Exosomal miR-137-3p targets UBE3C to activate STAT3, promoting migration and differentiation into endometrial epithelial cell of human umbilical cord mesenchymal stem cells under hypoxia

Abstract

BACKGROUND

Thin endometrium, leading cause of recurrent implantation failure and infertility, has been found to respond to exosomes.

AIM

To investigate the efficacy of exosomes in addressing the issue of thin endometrium.

METHODS

RNA sequencing and reverse transcription-quantitative polymerase chain reaction were employed to identify differentially expressed microRNAs (miRNAs) in human umbilical cord mesenchymal stem cell (hucMSC) treated with exosomes enriched with endometrial cell-derived components. Additionally, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to highlight significant enrichment in specific biological pathways, molecular functions, and cellular components. Transwell and wound healing assays were performed to assess migratory potential, and western blotting was detected protein level.

RESULTS

A total of 53 differentially expressed miRNAs were identified in hucMSC treated with exosomes enriched with endometrial cell-derived components, comprising 27 upregulated and 26 downregulated miRNAs, which includes miR-137-3p. Enhanced migratory potential was observed in the Transwell and wound healing assays, and western blotting confirmed the epithelial differentiation of hucMSC and the increased p-signal transducer and activator of transcription 3. These effects were attributed to the upregulation of miR-137-3p.

CONCLUSION

miR-137-3p in exosomes from hypoxia-affected endometrial epithelial cell stimulates the signal transducer and activator of transcription 3 signaling pathway, enhancing the migration and differentiation of hucMSC into endometrial epithelial cell.

Key Words: Endometrial epithelial cells; Exosomes; miR-137-3p; Ubiquitin protein ligase E3C; Signal transducer and activator of transcription 3; Human umbilical cord mesenchymal stem cells

Core Tip: This study uncovers that under hypoxic conditions, exosomal miR-137-3p, by targeting ubiquitin protein ligase E3C, activates signal transducer and activator of transcription 3, thereby enhancing the migration and differentiation of human umbilical cord mesenchymal stem cells (hucMSCs) into endometrial epithelial cells. Through microRNA sequencing and reverse transcription-quantitative polymerase chain reaction validation, it was observed that miR-137-3p is significantly upregulated in hucMSCs when co-cultured with endometrial epithelial cells. Additionally, exosome-based therapeutic system was established to evaluate the role of hucMSCs overexpressing miR-137-3p in therapeutic applications. This discovery offers novel insights into cell therapy under hypoxic environments.

INTRODUCTION

Human endometrium is characterized by remarkable plasticity, as evidenced by rapid tissue remodeling observed during early pregnancy and swift growth and differentiation that occur during menstrual cycle[1]. Development of thin endometrium, primary cause of failed embryo transfer and adverse obstetric outcomes, is underpinned by intrauterine adhesions, fibrosis formation, and disruption of endometrial regeneration[2]. To date, numerous therapeutic strategies have been proposed for treatment of thin endometrium[3], however, inconsistency in enhancing endometrial thickness remains pervasive limitation of these treatments.

Exosomes, distinct class of extracellular vesicles secreted by broad array of cells, including endometrial cells[4], facilitate intercellular communication through transport of diverse signaling molecules, such as DNA, mRNAs, microRNAs (miRNAs), proteins, and lipids[5]. They have been implicated in endometrial diseases[6], with endometrial epithelial cell (EEC)-derived exosomes demonstrated to inhibit cell invasion and migration in ovarian endometriosis[7]. Moreover, exosomes from bovine EECs support development of trophoblast cells[8].

Recently, stem cells have emerged as novel therapeutic modality in human regenerative medicine, particularly for regeneration of endometrial disorders, including thin endometrium[9]. Mesenchymal stem cells (MSCs) discern microenvironment at injury site and secrete specific factors that are crucial for repair in multiple physiological processes. Prior research has highlighted efficacy of MSCs in treating thin endometrium, with transplantation of umbilical cord-derived MSCs in rats promoting endometrial recovery[10] and bone marrow-derived MSC transplantation in damaged uterus increasing endometrial thickness and improving fertility[11,12]. Additionally, it has been established that exosomes secreted by human umbilical cord MSCs (hucMSCs) repair hypoxia-induced EEC injury[13], underscoring beneficial effects of MSC-derived exosomes on EEC. Differentiation of human umbilical cord Wharton’s jelly-derived MSCs into EECs has also been documented[14]. Furthermore, our previous findings indicate that exosomes from hypoxia-damaged EECs enhance hucMSC migration and differentiation into EECs, although precise mechanisms governing this process require further elucidation.

MiRNAs, small non-coding RNAs that function as regulators of gene expression[15], have been identified as relevant to endometrial diseases[16] and differentiation of MSCs[17]. A study has conducted integrated miRNA sequencing analyses on adjacent normal endometrium and thin adhesive endometrium samples obtained from patients[18]. Utilising a combination of RNA sequencing and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), the present study sought to identify and validate the differential expression of miRNAs in the context of co-culture of hypoxia-injured endometrial epithelial cell (EECD)-derived exosome (EECD-ex) with hucMSC. The analysis revealed a pronounced alteration in the expression of miR-137-3p. Its expression was significantly up-regulated in the EECD-ex + hucMSC group, indicating that it may play a key role in exosome-mediated regulation of hucMSC function. Nonetheless, role of miRNAs in thin endometrium has been examined only sparingly, and mechanisms by which miRNAs modulate recovery of thin endometrium remain to be thoroughly elucidated. This study explores whether exosomes released from hypoxia-damaged EECs influence cellular functions of hucMSCs through delivery of specific signaling molecules, and it also delves into underlying mechanisms.

MATERIALS AND METHODS

Cell culture

Human primary EECs, acquired from Cellverse Bioscience Technology (Shanghai, China), were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; 11965092, Gibco, NY, United States) enriched with 100 U/mL streptomycin, 100 ng/mL penicillin, and 10% fetal bovine serum (FBS; SH30406.05, HyClone, Marlborough, MA, United States), within humidified incubator maintained at 37 °C and 5% CO₂. HucMSC, similarly sourced from Cellverse Bioscience Technology, were nurtured under equivalent conditions at 37 °C and 5% CO₂, utilizing DMEM/F12 medium (03.2001C, EallBio, Beijing, China) augmented with 100 U/mL streptomycin, 100 ng/mL penicillin, and 10% FBS. HucMSC from passages 4 to 6 were employed for subsequent experiments, in alignment with prior studies[19].

Hypoxia treatment in EEC

Prior to hypoxia exposure, EEC were maintained under conventional conditions. Thereafter, cells were transferred to a hypoxic environment within a modular incubator chamber (94% N₂, 5% CO₂, 1% O₂) at 37 °C for 4 hours. By contrast, control group cells were sustained in a normoxic atmosphere (78% N₂, 5% CO₂, 21% O₂) throughout experimental period.

Exosome extraction

Culture supernatant from both normal and hypoxia-affected EEC was collected for exosome isolation, referred to as EEC-ex or EECD-ex, respectively, via ultracentrifugation. EEC were initially incubated for 48 hours in exosome-depleted DMEM. Supernatant was then subjected to centrifugation at 2000 r/minute for 20 minutes at 4 °C. To remove cellular debris, a subsequent centrifugation was performed at 10000 r/minute for 30 minutes at 4 °C. Post filtration through a 0.2 μm filter, supernatant underwent two ultracentrifugation rounds at 100000 r/minute for 1 hour at 4 °C. The ultimate precipitates constituted exosomes.

Cell transfection and treatment

For exosome treatment, hucMSC were first pre-incubated for 24 hours with either EECD-ex or EEC-ex at a concentration of 5 μg/mL, thus forming EECD-ex + hucMSC and EEC-ex + hucMSC groups, respectively. Mimic negative control (NC) and miR-137-3p mimic were sourced from Sangon Biotech (Shanghai, China) and diluted to concentration of 30 nmol/L. Additionally, ubiquitin protein ligase E3C (UBE3C) overexpression plasmid (OE-UBE3C) and Vector-PcDNA3.1(+) were procured. Transfection of hucMSC with OE-NC, OE-UBE3C, mimic NC, or miR-137-3p mimic was performed using jetPRIME^® reagent (101000046, Polyplus, Illkrich, France), in strict adherence to manufacturer’s protocol. To suppress function of signal transducer and activator of transcription 3 (STAT3), STAT3 inhibitor Stattic (HY-13818, MedChemExpress, NJ, United States) was introduced into hucMSC cultures.

MiRNA sequencing

Upon extraction of total RNA from cells, its quality was assessed using an Agilent 2100 Bioanalyzer. Small RNA libraries were constructed with TruSeq Small RNA Library Prep Kit (RS-200-0036, Illumina, CA, United States). These libraries were then equimolarly combined, denatured, and loaded onto GAIIx flow cell lanes using cBot station and Illumina cluster generation kits. Differentially expressed miRNAs (DEMs) with raw reads ≥ 5 and a P value < 0.05 were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to evaluate representation of DEM target genes in GO terms and KEGG pathways, with a significance threshold of P value < 0.05.

RT-qPCR

Total RNA was extracted from cells using TRIzol reagent (R1100, Solarbio, Beijing, China). Reverse transcription was achieved using iScript cDNA Synthesis Kit (KR118, TIANGEN, Beijing, China). SYBR Green (FP205, TIANGEN, Beijing, China) was utilized for RT-qPCR analysis. Relative gene expression was quantified using 2^-ΔΔCt method, with U6 (for miRNAs) and GAPDH (for mRNAs) serving as endogenous reference genes. Primer sequences are summarized in Table 1.

Table 1 The primer sequences used in reverse transcription-quantitative polymerase chain reaction.

Target gene	Primer (5’-3’)
U6-F	ACGATACAGAGAAGATTAGCATGG
U6-R	AAATATGGAACGCTTCACGAA
hsa-miR-137-3p-F	CTCAACTGGTGTCGTGGAGT
hsa-miR-137-3p-R	TCGGCAGGTTATTGCTTAAGAATAC
hsa-miR-137-3p-RT	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCTACGC
UBE3C-F	AGCTATGTGGTGTCAGTGATTG
UBE3C-R	GAAGGCTGTAAAAACTTGTTGCC
STAT3-F	ACCAGCAGTATAGCCGCTTC
STAT3-R	GCCACAATCCGGGCAATCT
GAPDH-F	ACGGATTTGGTCGTATTGGG
GAPDH-R	GGGATCTCGCTCCTGGAAG

UBE3C: Ubiquitin protein ligase E3C; STAT3: Signal transducer and activator of transcription 3.

Transwell assay

Transwell assay was employed to evaluate migratory potential of hucMSC. Cells in logarithmic growth phase were harvested, washed in phosphate buffered saline, and resuspended in serum-free DMEM/F12. Upper chamber was seeded with 4 × 10⁴ hucMSC, while lower chamber was filled with culture medium rich in 10% FBS. Following a 24-hour incubation, migrated cells were fixed with 2% methanol and stained using a 0.5% crystal violet solution. Cell migration was visualized and documented using a microscope (2103012, AOSVI, Shenzhen, China).

Wound healing assay

HucMSC were seeded in 6-well plates and incubated in medium devoid of FBS for 24 hours. A linear scratch was created on cell monolayer using a 10 μL pipette tip. Cell migration was observed and imaged at various time points (0 hour, 48 hours) under an inverted microscope. Cell migration rate was quantified using Image J software.

Western blot

Total protein extraction from hucMSC was achieved through use of RIPA buffer containing protease inhibitor (P0013B, Beyotime, Shanghai, China), with mixture incubated on ice for 30 minutes. Protein samples were subjected to sodium-dodecyl sulfate gel electrophoresis and then transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore, Burlington, MA, United States). After a 2-hour incubation with 5% nonfat milk as a blocking agent, membranes were incubated overnight with primary antibodies targeting CD9 (ab236630, Abcam, United Kingdom), CK19 (ab76539, Abcam, United Kingdom), vimentin (5741, CST, Danvers, MA, United States), CD13 (ab108310, Abcam, United Kingdom), β-Tubulin (CW0098M, CWBIO, Beijing, China), p-STAT3 (9138, CST, Danvers, MA, United States), STAT3 (4904, CST, Danvers, MA, United States), mitogen-activated protein kinase (MAPK; 4695, CST, Danvers, MA, United States), p-MAPK (4370, CST, Danvers, MA, United States), protein kinase B (AKT; 4691, CST, Danvers, MA, United States), p-AKT (13038, CST, Danvers, MA, United States), and UBE3C (ab226173, Abcam, United Kingdom). Membranes were subsequently incubated for 2 hours with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using an ECL reagent (MA0186-2, Meilunbio, Dalian, China).

Luciferase reporter assay

Potential binding of miR-137-3p to UBE3C, as predicted by miRDB, was investigated. A fragment of wild-type (WT) or mutant UBE3C-3’-untranslated region (UBE3C-3’-UTR), encompassing predicted binding site of miR-137-3p, was subcloned into psiCHECK-2 vector (GENERAL BIO, Anhui, China) to generate UBE3C-3’-UTR WT or Mutant constructs. Cells were plated in 24-well plates and co-transfected with miR-137-3p mimic or mimic NC and aforementioned vectors, using jetPRIME^® reagent (101000046, Polyplus, Illkrich, France). Forty-eight hours after transfection, luciferase activity was measured using a luciferase reporter assay system (RG028, Beyotime, Shanghai, China).

Statistical analysis

All experimental data are presented as mean ± SD. One-way ANOVA was employed to compare differences among multiple groups, while Student’s t-test was used for comparisons between two groups. P value < 0.05 was deemed statistically significant. Statistical analyses were conducted using Prism 7.0.

RESULTS

miR-137-3p was highly expressed in EECD-ex-cocultured hucMSC

We have previously established that EECD-ex significantly enhances hucMSC migration and differentiation into EECs. Nonetheless, precise underlying mechanisms have yet to be elucidated. In this study, culture media from both normal EECs and hypoxia-induced EEC injury (hypoxia-damaged EEC) were collected for exosome extraction, and these exosomes were subsequently introduced to hucMSCs for coculture. Through miRNA sequencing, we uncovered DEMs in hucMSCs exposed to EEC-ex and EECD-ex. Our analysis revealed identification of 53 DEMs, comprising 26 downregulated and 27 upregulated miRNAs, in EECD-ex + hucMSC group relative to EEC-ex + hucMSC group (Figure 1A). GO and KEGG databases were harnessed to conduct functional analysis of DEM targets, indicating specific biological pathways, molecular functions, and cellular components (Figure 1B and C). Furthermore, KEGG pathway enrichment analysis discerned several pertinent signaling pathways (Figure 1D). RT-qPCR corroborated that, in comparison with hucMSCs and EEC-ex + hucMSC groups, miR-137-3p was upregulated in EECD-ex + hucMSC group, aligning with findings from RNA sequencing (Figure 1E). Consequently, it is proposed that miR-137-3p, released by EECD-ex, may play regulatory role in functional dynamics of hucMSCs.

Figure 1 miR-137-3p was markedly elevated in human umbilical cord mesenchymal stem cell co-cultured with hypoxia-compromised endometrial epithelial cell-derived exosomes. The culture medium from healthy endometrial epithelial cell (EEC-ex) or hypoxia-compromised EEC (EECD-ex) was gathered for the extraction of exosomes, which were subsequently incorporated into a co-culture system with human umbilical cord mesenchymal stem cell. A: A volcano plot illustrating the differentially expressed microRNAs in human umbilical cord mesenchymal stem cell subjected to EEC-ex or EECD-ex; B-D: Gene Ontology analysis (B and C) and Kyoto Encyclopedia of Genes and Genomes pathway analysis (D) of the target genes associated with differentially expressed microRNAs, as assessed by RNA sequencing; E: The quantification of miR-137-3p expression levels via reverse transcription-quantitative polymerase chain reaction. Data are represented as mean ± SD. ^aP < 0.0001 vs human umbilical cord mesenchymal stem cell group, ^bP < 0.0001 vs healthy endometrial epithelial cell + human umbilical cord mesenchymal stem cell group, n = 3. EEC: Endometrial epithelial cell; GO: Gene Ontology; HucMSC: Human umbilical cord mesenchymal stem cell; EEC-ex: Healthy endometrial epithelial cell; EECD-ex: Hypoxia-compromised endometrial epithelial cell.

miR-137-3p facilitated hucMSC migration and differentiation into EEC

To elucidate role of miR-137-3p in migratory abilities of hucMSCs, we enhanced expression of miR-137-3p via transfection with a miR-137-3p mimic. Transwell assay illustrated that hucMSC migration was markedly amplified in miR-137-3p mimic group when juxtaposed with mimic NC group (Figure 2A). Moreover, wound healing assay indicated that miR-137-3p mimic significantly enhanced migratory distance of hucMSCs (Figure 2B). Aforesaid data suggest facilitative effect of miR-137-3p on migration of hucMSCs. Subsequently, we examined if miR-137-3p modulates differentiation of hucMSCs into EECs. It was observed that, as duration of transfection progressed, miR-137-3p mimic upregulated expression levels of epithelial lineage markers (CK19 and CD9) while simultaneously downregulating levels of stromal lineage markers (CD13 and vimentin) in hucMSCs (Figure 2C). Expression levels of CK19 and CD9 proteins were elevated, whereas those of vimentin and CD13 were reduced in hucMSCs after transfection with miR-137-3p mimic (Figure 2D). These findings further corroborate that miR-137-3p facilitates differentiation of hucMSCs into EECs.

Figure 2 miR-137-3p augmented the migratory and differentiation capabilities of human umbilical cord mesenchymal stem cell into endometrial epithelial cell. Human umbilical cord mesenchymal stem cells were subjected to transfection with miR-137-3p mimic or negative control. A: Transwell assays were employed to assess cellular migration. Scale bar = 100 μm, n = 3; B: Wound healing assays quantified the migratory capacity of human umbilical cord mesenchymal stem cell. Scale bar = 200 μm, n = 3; C: The protein levels of CD9, CK19, vimentin, and CD13 were elucidated using western blotting; D: Western blot analysis elucidating the protein levels of CD9, CK19, vimentin, and CD13. The results are depicted as mean ± SD. ^aP < 0.0001 vs negative control mimic group, n = 3. HucMSC: Human umbilical cord mesenchymal stem cell; NC: Negative control.

miR-137-3p activated STAT3 signaling

Expressions of p-AKT and p-MAPK proteins in hucMSCs were, respectively, elevated and diminished following transfection with miR-137-3p mimic (Figure 3A). This suggests that miR-137-3p may exert its influence through AKT pathway rather than MAPK pathway. Additionally, miR-137-3p mimic augmented expression of p-STAT3 protein in hucMSCs (Figure 3B), indicating possible involvement of STAT3 signaling in role of miR-137-3p.

Figure 3 miR-137-3p invigorated the signal transducer and activator of transcription 3 signaling cascade. Human umbilical cord mesenchymal stem cells were transfected with miR-137-3p mimic or negative control. A: Western blot analysis of p-protein kinase B and p-mitogen-activated protein kinase protein expressions; B: Western blot evaluation of p-signal transducer and activator of transcription 3 protein levels. The findings are presented as mean ± SD. AKT: Protein kinase B; MAPK: Mitogen-activated protein kinase; STAT3: Signal transducer and activator of transcription 3; NC: Negative control.

miR-137-3p promoted hucMSC migration and differentiation into EEC, which was antagonized after inhibition of STAT3

To ascertain whether miR-137-3p mediated functional modifications of hucMSCs by modulating STAT3, hucMSCs were transfected with miR-137-3p mimic, followed by administration of STAT3 inhibitor (Stattic). Transwell assay demonstrated that miR-137-3p mimic potentiated hucMSC migration, response that was attenuated upon introduction of STAT3 inhibitor (Figure 4A). In accordance with these findings, wound healing assay revealed that while miR-137-3p mimic augmented migratory distance of hucMSCs, this effect was negated by STAT3 inhibitor (Figure 4B). Western blot analysis of epithelial and stromal lineage markers further supported notion that miR-137-3p mimic enhanced hucMSC differentiation into EECs, an effect that was inhibited by STAT3 inhibitor (Figure 4C). Collectively, these data suggest that STAT3 inhibition can counteract stimulatory effects of miR-137-3p on hucMSC function.

Figure 4 miR-137-3p bolstered the migratory and differentiation propensities of human umbilical cord mesenchymal stem cell into endometrial epithelial cell, an effect mitigated by signal transducer and activator of transcription 3 inhibition. Human umbilical cord mesenchymal stem cell, transfected with miR-137-3p mimic or negative control (mimic NC), were exposed to the STAT3 inhibitor Stattic. A: Transwell assays were conducted to evaluate migratory ability. Scale bar = 100 μm, n = 3; B: Wound healing assays were utilized to assess the migratory response of human umbilical cord mesenchymal stem cell. Scale bar = 200 μm, n = 3; C: The protein levels of CD9, CK19, vimentin, and CD13. The results are illustrated as mean ± SD. ^aP < 0.0001 vs negative control mimic group, ^bP < 0.0001 vs negative control mimic group, ^cP < 0.0001 vs miR-137-3p mimic group, ^dP < 0.0001 vs negative control mimic + Stattic group, n = 3. HucMSC: Human umbilical cord mesenchymal stem cell; NC: Negative control.

miR-137-3p targeted UBE3C to promote activation of STAT3

We posited that miR-137-3p might target UBE3C, hypothesis supported by predictions from miRDB (Figure 5A). Subsequent dual-luciferase reporter assays confirmed that miR-137-3p specifically binds to 3’-UTR of UBE3C; however, this interaction was markedly weakened when UBE3C-3’-UTR was mutated (Figure 5B). Transfection of HucMSCs with miR-137-3p mimic led to reduction in UBE3C mRNA levels (Figure 5C). Notably, protein levels of UBE3C were observed to decrease, whereas levels of STAT3 were seen to increase in hucMSCs following miR-137-3p mimic transfection (Figure 5D). This evidence implies that miR-137-3p downregulates UBE3C to promote STAT3 activation.

Figure 5 miR-137-3p facilitated signal transducer and activator of transcription 3 activation through targeting ubiquitin protein ligase E3C. A: The potential interaction between miR-137-3p and ubiquitin protein ligase E3C (UBE3C), as predicted by miRDB; B: The comparative luciferase activities; C: Human umbilical cord mesenchymal stem cell were transfected with miR-137-3p mimic, and the mRNA expressions of UBE3C were quantified; D: Western blot analysis to ascertain the protein levels of UBE3C and signal transducer and activator of transcription 3. ^aP < 0.001 vs negative control mimic group, ^bP < 0.01 vs negative control mimic group, n = 3. Results are presented as mean ± SD. UBE3C: Ubiquitin protein ligase E3C; NC: Negative control; UTR: Untranslated region.

Overexpression of UBE3C reversed promotive effects induced by miR-137-3p on hucMSC function

In a subsequent experiment, to explore if UBE3C is implicated in miR-137-3p-regulated functional variations of hucMSCs, hucMSCs were transfected with combination of miR-137-3p mimic and overexpression vector of UBE3C. Transwell assay revealed that migratory capacity of hucMSCs, when overexpressing UBE3C, was significantly impaired in miR-137-3p mimic + OE-UBE3C group compared to miR-137-3p mimic alone group (Figure 6A). Wound healing assay provided corroborative results, showing that hucMSC migration was effectively restrained in miR-137-3p mimic + OE-UBE3C group (Figure 6B). Moreover, in miR-137-3p mimic + OE-UBE3C group, expression of p-STAT3 protein was observed to decrease (Figure 6C). These outcomes suggest that overexpression of UBE3C reverses facilitative effects of miR-137-3p on hucMSC function.

Figure 6 Overexpression of ubiquitin protein ligase E3C negated the stimulatory influences of miR-137-3p on human umbilical cord mesenchymal stem cell performance. Human umbilical cord mesenchymal stem cells were transfected with miR-137-3p mimic or ubiquitin protein ligase E3C overexpression construct. A: Transwell assays to examine human umbilical cord mesenchymal stem cell migration. ^aP < 0.01, negative control (NC) mimic group vs miR-137-3p mimic group, ^bP < 0.01 miR-137-3p mimic group vs miR-137-3p mimic + overexpression of ubiquitin protein ligase E3C (UBE3C-OE) group. Scale bar = 100 μm, n = 3; B: Wound healing assays to gauge cell migration. ^aP < 0.0001, NC mimic group vs miR-137-3p mimic group, ^bP < 0.0001, miR-137-3p mimic group vs miR-137-3p mimic + UBE3C-OE group. Scale bar = 200 μm, n = 3; C: Western blot analysis of p-signal transducer and activator of transcription 3 protein levels. ^aP < 0.05, NC mimic group vs miR-137-3p mimic group, ^bP < 0.05, miR-137-3p mimic group vs miR-137-3p mimic + UBE3C-OE group. n = 3. The data are expressed as mean ± SD. HucMSC: Human umbilical cord mesenchymal stem cell; NC: Negative control; UBE3C-OE: Overexpression of ubiquitin protein ligase E3C; STAT3: Signal transducer and activator of transcription 3.

DISCUSSION

Thin endometrium is prevalent cause of chronic infertility and is associated with adverse outcomes[20]. It has been substantiated that exosomes derived from hucMSCs ameliorate hypoxia-induced injury in EECs[13]. Notably, our prior research has elucidated that EECD-ex augment hucMSC migration and differentiation into EECs. In this contribution, we have further unveiled that miR-137-3p is upregulated in hucMSCs co-cultured with EECD-ex. miR-137-3p facilitates hucMSC migration and differentiation into EECs. Furthermore, miR-137-3p targets UBE3C to promote STAT3 activation, thereby enhancing hucMSC migration and differentiation into EECs.

The genesis of exosomes commences with biogenesis of multi-vesicular bodies within cellular interior. Organelles within EECs damaged by hypoxia can encapsulate molecular cargoes within intra-luminal vesicles of exosomes, which are subsequently liberated into extracellular milieu upon multi-vesicular body fusion with plasma membrane[21]. Exosomes, once disseminated into extracellular environment, can be conveyed to distant tissues or cells via circulatory system, lymphatic system, or other bodily fluids. Exosomal surface is adorned with specific proteins and lipids, which facilitate their recognition and binding to target cells, thereby influencing tropism of exosomes. Target cells can internalize exosomes through variety of mechanisms, including phagocytosis, endocytosis, and receptor-mediated endocytosis. Once internalized, miRNAs harbored by exosomes are released into cytoplasm and modulate gene expression within target cell. MiRNAs exert regulatory control over biological functions, such as cell proliferation, differentiation, and apoptosis, by modulating stability or translational efficiency of specific mRNAs through binding[22].

Within scope of this investigation, exosomes characterized by high expression of miR-137-3p catalyze repair of endometrium through multitude of processes. These exosomes facilitate differentiation of stem cells into endometrial cells, critical step in regeneration of endometrium. Exosomes, enriched with elevated levels of miR-137-3p, convey molecular signals that induce differentiation to their target sites, guiding transformation of stem cells into necessary cell types and expediting repair of endometrium. By transferring miRNAs to umbilical cord MSCs, exosomes modulate gene expression profiles of these target cells, either promoting or inhibiting expression of specific genes. This capability is of paramount importance in exosomes’ role in regeneration of endometrium. By modulating expression of genes associated with repair and regeneration, exosomes contribute to enhancing functional status of endometrium. The bioactive molecules borne by exosomes with high miR-137-3p expression stimulate proliferation and migration of endometrial cells, thereby accelerating repair process. This is exceedingly beneficial for restoring integrity and thickness of endometrium, particularly in scenarios of endometrial damage, such as those caused by Asherman syndrome or endometriosis. It is conceivable that endometrial repair may also occur through alternative pathways. Exosomes contain growth factors and miRNAs that foster angiogenesis, facilitating formation of new blood vessels within endometrium to provide essential nutrients and oxygen, thus promoting its regeneration. Specific constituents within exosomes modulate inflammatory responses, mitigating harmful inflammation while fostering beneficial inflammation that participates in repair process. miR-137-3p derived from exosomes can enter umbilical cord blood MSCs (UCB-MSCs), enhancing their migratory capacity and promoting their differentiation into epithelial cells, while simultaneously inhibiting differentiation of mesenchymal cells. In UCB-MSCs with elevated miR-137-3p expression, overexpression of UBE3C further inhibits enhancement of migratory ability and differentiation potential toward epithelial cells. Post administration of a STAT3 inhibitor, migratory capacity and differentiation potential of UCB-MSCs overexpressing miR-137-3p into epithelial cells are also attenuated. It is plausible that exosomal miR-137-3p regulates biological functions of UCB-MSCs through downstream molecules, such as UBE3C and STAT3.

In total, 72 DEMs have been identified in thin, adhered endometrium of patients, as compared to adjacent normal endometrium, through miRNA sequencing analysis[18]. MiRNA microarray chip analysis reveals that in damaged endometrium of rats, 39 miRNAs are upregulated, and 45 miRNAs are downregulated, effect which is overturned by transplantation of hucMSCs[10]. In current study, miRNA sequencing was conducted to screen for DEMs in hucMSCs incubated with EEC-ex or EECD-ex. We observed that 53 DEMs (27 upregulated and 26 downregulated) were identified in hucMSCs pre-treated with EECD-ex. GO analysis demonstrated enrichment of DEM targets in specific cellular components, molecular functions, and biological pathways. Moreover, several pertinent signaling pathways were elucidated through KEGG analysis. DEMs were further validated using RT-qPCR, and our findings revealed pronounced upregulation of miR-137-3p in hucMSCs co-cultured with EECD-ex.

Compared to other delivery methods, such as transfection, exosome-mediated miRNA delivery exhibits several advantages: (1) Superior biocompatibility and relatively diminished immunogenicity. Exosomes, derived from living cells, inherit numerous intrinsic attributes of their parent cells, including specific protein markers on cell surface. This property enables exosomes to be tailored for targeted delivery, thereby enhancing selectivity for target cells. They demonstrate excellent biocompatibility in vivo and present a reduced risk of eliciting immune responses compared to synthetic carriers, such as liposomes or polymer nanoparticles[23]; (2) Enhanced permeability through biological barriers and markedly low RNA degradation rate. Exosomes possess capability to penetrate stringent biological barriers, such as blood-brain barrier, rendering them highly promising vehicle for treatment of central nervous system disorders. Internal microenvironment of exosomes shields carried RNA from degradation by cellular RNA enzymes, ensuring RNA integrity upon arrival at target cells[24]; and (3) Efficient uptake by target cells and a minimal side effect profile. Exosomes can be internalized by target cells via various mechanisms, including phagocytosis, endocytosis, and receptor-mediated endocytosis. In contrast to certain chemical carriers, exosomes exhibit lower toxicity and side effects, which enhances their safety as therapeutic vehicles[25].

It has been reported that in HTR-8/SVneo trophoblast cells stimulated by high glucose, miR-137-3p plays a role in regulating cell viability, migration, and invasion[26], osteogenic differentiation of bone marrow-derived MSCs[27], and myogenic differentiation of endometrial mesenchymal stem/stromal cells[28]. Here, we have revealed that overexpression of miR-137-3p not only facilitated migration of hucMSCs but also promoted their differentiation into EECs. To best of our knowledge, this study is first to validate biological function of miR-137-3p in modulating differentiation of hucMSCs into EECs.

STAT proteins are pivotal in a myriad of biological functions, including migration, proliferation, and differentiation[29]. Augmenting phosphorylation of STAT3 in bone marrow-derived MSCs can bolster osteogenic differentiation[30]. Icaritin enhances both proliferation and osteogenic differentiation of MSCs via activation of STAT3 pathway[31]. Hypoxia-induced migration and osteogenic differentiation of bone marrow-derived MSCs occur through STAT3 phosphorylation[32]. Myofibroblast differentiation of endometrial MSCs, driven by activin A, is also STAT3-dependent[33]. In this study, we identified that overexpression of miR-137-3p increased expression of p-STAT3 in hucMSCs, indicative of STAT3 signaling pathway activation. Notably, miR-137-3p accentuated migration and differentiation of hucMSCs into EECs, effect that was mitigated by STAT3 inhibitor, Stattic. Our findings thus conclude that miR-137-3p activates STAT3 signaling pathway to enhance migration and differentiation of hucMSCs into EECs. In this paper, although the mRNA level of STAT3 was not significantly up-regulated, the protein expression was remarkably elevated. It was found that miR-137-3p targeted the expression of ubiquitination-related proteins. We questioned whether miR-137-3p could inhibit the ubiquitination of STAT3 by suppressing the expression of ubiquitinated proteins. Moreover, we wondered if ubiquitination could occur, leading to a phenomenon where the antagonistic sites of STAT3 phosphorylation show higher-intensity phosphorylation. Subsequently, whether STAT3 with such modified phosphorylation status could enter the nucleus and influence downstream transcription factors was also under consideration. This line of speculation was repeated as we further explored if ubiquitination might take place, resulting in the appearance of the antagonistic site phenomenon of STAT3 phosphorylation with higher-intensity phosphorylation, and then enabling STAT3 to enter the nucleus and exert an effect on downstream transcription factors.

UBE3C is a member of HECT ubiquitin protein ligase family, which modulates crucial cellular signaling pathways[34]. UBE3C is essential in maintaining stem cell properties in lung cancer[35]. Particularly, UBE3C is key mediator in miRNA-regulated cell migration and proliferation. Prior studies have established that UBE3C is targeted by miR-542-3p and miR-30a-5p, which influences gene expression in hepatocellular carcinoma and breast cancer[36]. In present investigation, miRDB predicted that miR-137-3p might bind to UBE3C, and this direct interaction was subsequently confirmed using luciferase reporter assay. Our research unveiled that miR-137-3p targets UBE3C to potentiate STAT3 activation. Moreover, overexpression of UBE3C counteracted beneficial effects induced by miR-137-3p on hucMSC function. In future endeavors, we aim to explore intricate role of downstream gene UBE3C within ubiquitination process. UBE3C, which mitigates inhibitory factors that obstruct differentiation of umbilical cord MSCs into EECs, will undergo extensive evaluation through predictive database analysis and rigorous experimental validation to assess changes in its critical downstream targets. Thereafter, an inquiry into variation of its ubiquitination sites will be undertaken, with point mutations introduced at key residues to determine if such alterations can impact protein binding and, consequently, result in significant functional shifts. Alternatively, we envision developing an innovative ex vivo exosome-based therapeutic system, involving construction of a thin endometrial model treated in co-culture with UCB-MSCs overexpressing miR-137-3p. Following this treatment, therapeutic efficacy of exosomes will be rigorously assessed.

Although in vitro experiments can reveal the mechanisms of miR-137-3p at the cellular level, their results may not fully reflect the complex in vivo environment. Therefore, in vivo experiments are crucial for validating the actual effects of miR-137-3p in animal models, particularly in tissue repair, regeneration, or disease models. By injecting miR-137-3p mimics or inhibitors into animal models, one can observe their impact on tissue repair, inflammatory responses[37], or tumor growth[38], while also assessing their stability, biodistribution, and potential toxicity or side effects in living organisms. Additionally, in the in vivo environment, miR-137-3p may interact with other miRNAs, signaling pathways, or cytokines, forming a complex regulatory network, which further highlights the importance of in vivo experiments. Clinical trials are the critical step in evaluating the therapeutic potential of miR-137-3p, such as in wound healing, tissue regeneration, or the treatment of certain diseases, where miR-137-3p may serve as a potential biomarker or therapeutic target. Through clinical trials, the safety, efficacy, and optimal delivery methods of miR-137-3p in patients can be assessed. To successfully apply miR-137-3p in clinical settings, it is also necessary to develop effective delivery systems[39] to ensure its targeted delivery to specific tissues or cells and its functional efficacy.

CONCLUSION

In summary, our study has elucidated that miR-137-3p, sourced from EECD-exosomes, specifically targets UBE3C, thereby activating STAT3 signaling pathway. This activation notably facilitated migration and differentiation of hucMSC into EEC. These findings may serve as a foundational cornerstone for innovative therapeutic strategies aimed at addressing endometrial injury (Figure 7).

Figure 7 Under hypoxic injury, endometrial epithelial cells secrete exosomes rich in miR-137-3P, which act on umbilical cord blood mesenchymal stem cells. Through the sponge adsorption effect, miR-137-3P in these exosomes adsorbs ubiquitin protein ligase E3C, thereby promoting the phosphorylation of mitogen-activated protein kinase/protein kinase B. It also promotes the signal transducer and activator of transcription 3-mediated epithelial markers CD9/CD13, which in turn promotes the differentiation of umbilical cord blood mesenchymal stem cells into endometrial epithelial cells. HucMSC: Human umbilical cord mesenchymal stem cell; UBE3C: Ubiquitin protein ligase E3C; STAT3: Signal transducer and activator of transcription 3; MAPK: Mitogen-activated protein kinase; AKT: Protein kinase B.