MiR-455-5p upregulation in umbilical cord mesenchymal stem cells attenuates endometrial injury and promotes repair of damaged endometrium via Janus kinase/signal transducer and activator of transcription 3 signaling

ABSTRACT Umbilical cord mesenchymal stem cells (UCMSCs) are regarded as an ideal source for clinical use. Increasing evidence has suggested that microRNAs (miRNAs) work as a crucial regulator in the development of plentiful diseases, including intrauterine adhesions (IUA). Herein, we investigated the specific impacts of UCMSCs overexpressing miR-455-5p in IUA. UCMSCs were cocultured with endometrial stromal cells (ESCs). Thirty-two female mice were divided into four different treated groups: sham, model, model + UCMSC-miR-NC and model + UCMSC-miR-455-5p. Mice in model groups were induced by uterine curettage. MiR-455-5p overexpressed UCMSCs facilitated the proliferation and cell cycle progression of ESCs according to 5-ethynyl-2′-deoxyuridine assay and flow cytometry analysis. Hematoxylin-eosin and Masson staining revealed that miR-455-5p upregulation in UCMSCs increased the number of endometrial glands and suppressed endometrial fibrosis in murine uterine tissues. Western blotting displayed that miR-455-5p overexpressed UCMSCs promoted the activation of Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling in ESCs and murine uterine tissues. Mechanistically, miR-455-5p targeted 3’ untranslated region of suppressor of cytokine signaling 3 (SOCS3), which was confirmed by luciferase reporter assay. Reverse transcription quantitative polymerase chain reaction demonstrated that miR-455-5p was lowly expressed and SOCS3 was highly expressed in murine uterine tissues of IUA model. Moreover, Pearson correlation analysis showed that their expression was inversely correlated. Rescue assays suggested that inhibiting JAK/STAT3 signaling reversed effects of miR-455-5p on the behaviors of ESCs. The results indicated that miR-455-5p overexpression in UCMSCs helps to attenuate endometrial injury and repair damaged endometrium by activating SOCS3-mediated JAK/STAT3 signaling.


Introduction
Intrauterine adhesions (IUA), also known as Asherman's syndrome, results from damage to the basal layer of the endometrium [1]. Patients with moderate-to-severe IUA may struggle with pelvic pain, abnormal menstruation, recurrent miscarriages and even infertility [2,3]. IUA was reported to be the most common cause of uterine infertility, with approximately 25%-30% of infertile women suffering from IUA [4]. Hysteroscopy has been regarded as the mainstay for diagnosis of IUA and also as the most commonly used approach for IUA treatment followed by postoperative management [5]. Despite alternative strategies have been applied to limit or prevent re-adhesion, the recurrence rate after surgery is still very high, with 23% for moderate adhesion and 62% for severe adhesion [6]. Hence, more effective methods for IUA treatment are required to be found out.
Mesenchymal stem cells (MSCs) are multipotent stromal cells from adult tissues that have the capability of differentiating in different cell lineages [7]. Umbilical Cord MSCs (UCMSCs) is one of the ideal sources of MSCs, which are attractive for clinical use [8,9]. Compared to other kinds of MSCs, UCMSCs possess a superior capacity in differentiation, migration and self-renewal and can be invasively collected [10]. Increasing evidence has indicated that UCMSCs contribute to the repair of damaged endometrium [11]. For example, UCMSC transplantation restores endometrial thickness and attenuates excessive fibrosis in a rat model [12]. UCMSCs enhance the response of endometrium to hormones and improve endometrial proliferation as well as angiogenesis [13]. Furthermore, endometrial stromal cells (ESCs) function as a crucial cellular component in the endometrium, which is indispensable for proper physiological activities of the endometrium [14]. ESCs are implicated in the implantation and maintenance of pregnancy [15].
MicroRNAs (miRNAs) are small noncoding RNAs (21-24 nucleotides) which work as a critical post-transcriptional gene regulator [16]. Increasing evidence has revealed that miRNAs have great potential to be promising biomarkers with clinical utility [17]. Dysregulation of miRNAs are strongly associated with various human diseases, including IUA [18,19]. Recently, miR-455-5p has attracted increasing attention and has been reported to participate in multiple disorders. For example, miR-455-5p upregulation is related to the poor prognosis of patients with neonatal sepsis [20]. Moreover, miR-455-5p inhibits the proliferation and migration of vascular smooth muscle cells [21]. These suggest that miR-455-5p might be a biomarker in many diseases. Importantly, an IUA cell model was established in a previous study by treating ESCs with TGF-β1 [22]. ESCs were isolated from tissues of rats, and it was showed that rno-miR-455-5p overexpression could promote the proliferation of TGF-β1-treated ESCs [22]. However, the potential mechanism of hsa-miR-455-5p in IUA remains unanswered and there is no available evidence for the role of miR-455-5p in regulating the pathogenesis of IUA in vivo. Additionally, it has been indicated that miR-455-5p exerts its regulatory functions in some biological processes by targeting suppressor of cytokine signaling 3 (SOCS3) [23,24]. SOCS3 encodes a gene that can bind to Janus kinase 2 (JAK2) and inhibit the activity of JAK2 kinase [25]. Furthermore, SOCS3 is the main inhibitor of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling pathway [26]. The JAK/STAT3 signaling pathway has been reported to be implicated in the fibrosis of diseases, such as cardiac fibrosis, subepithelial fibrosis, and hepatic fibrosis [27][28][29].
This study intended to figure out the role and mechanism of miR-455-5p in UCMSC-treated ESCs and a IUA mouse model. We hypothesized that miR-455-5p might impact the proliferation and cell cycle progression of ESCs as well as the repair of damaged endometrium by targeting certain downstream gene and regulating the signaling pathway. This study might provide new ideas for the treatment of IUA.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from ESCs and murine uterine tissues utilizing TRIzol reagent (Invitrogen). The synthesis of cDNA for miRNA and mRNAs was achieved by reverse transcription of 1 µg RNA using miRNA First-Strand cDNA Synthesis kit (Sangon, Shanghai, China) and PrimeScript TM RT reagent Kit (Takara, Dalian, China), respectively. RT-qPCR was then implemented with Power SYBR Green RT-PCR reagents (Applied Biosystems, Foster City, CA, USA). The relative gene expression of miR-455-5p and messenger RNAs (mRNAs) was normalized to U6 and GAPDH, respectively, and analyzed with the 2 −ΔΔCt method [33]. Primer sequences are listed in Table 1.

Flow cytometry analysis
To analyze cell cycle, ESCs in each treatment group were harvested and fixed in 70% ethanol. Then the cells (1 × 10 6 ) were washed with PBS and incubated with PI/RNase Staining Buffer (BD Biosciences, San Jose, CA, USA) in the dark at 37°C. The distribution of cell cycle was analyzed with a FACSCanto II flow cytometer (BD Biosciences) [36].

Luciferase reporter assay
The fragment of wild type or mutant SOCS3 3'untranslated region (3'UTR) containing predicted binding site of miR-455-5p was synthesized and subcloned into pmirGLO vectors (Promega, Madison, WI, USA) to establish SOCS3-Wt/Mut. Phusion Site-Directed Mutagenesis Kits (Thermo Fisher Scientific) were used to mutate the predicted binding site. The above vectors were then co-transfected with miR-455-5p mimics or miR-NC into ESCs using Lipofectamine 2000 (Invitrogen). The luciferase activity was measured with a dual luciferase® reporter assay system (Promega) after 48 h of co-transfection [37].

Animal models
All the animal experiments were approved by the institutional Animal Care and Use Committee of Wuhan University of Science and Technology (Hubei, China) and were conducted strictly following the National Research Council Guide for the Care and Use of Laboratory Animals. Female six-week-old mice were purchased from Wuhan University of Science and Technology (Hubei, China). The establishment of IUA models was implemented according to the previous study [38,39]. Briefly, the mice were randomly divided into four groups: sham group, model group, model + UCMSC-miR-NC group and model + UCMSC-miR-455-5p mimics group (n = 8 per group). After anesthesia by intraperitoneal injection of 1% sodium pentobarbital (0.5 mL/kg), the abdominal cavity was opened to expose the uterus. Then, an incision was made at the bilateral uterus junctions. A mini-endometrial curette was inserted into uterus cavity through the incision for scraping until the uterus was hyperemic to the naked eye. After that, the abdominal cavity was closed. Mice in the sham group had the same laparotomy without any treatment for the uteri. In the model + UCMSC-miR-NC group and model + UCMSC-miR-455-5p mimics group, UCMSCs transfected with miR-NC or miR-455-5p mimics were injected into the mice, respectively. After treatment for 4 weeks, the mice were sacrificed by cervical dislocation, and uterine tissues were harvested.

Hematoxylin-eosin (HE) staining and Masson staining
Murine uterine tissues were fixed with 4% paraformaldehyde for 24 h before paraffin embedding. After being deparaffinized and rehydrated, the slides were stained with hematoxylin and eosin. The pathological changes of uterine tissues were observed under a Leica DMLB2 microscope (Nussloch, Germany) [40]. The slides were subjected to Masson staining and four high-power fields were selected for each slide. The results of endometrial fibrosis were analyzed with Image-pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA).

Statistical analysis
Data were analyzed using Statistical Product and Service Solutions (SPSS) 19.0 (IBM Corp, Armonk, NY, USA) and are expressed as the mean ± standard deviation (SD). Each experiment was repeated at least three times. Significant differences between two groups were examined by Student's t-test, while those among more groups were evaluated by analysis of variance (ANOVA) followed by Tukey's post hoc analysis [41]. The value of p < 0.05 was regarded as statistically significant.

Results
MiR-455-5p was reported to exacerbate the process of IUA in a previous study; however, the specific impact and mechanism of miR-455-5p in UCMSCs underlying the progression of IUA are obscure. This study aimed to explore the role of miR-455-5p in UCMSCs-treated ESCs and murine IUA models. We hypothesized that miR-455-5p in UCMSCs might impact the phenotypes of ESCs and damaged endometrium by regulating the downstream target and signaling pathway. The results indicated that miR-455-5p upregulation in UCMSCs promotes the proliferation and cell cycle progression of ESCs, increases the number of endometrial glands and suppresses endometrial fibrosis in murine uterine tissues by activating SOCS3-mediated JAK/STAT3 signaling.

Identification of UCMSCs
First, we identified UCMSCs from three aspects. Using phase-contrast microscopy, the cells were observed to show a fibroblast-like morphology, adherent growth and a spiral arrangement (Figure 1(a)). Afterward, differentiation induction media were used to examine the multipotency of UCMSCs. The vacuoles of cells cultured in adipogenic medium gathered together to form lipid droplets in cytoplasm and displayed Oil Red O staining (Figure 1(b)). In parallel, the cells cultured in osteogenic medium exhibited a calcium nodule shape and Alizarin Red staining (Figure 1 (c)). Moreover, flow cytometry analysis indicated that the cells were positive for CD90, CD73 and CD105 ( 95%), while were negative for CD19, CD45, CD34, CD11b and HLA-DR ( 5%) (Figure 1(d)). The characteristics of cells from three aspects were all in accord with MSCs.

MiR-455-5p promotes cell proliferation and cell cycle progression
The primary ESCs were observed to adhere to the plastic culture dishes and show a spindle-shaped and fibroblast-like morphology (Figure 2(a)). To investigate the impact of miR-455-5p in UCMSC-treated ESCs, we cocultured ESCs with blank control medium, UCMSCs with miR-NC or UCMSCs with miR-455-5p mimics in a Transwell system (Figure 2(b)). After coculture, ESCs were collected from Transwell inserts for further experiments. Then, we detected  transfection efficiency of miR-455-5p mimics in UCMSCs and as shown by RT-qPCR, miR-455-5p level was obviously raised after transfection of miR-455-5p mimics (Figure 2(c)). Afterward, EdU assay was implemented to examine impact of miR-455-5p on cell proliferation. Compared to the control group, UCMSC-miR-NC group showed an increased number of EdU positive cells, while UCMSC-miR-455-5p mimics group displayed a higher level of EdU positive cells (Figure 2(d,e)). Furthermore, a similar trend was observed in flow cytometry analysis. The number of ESCs at S phase evidently increased in UCMSC-miR-NC group, proving the promotive influence of UCMSCs on cell cycle (Figure 2(f,g)). Additionally, miR-455-5p mimics evidently induced an increase in the G2 phase cell cycle populations (Figure 2(f,g)), suggesting that miR-455-5p promoted S/G2 transition in ESCs. Hence, the above results indicate that miR-455-5p facilitates the proliferation and cell cycle progression of ESCs cocultured with UCMSCs.

MiR-455-5p attenuates endometrial injury and promotes repair of damaged endometrium
To verify whether miR-445-5p affects the progression of IUA, we established a murine model of IUA and collected the uterine tissues at the 4 th week after modeling. As revealed by HE staining, in the sham group, the uterine cavity surface was covered with columnar epithelium and the endometrial glands were abundant, arranged in round or oval shapes (Figure 3(a,  b)). In the model group, tissues were observed to be adherent, and the number of glands was markedly decreased (Figure 3(a,b)). In comparison to the model group, the number of glands in the model + UCMSC-miR-NC group displayed was elevated (Figure 3(b)). Notably, miR-455-5p treatment significantly led to an enhancement in the number of glands compared with the model + UCMSC-miR-NC group (Figure 3(b)). These results indicated that both UCMSCs and miR-455-5p could increase the number of glands. Furthermore, Masson staining was performed to investigate the impact of miR-455-5p on endometrial fibrosis. As shown by the results, compared to that in the sham group, the fibrotic area in the endometrium of model group was remarkably enhanced, but UCMSC treatment resulted in a slight reduction in fibrosis (Figure 3(c,d)). Notably, an evident reduction in fibrotic area was displayed in miR-455-5p mimics-treated group compared with the miR-NC-treated group (Figure 3(c,d)), suggesting that miR-445-5p contributed to the improvement of endometrial fibrosis. Collectively, miR-455-5p increases the number of endometrial glands and suppresses endometrial fibrosis, thereby helping to attenuating endometrial injury and promoting repair of damaged endometrium.

MiR-455-5p expression is negatively correlated with SOCS3 expression in murine uterine tissues
The expression correlation between miR-455-5p and SOCS3 was further analyzed. Obviously, in comparison to that in the sham group, the level of miR-455-5p was reduced in the model group (n = 8/group), as shown by RT-qPCR ( Figure 5(a)). A higher level of miR-455-5p was observed in UCMSC-miR-455-5p mimics-treated group than that in UCMSC-miR-NCtreated group (n = 8/group) ( Figure 5(a)). Moreover, SOCS3 displayed a higher level in the model group than it was in the sham group, and SOCS3 was significantly downregulated in UCMSC-miR-455-5p mimics-treated group compared to UCMSC-miR-NC-treated group (n = 8/group) ( Figure 5(b)). Additionally, Pearson correlation analysis suggested that miR-455-5p expression had a negative correlation with SOCS3 expression in murine uterine tissues (n = 24) ( Figure 5(c)).

MiR-455-5p activates the JAK/STAT3 signaling pathway
SOCS3 is known to be an inhibitor of the JAK/ STAT3 signaling pathway [26]. Subsequently, we investigated how miR-455-5p regulated the SOCS3mediated JAK/STAT3 signaling pathway. Western blotting was used for evaluating the levels of JAK/ STAT3 signaling pathway-associated proteins. As shown by the results, miR-455-5p mimics remarkably enhanced protein levels of phosphorylated (p)-JAK2 and p-STAT3 in ESCs (Figure 6(a)). Additionally, SOCS3 protein expression was obviously upregulated in the model group and model + UCMSC-miR-NC group and was slightly upregulated in miR-455-5p mimics-treated group (Figure 6(b)). With respect to those in the sham group, the protein levels of p-JAK2 and p-STAT3 were reduced in the model and model + UCMSC-miR-NC groups, and this effect was reversed after treatment of miR-455-5p mimics (Figure 6(b)). Hence, miR-455-5p facilitates the phosphorylation of JAK2 and STAT3 by targeting SOCS3 in ESCs and endometrial tissues, thereby activating the JAK/ STAT3 signaling pathway.

The influence of miR-455-5p on the phenotypes of ESCs is reversed by AG490
To further elucidate that miR-455-5p influences the phenotypes of ESCs by regulating JAK/STAT3 signaling, AG490 was used to inhibit the JAK/STAT3 signaling pathway. EdU assays demonstrated that the proliferation of ESCs promoted by miR-455-5p was attenuated after AG490 treatment (Figure 7(a,b)). Likewise, flow-cytometry analysis indicated that miR-455-5p mimics promoted the cell cycle progression of ESCs, but this effect was reversed by AG490, which led to the decrease of cell cycle at G2/ M (Figure 7(c,d)). Thus, the inactivation of JAK/ STAT3 signaling induced by AG490 reverses the influence of miR-455-5p on the phenotypes of ESCs.

Discussion
One of the most important pathological process of IUA is endometrial fibrosis [42]. Considerable studies have demonstrated that MSCs exert an indispensable role in IUA. For example, MSC treatment suppresses inflammation and fibrosis in a rat model, thereby improving the condition of IUA [43]. Moreover, MSCs were reported to be a therapeutic agent of endometrial fibrosis [44]. MSCs are able to limit endometrial fibrosis via inhibiting TGF-β that is involved in the formation of IUA [44]. UCMSCs, one of the best sources of MSCs, was once reported to ameliorate the severity of liver fibrosis [45]. Moreover, UCMSCs contribute to endometrial regeneration and repair [11]. In the present study, we cocultured ESCs with UCMSCs and confirmed that UCMSCs could promote the proliferation and cell cycle progression of ESCs and help to repair the damaged endometrium. Emerging evidence has suggested that miRNAs are associated with a multitude of human disorders [46]. MiR-455-5p has been reported to be a potential biomarker in many diseases. For example, miR-455-5p upregulation influences retinol absorption in a lung hypoplasia model by regulating STRA6 [47]. Moreover, miR-455-5p is considered to be a diagnostic and prognostic marker for neonatal sepsis [20]. A previous study has indicated that rno-miR -455-5p contributes to the proliferation of ESCs that are treated with TGF-β for establishing an IUA cell model [22]. MiR-455-5p was reported to be involved in the regulation of cell cycle in multiple diseases. For example, miR-455-5p is sponged by HOXA-AS3 in atherosclerosis and can reverse HOXA-AS3 knockdown-mediated promotive effect on cell cycle [48]. In the current study, miR-455-5p was overexpressed in UCMSCs that were cocultured with ESCs or implanted into the mice. It was found that miR-455-5p overexpression facilitated the proliferation and cell cycle progression of ESCs. Additionally, it was shown that after overexpressing miR-455-5p, the number of endometrial glands was increased and endometrial fibrosis was limited in murine uterine tissues, suggesting that miR-455-5p was favorable for the regeneration and repair of damaged endometrium.
MiRNAs are recognized to regulate the stability and translation of mRNAs by targeting their 3'UTR [49]. Bioinformatics tool ENCORI was utilized for predicting the downstream targets containing binding site for miR-455-5p, and SOCS3 was singled out. SOCS3, a member of SOCS family, was reported to affects the fibrosis of some diseases. For example, absence of SOCS3 leads to the liver fibrosis by increasing the production of TGF-β that was mediated by STAT3 [50]. In our study, a series of assays were implemented to verify the interaction between SOCS3 and miR-455-5p. Notably, miR-455-5p expression was shown to have a negative correlation with SOCS3 expression in murine uterine tissues. Furthermore, previous investigations have elucidated that SOCS3 works as an inhibitor of the JAK/STAT3 signaling pathway [26,51]. Here, we investigated the impact of miR-455-5p on SOCS3-mediated JAK/STAT3 signaling pathway.
It was found that miR-455-5p downregulated SOCS3 and facilitated the phosphorylation of JAK2 and STAT3 in ESCs and murine uterine tissues, indicating that miR-455-5p exerted its influences by activating the JAK/ STAT3 signaling. To further confirm this effect, rescue assays were carried out. AG490 was used as an inhibitor of the JAK/STAT3 signaling pathway. As anticipated, after inhibiting the JAK/STAT signaling, the promotive influence on the proliferation and cell cycle progression of ESCs induced by miR-455-5p was attenuated.

Conclusion
In conclusion, this study explored the functions and mechanism of miR-455-5p overexpressed UCMSCs in the proliferation and cell cycle progression of ESCs. Moreover, we investigated the impacts of miR-455-5p on endometrial glands and endometrial fibrosis in a mouse model. Overexpression of miR-455-5p in UCMSCs promotes ESC proliferation and cell cycle progression, attenuates endometrial injury and facilitates repair of damaged endometrium by regulating SOCS3-mediated JAK/STAT3 signaling pathway. These findings might provide new ideas for treating IUA.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The work was supported by Health Commission of Hubei Province (WJ2018H0146) and the 4th Wuhan Young and Middle-aged Backbone Talent of Medical Training Project.