, and heterogeneity of study group. Fourth, a small number of miRNAs were assessed by at least two independent studies. Moreover, most of the studies that assessed the correlation of miRNA level and clinical parameters relied on a small group of patients and many of the included articles did not report relevant statistical parameters. Therefore, a meta-analysis could not be performed. Despite these limitations, our article is the first systematic review that comprehensively discusses data regarding the role of miRNAs in EC invasiveness and metastasis.

Conclusions
In this systematic review, we comprehensively reviewed miRNAs that are crucial regulators of EC invasiveness and metastasis. Extensive research revealed a complex regulatory network of tumor suppressors and oncomiRNAs that orchestrate tumor progression. Identified miRNAs control EC cell migration, invasion, and EMT in vitro, as well as tumor growth and lymph node metastases in vivo. These miRNAs regulate EC by targeting various members of pathways involved in diverse steps of cancer progression. That makes miRNAs promising candidates for diagnostic and prognostic biomarkers and potential therapeutic targets. Nonetheless, miRNAs that can be useful in clinical practice remain to be identified yet. Further large translational and clinical studies are needed to assess the clinical utility of miRNAs.