Current and Future Roles of Circular RNAs in Normal and Pathological Endometrium

The endometrium is a layer of cells that forms the lining of the mammalian uterus. It responds to changes in the levels of both estrogen and progesterone ( 1 ). The endometrium consists of two layers: the functional layer and the basal layer. The functional layer, which comprises a dense layer and a sponge layer, changes and is shed during the ovarian cycle. The basal layer is not affected by ovarian hormones and does not undergo periodic changes. High-throughput sequencing has enabled the discovery of a wide range of non-coding RNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), that function in various biological processes ( 2 ). In some cases, these non-coding RNAs exhibit abnormal expression patterns that may lead to multiple diseases ( 3 ). Unlike conventional linear RNAs, circRNAs are a unique class of RNAs whose 3′ and 5′ ends are covalently bonded to form a closed continuous loop that is resistant to exonuclease digestion ( 4 ). The high abundance, stability, and evolutionary conservation of circRNAs suggest that they play important regulatory roles ( 5 ). For example, as competing endogenous RNA (ceRNA), circRNAs may use miRNA response elements (MREs) to sponge miRNAs and strongly inhibit miRNA activity, resulting in the upregulation of miRNA targets, which ultimately affects cellular processes such as cell differentiation, proliferation, and apoptosis as well as other cellular functions ( 6 ). These properties make circRNAs important bioregulators of molecular mechanisms underlying various diseases, indicating that circRNAs may be useful as potential diagnostic biomarkers and therapeutic targets ( 5 ). However, studies related to the activity of circRNAs in the endometrium, including those involving endometrial development and endometrium-related diseases, such as endometrial cancer (EC) and endometriosis, are currently in the formative stages. Investigating circRNA function in the endometrium as well as mechanisms underlying such functions may enhance our understanding of the molecular processes associated with the physiological development of the endometrium and provide new opportunities to develop more effective diagnostics and treatments for endometrium-related diseases. The current study attempted to review the properties and functions of circRNAs in the endometrium under both physiological and pathological conditions with a view toward the future of circRNA research.

JT and YC (3rd author) drafted the manuscript. HY, YC (4th author), HC, ZL, LL, YZ, XC, and ZY revised the article. All authors contributed to the article and approved the submitted version.

Other endometrial-related disorders, such as endometrial damage and implantation failure, also greatly affect the quality of life of women ( 49 ). Wharton’s jelly derived mesenchymal stem cells (WJ-MSCs) have shown great potential for repairing different diseases. Human endometrial stromal cells (ESCs) damaged by culturing with mifepristone were repaired by WJ-MSCs ( 50 ). The repairing of damaged ESCs in the co-culture group by WJ-MSCs, improved cell morphology, increased proliferation and decreased apoptosis. The circRNA microarray analyses indicated that 7,757 circRNAs showed differential expression in ESCs co-cultured with WJ-MSCs. In addition, researchers focused on hsa_circRNA_0111659 and predicted it to be related to miR-17/20b/93 and the target mRNA, VEGF. The circRNA-miRNA-mRNA combination may be involved in regulating endometrial damage repair. The results showed that abundant circRNAs were expressed during repair of damaged endometria by WJ-MSC, providing a new perspective in regard to the mechanism of endometrial repair by WJ-MSCs ( Figure 1C ). The specific role of circRNAs in the pathogenesis of repeated implantation failure remains unclear ( 51 ). CircRNA expression in the endometrial biopsies of six women showing repeated implantation failure and a control group (six healthy women) was screened using microarrays ( 52 ). Data from this circRNA microarray assay showed that 856 unique circRNAs were significantly altered. Subsequently, qRT-PCR verified the upregulation of hsa_circRNA_070616, hsa_circRNA_103716, hsa_circRNA_104001, and hsa_circRNA_104854 and the downregulation of hsa_circRNA_ 004183, hsa_circRNA_044353, and hsa_circRNA_404686. Differentially expressed circRNAs provided new target molecular candidates for the diagnosis and clinical treatment of patients with repeated implantation failures. Endometrial receptivity is defined as the ability of the endometrium to accept an embryo implantation ( 53 , 54 ). Studies directed at the potential molecular mechanisms that may be involved, centered on protein-coding genes in the field of assisted reproduction ( 55 ). In addition, circRNA microarray assays, used to compare circRNA expression in the early secretory phase and mid-secretory phase endometrium, identified a number of circRNAs (hsa_circRNA_101280, hsa_circRNA_102293, hsa_circRNA_104789, hsa_circRNA_104791, hsa_circRNA_101263, hsa_circRNA_103493, hsa_circRNA_104625, has_circRNA_400019 and hsa_circRNA_104700), which may be useful as potential biomarkers of endometrial receptivity ( 56 ). Adenomyosis is a benign uterine disorder characterized by presence of endometrium in the myometrium ( 57 ). The down-regulation of hsa_circRNA_101280 was validated in adenomyosis samples (n=11) compared with that in the control samples (n=11), suggesting a potential mechanism underlying decreased implantation rates observed in women with adenomyosis ( 56 ). This study not only expands the knowledge of circRNAs in human endometrium but also provides useful clues for understanding the role of circRNAs in endometrial receptivity. Researchers used Illumina Solexa techniques to analyze circRNAs in the endometria of three goats at gestational day 5 (pre-receptive endometrium, PE) and three goats at gestational day 15 (receptive endometrium, RE) ( 58 ). Overall, 21,813 circRNAs were identified, of which 5,925 circRNAs were RE- and 9,078 circRNAs were PE-specific, indicating the high stage-specificity of circRNAs. Further analyses indicated that there were 334 differentially expressed circRNAs in the RE stage compared with those in the PE stage. Analyses of the cyclic RNA-miRNA interaction network further supported the contention that circRNAs may be used as miRNA sponges to regulate gene expression. In addition, estrogen/progesterone regulated some circRNAs in the endometrial epithelial cells (EECs) and ESCs. These data were used to compile a circRNA atlas of the goat endometrium during embryo implantation. CircRNA-miRNA-mRNA networks were constructed to explore the involvement of ceRNA in the development of RE in goats ( 59 ). Cyclic RNA8073 (ciR-8073) reduced miR-181a levels in a manner similar to that of a miRNA sponge. This effect indirectly increased the expression of neurotensin in EECs. Neurotensin promotes BCL-2 expression via the MAPK pathway and induces expression of leukemia inhibitor factor, cyclooxygenase 2 (COX-2), vascular endothelial growth factor A (VEGFA), and homeobox A10 (HOXA10). This indicated the presence of a ciR-8073-miR181a-neurotensin pathway in the endometrium of goats. CiR-8073 functions as a ceRNA sequestering miR-181a, thereby protecting neurotensin from miR-181a-mediated suppression in EECs. Circ-8073 directly binds miR-449a and inhibits its activity ( 60 ). Centrosomal protein 55 (CEP55) is a direct target of miR-449a. Circ-8073 improves the expression of CEP55 by absorbing miR-449a in EECs in vitro . Circ-8073/miR-449a/CEP55 promotes EC proliferation via the PI3K/AKT/mTOR pathway. In addition, CEP55 regulates the expression of VEGF and FOXM1 in EECs, thereby contributing to the formation of endometrial receptors. These findings in goats suggest that circ-8073 regulates endometrial receptivity via miR-449a/CEP55 and PI3K/AKT/mTOR pathways. Another study also validated the promotive effects of circ-8073 on EEC proliferation via competitive sponging of miR-34a/c via CEP55 ( 61 ) ( Figure 2A ). CircRNAs in endometrial receptivity. (A) CiR-8073 functions as a ceRNA to sequester miR-181a, thereby protecting neurotensin from miR-181a-mediated suppression in EECs. Circ-8073 regulates endometrial receptivity via miR-181a/neurotensin and miR-449a/CEP55 and miR-34/CEP55 axes. (B) Modulation of the circRNA-9119-miR-26a-PTGS2 axis in EECs may be a potential target for regulating RE development. (C) The ciR3175-miR182-testin axis is essential for development of pre-receptive endometrium in goats. A similar study detected high expression of circRNA-9119 and prostaglandin-endoperoxide synthase 2 (PTGS2) and low levels of miR-26a at the RE stage of goats ( 62 ). Further studies showed that circRNA-9119 reduces miR-26a by acting as a miRNA sponge, and it is known that miR-26a reduces the expression of PTGS2 in goat EECs. In addition, PTGS2 is involved in the regulation of certain protein markers of endometrial receptivity in goat EECs. Therefore, modulation of the circRNA-9119-miR-26a-PTGS2 pathway in EECs may be a potential target in the regulation of RE development ( Figure 2B ). The levels of circRNA3175 (ciR3175) and testin in the goat pre-receptive endometrium were high, whereas the expression level of miR-182 was low ( 63 ). Further studies showed that ciR3175 and testin functioned as ceRNAs by competitively sponging miR-182 in EECs. In addition, testin inhibited EEC apoptosis by reducing the expression levels of BCL-2/BAX via the MAPK pathway. Therefore, the ciR3175-miR182-testin may be essential for the development of pre-receptive endometrium in goats. High-quality circRNA expression profiles were obtained from endometrial tissue in these studies ( Figure 2C ). A better understanding of the conditions associated with endometrial receptivity may help in improving the rate of embryonic bedding, which positively affects the treatment of female infertility ( 64 ). Mechanistic research has concentrated on ceRNA regulatory networks, such as the ciR-8073-miR181a-neurotensin, circ-8073/miR-449a/miR-34/CEP55, circRNA-9119-miR-26a-PTGS2, and ciR3175-miR182-testin networks. Many circRNAs are differentially expressed between PE and RE. Following GO and KEGG pathway analysis, interaction network analysis of circRNA-miRNA-mRNA and analysis of circRNAs and their host genes may improve our understanding of how circRNAs mediate the regulation of target genes in the development of endometrial receptivity. The above studies have demonstrated that some circRNAs exert their biological effects on endometrial receptivity by competitively sponging miRNA, thus inducing the expression of miRNA’s targets. In the studies of ciR8073, this circRNA was validated as target for three miRNAs (miR-181a, miR-449 and miR-34a/c), suggesting that a circRNA was regulated by multiple miRNAs, and further analysis should investigate whether a miRNA could be targeted for multiple circRNAs in endometrial receptivity. However, in addition to ceRNA regulatory mechanisms, other underlying mechanisms, such as circRNA-protein interactions that also lead to significant changes in circRNA expression as well as the function of individual circRNAs in endometria, need further investigation. These findings will expectedly increase the diversity of the endometrial transcriptome for circRNAs and yield new insights into the development of endometrial receptivity.

Thousands of circRNAs are encoded by the human genome in a context-dependent manner ( 65 ). Due to their abundant expression, stability, and multiple MREs, a large number of circRNAs function via ceRNA regulatory mechanisms ( 66 ). circRNAs interact with RNA-binding proteins via direct binding, which constitutes another major regulatory mechanism of circRNAs ( 67 ). In addition, inflammation of endometrium is a complex condition, which is almost involved in all endometrial disorders ( 68 – 70 ). Further research is needed for a better understanding of circRNAs in inflammation of reproductive tract. Certain properties of circRNAs, such as small size and stability, are ideally suited for a role as biomarkers. Furthermore, tissue-specific expression patterns of circRNAs are closely associated with clinical phenotypes. In addition, circRNAs, which are not affected by endonuclease degradation and are stable in formalin-fixed paraffin-embedded tissues ( 71 ), can be detected in vaginal secretions ( 72 ), and therefore should be investigated further for the purpose of diagnosing related diseases. Due to being considered as a “hotspot” of RNA research, an increasing number of circRNAs are identified via effective high-throughput sequencing techniques and bioinformatics. However, studies pertaining to the emerging role of circRNAs in the endometrium are yet in the formative stages. Advanced databases, testing tools, and research techniques have enabled circRNAs to be recognized as potential non-invasive biomarkers of reproductive as well as gynecological diseases. In addition, there is also a lack of studies evaluating the panoramic view of circRNAome and individual circRNA along the entire menstrual cycle in human endometrium. Considered together, circRNA research allows researchers to enter a new level in epigenetic regulatory networks, whereby future research may lead to a better understanding of the mechanisms regulating circRNAs in the endometrium.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This study was supported by grants from the Sanming Project of Medicine in Shenzhen (SZSM201812041) and Clinical Research Funding from Shenzhen Second People’s Hospital (4001023).