Hypoxic endometrial epithelial cell-derived microRNAs effectively regulate the regenerative properties of mesenchymal stromal cells

Abstract

Endometrial thickness plays an important role in successful embryo implantation and normal pregnancy achievement. However, a thin endometrial layer (≤ 7 mm) may have a significant effect on microenvironment tolerance, which is further related to successful embryo implantation or conception, either naturally or after assisted reproductive technology. Moreover, this microenvironment tolerance shift induces hypoxic damage to endometrial epithelial cells (EECs), which results in altered signaling biomolecule secretion, including exosome content. In the context of endometrium regeneration, mesenchymal stromal cells (MSCs) and umbilical cord (UC)-derived stem cells have been applied in clinical trials with promising results. It has been recently shown that exosomes derived from hypoxic damaged EECs directly contribute to the increased migratory and regenerative abilities of UCs and MSCs. Specifically, microRNAs in exosomes secreted by the hypoxic damaged EECs, such as miR-214-5p and miR-21-5p, play a crucial role in the migratory capacity and differentiation ability of MSCs to EECs mediated through the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Taking into consideration the above information, UC-MSCs may be considered as a modern intervention for endometrial regeneration.

Key Words: MicroRNAs; Mesenchymal stromal cells; Endometrial tissue; Wound healing; Tissue regeneration; MiR-214-5p; MiR-21-5p

Core Tip: Endometrial tissue damage is the primary reason for unsuccessful embryo implantation. Hypoxic damaged endometrial epithelial cells can secrete miRNAs through exosomes, which can efficiently regulate endometrial microenvironment homeostasis. Specifically, miR-21-5p and miR-214-5p can regulate the migration, wound healing and differentiation of umbilical cord-derived mesenchymal stromal cells. Therefore, the latter favors endometrial tissue regeneration. By understanding the primary endometrial defect, alternative therapeutic protocols such as advanced cellular therapies may be used to restore women’s fertility.

TO THE EDITOR

MicroRNAs (miRNAs) have been shown to play a pivotal role in regulating transcriptional machinery and are actively associated with protein expression levels [1]. MiRNAs are small non-coding highly conserved RNAs with an average length of 22 nucleotides. They are complementary with the target mRNA primarily to the 3’-untranslated region (UTR) where they regulate mRNA transcription in a cell type-specific context [2]. MiRNAs are produced from various DNA regions located within an actively transcribed gene (such as introns), followed by a maturation process orchestrated by Drosha and Dicer complexes[2,3]. Besides their complementarity with the 3’-UTR, miRNAs can also target the 5’-UTR, coding sequences and gene promoters[1-4]. MiRNAs can be transported between cells through various mechanisms, such as direct secretion to extracellular fluid, interaction with the Argonaute protein complexes or through vesicle production like exosomes[3,5]. The first evidence of miRNA function was reported by Victor Ambros and Gary Ruvkun in 1993, who were awarded the Nobel Prize in 2024[5-11]. In addition to their important role in cellular homeostasis, miRNAs are actively associated with various diseases; therefore, they have been thoroughly studied by different research groups worldwide[12-15]. In a recent publication, Zhang et al[16] elucidated the role of exosomal miR-214-5p and miR-21-5p secreted by hypoxic endometrial epithelial cells (EECs) in regulating human umbilical cord-derived mesenchymal stromal cell (UC-MSC) function.

Endometrial tissue damage induced by various pathological issues, such as radiation, infection, surgery-induced trauma and Asherman syndrome, significantly affects women’s fertility, often leading to unsuccessful embryo implantation[17]. For this purpose, different therapeutic approaches like exogenous hormones have been applied to restore primary endometrial damage, thus increasing the possibility for a successful pregnancy[18]. However, the results for women who receive hormone therapy require further clarification[18-21]. On the other hand, as proposed by Zhang et al[16], cellular therapies may represent an alternative therapeutic approach in such conditions.

MSCs are a mesodermal progenitor cell population that was first observed in bone marrow aspirate cultures. They possess significant immunoregulatory and multipotential differentiation ability[22,23]. MSCs can also be isolated from fetal-derived origins such as amniotic fluid, placenta and umbilical cord (UC). Compared to MSCs from adult origin, fetal-derived MSCs carry fewer mutations and epigenetic alterations in their genome[24]. Additionally, MSCs are considered immune privileged cells, characterized by low expression of human leukocyte antigens (HLA) class II and lack of expression of co-stimulatory molecules (CD40, B7-1, etc.)[24]. Therefore, MSCs are characterized by their safety and tolerability to recipients, without serious adverse reactions, as has been reported in the currently performed clinical trials for different disease conditions[22,25,26].

Moreover, it has been shown that MSCs can be primed to exert their immunomodulatory properties or can actively participate in tissue’s resident progenitor cell differentiation, in part through specific microenvironment signaling cues [24]. Specifically, primed MSCs can produce high levels of anti-inflammatory cytokines (interleukin (IL)-1Ra, IL-6, IL-10, IL-13) and immunoregulatory biomolecules (nitric oxide, indoleamine 2,3 dioxygenase, galectins, etc.), which are responsible for tolerance against overactivated immune responses[24]. In addition, as recipients of differentiation signaling cues such as growth factors (transforming growth factor-β1, endothelial growth factor, platelet derived growth factor, vascular endothelial growth factor, insulin-like growth factor) and miRNAs, MSCs could potentially differentiate into specific cell types or adopt an activated state to further promote the differentiation of the tissue’s resident progenitor cells to participate in damaged tissue regeneration process[27].

In the context of tissue regeneration ability, the study of Zhang et al[16] entitled “Effects of miR-214-5p and miR-21-5p in hypoxic endometrial epithelial-cell-derived exosomes on human umbilical cord mesenchymal stem cells” represented very interesting work in the field, as it demonstrated that UC-MSCs are a promising therapeutic approach for endometrial regeneration. Specifically, this study demonstrated that endometrial damage is actively related to hypoxic EEC induction, which is characterized by an altered secretory profile. Moreover, the authors demonstrated that EEC-derived exosomes contained a great number of miRNAs, which were analyzed utilizing miRNA sequencing. Interestingly, they identified 32 differentially expressed miRNAs between the normal EEC group and hypoxic damaged EECs. Among them, miR-214-5p and miR-21-5p were the most important for modulating MSC function. They were depleted from hypoxic damaged EEC-derived exosomes, as confirmed by real-time reverse transcriptase-polymerase chain reaction analysis. Specifically, miR-214-5p and miR-21-5p efficiently regulated the migratory capacity and differentiation ability of UC-MSCs through the STAT3 signaling pathway. Zhang et al[16] further explored the implication of miR-21-5p and miR-214-5p in MSC function such as their migratory and differentiation ability by utilizing specific inhibitors against the aforementioned miRNAs. When miR-214-5p and miR-21-5p inhibitors were applied, UC-MSCs exhibited enhanced migratory and wound healing capacity, in assays performed in transwell plates. These are important properties of MSCs in promoting endometrial regeneration. In the same way, the low abundance of miR-214-5p and miR-21-5p further promoted UC-MSC differentiation to EEC-like cells, as was confirmed by high expression of CD9 and CK19. Indeed, CD9 is a member of the transmembrane 4 superfamily and is directly associated with integrin beta(1), alpha(3) and alpha(6) subunits[28]. CK19 is a low molecular weight cytokeratin responsible for the arrangement of myofibers, and also is used as a diagnostic biomarker with prognostic significance in proper endometrial function[29]. Therefore, they are both considered typical markers for EEC differentiation. Moreover, the differentiation of MSCs to EECs was confirmed by low vimentin and CD13 expression, which can be considered among the typical MSC markers. In the same way, miR-21-5p and miR-214-5p inhibitors also impaired MSC differentiation towards “osteocytes” and “adipocytes”. Regarding osteogenic differentiation, alizarin Red S staining was weak compared to the control group, accompanied by low expression of osteoprotegerin, bone morphogenetic protein 2, osteocalcin, and Runx2. Similarly, in adipogenic differentiation, the size and the number of lipid droplets were significantly decreased compared to the control group, confirmed by weak Oil Red O staining. The above results showed the impact of miR-21-5p and miR-214-5p in regulating EEC differentiation from MSCs.

This is among the first studies that showed the direct association between miRNA and the STAT3 signaling pathway. Interestingly, inhibition of miR-214-5p and miR-21-5p related to STAT3 signaling pathway upregulation mediated EEC-like cell differentiation. Through a comprehensive analysis of miRNAs using databases such as MirDB and TargetScan, the authors demonstrated that both miR-21-5p and miR-214-5p were actively associated with the function of protein inhibitor of activated STAT3 (PIAS3) and STAT3. Interestingly, Zhang et al[16] showed that silencing of miR-21-5p was related to the downregulation of PIAS3 and upregulation of STAT3 (Figure 1). On the contrary, silencing of miR-214-5p resulted in upregulation of both PIAS3 and STAT3, favoring EEC differentiation from MSCs. STAT3 comprises an important transcription factor, exerting a pleiotropic action in most cells. In the study herein, abolishment of STAT3 signaling using a direct inhibitor against STAT3 blocked MSC migration and differentiation to ECCs (Figure 1). Therefore, the interaction of miR-21-5p and miR-214-5p with STAT3 is considered of paramount importance for MSC migratory and differentiation capacity (Figure 1).

Figure 1 Implication of miR-21-5p and miR-214-5p in regulation of signal transducer and activator of transcription 3 signaling pathway. A: Normal endometrial epithelial cells produce a high amount of exosomes enriched in miR-12-5p and miR-214-5p, followed by adaptation by mesenchymal stromal cells (MSCs). MiR-21-5p and miR-214-5p then stably connect with the 3’-untranslated region of signal transducer and activator of transcription 3 (STAT3), inducing the mRNA degradation. This event is directly associated with the total absence or low expression of STAT3, blocking MSC migration and differentiation; B: Hypoxic damaged-endometrial epithelial cells are responsible for exosome production with low content of miR-21-5p and miR-214-5p. The absence of the aforementioned miRNAs induces proper mRNA transcription and STAT3 production. Eventually, p-STAT3 is translocated to the MSC nucleus, thus enhancing the migratory and differentiation capacity of the cells. EEC: Endometrial epithelial cell; MSC: Mesenchymal stromal cell; STAT3: Signal transducer and activator of transcription 3.

It also should be noted that the results obtained from the study conducted by Zhang et al[16] relied on in vitro approaches, thus further clarification in an in vivo system is required. Future steps of this study may include a comprehensive understanding of the underlying differentiation mechanism of MSCs to EECs. It is important to shed light on the interplay of other transcription factors responsible for MSC stemness. Interestingly, the alerted expression of REX1, SOX2, NANOG and octamer-binding transcription factor 4 with the parallel activation of p38 mitogen-activated protein kinase is considered the initial step for MSC differentiation to other cell types[30,31]. In addition, the impact of the miR-21-5p and miR-214-5p in the immunomodulatory abilities of MSCs represents an interesting research area, which demands the appropriate scientific attention. It is crucial to mention that MSCs, especially those of fetal origin, exert key immunoregulatory properties when located in a microenvironment with overactivated immune responses. For this purpose, MSCs are considered a promising therapeutic approach for human immune-related disorders and therefore have been used in clinical trials with promising results. Endometriosis may cause inflammation, accompanied by scar tissue formation in the pelvic region. Considering this, MSCs could be locally administrated to exert their key immunoregulatory role and to ameliorate scar tissue formation. However, the impact of damaged EECs to to alter the immunobiology of infused MSCs through miRNA interplay still remains unknown and the potent underlying mechanism requires further clarification.

Another thing that should be clarified is the HLA class II expression during EEC phenotype adaptation by MSCs. It is widely known that undifferentiated MSCs do not express HLA class II and co-stimulatory molecules, and are thus considered non-immunogenic cells[24]. However, Le Blanc et al[32] showed the elevated expression of HLA class II in differentiated MSCs. Because miR-21-5p and miR-214-5p play important roles in MSC migration and differentiation, these biomolecules may also be associated with altered HLA gene expression. In a similar way, endometrial cancer cells are characterized by low expression of miR-21-5p and miR-214-5p, which could be potentially associated with altered HLA gene expression, and thus may explain the immune unresponsiveness and tumor invasion. Therefore, miR-21-5p and miR-214-5p expression levels should be evaluated as biomarkers of tumor invasion in endometrial cancer. Lastly, the study performed by Zhang et al[16] should further explore the role of miR-21-5p and miR-214-5p in animal models of endometriosis.

In conclusion, the study performed by Zhang et al[16] insightfully explored the potential use of UC-MSCs as an alternative therapeutic approach in restoring endometrial tissue damage. The signal cues exerted by the hypoxic damaged EECs can efficiently regulate the fate of applied MSCs, favoring their differentiation to EEC-like cells and further contributing to endometrial regeneration. Additionally, the proper regulation of miR-21-5p and miR-214-5p may further be proven useful to restore primary defects of the endometrium, orchestrating the activation of endometrium epithelial progenitor cells to sites of injury. Moreover, the results presented by Zhang et al[16] showed the potential use of miR-21-5p and miR-214-5p as prognostic biomarkers in endometrial cancer invasion. However, further exploration is required using both in vitro and in vivo models. Undoubtedly, the understanding of intrinsic cell pathways leading to enhanced cell migration and differentiation through miRNA-mediated signaling is an important aspect, thus favoring the utilization of advanced stem cell therapies in endometrial regeneration.