circPTP4A2-miR-330-5p-PDK2 Signaling Facilitates In Vivo Survival of HuMSCs on SF-SIS Scaffolds and Improves The Repair of Damaged Endometrium

circPTP4A2-miR-330-5p-PDK2 Signaling Facilitates In Vivo Survival of HuMSCs on SF-SIS Scaffolds and Improves The Repair of Damaged Endometrium | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research circPTP4A2-miR-330-5p-PDK2 Signaling Facilitates In Vivo Survival of HuMSCs on SF-SIS Scaffolds and Improves The Repair of Damaged Endometrium Yuanyuan Zheng, Linhao Li, Xuewei Bi, Ruyue Xue This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-895708/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Human umbilical cord MSCs (HuMSC)-based therapy has shown promising results in the treatment of intrauterine adhesions (lUA). In this study, our aim was to construct a HuMSC-seeded silk fibroin small-intestinal submucosa (SF-SIS) scaffold and evaluate the impact of repairing the damaged endometrium in an lUA mouse model. Methods To identify the functional effect of HuMSCs-silk cellulose (SF)- small-intestinal submucosa (SIS) scaffolds on the repair of damaged endometrium, a mouse lUA model was established in this study. The uterine morphology and fibrosis were evaluated by hematoxylin - eosin (H&E) staining and Masson staining. CircRNA sequencing, real-time PCR and RNA fluorescence in situ hybridization were used to screen and verify the potential circRNAs that involved in the repair of damaged endometrium by HuMSCs. Real time integrated cellular oxygen consumption rate (OCR) was measured using the Seahorse XF24 Extracellular Flux Analyser. The potential down-stream miRNAs and proteins of circRNAs were analyzed dual-luciferase report and Western Blot. Results We found that HuMSCs-SF-SIS not only increased the number of glands, but also reduced the ulcer area in the IUA model. Furthermore, we demonstrated that circPTP4A2 was elevated in the HuMSCs seeded on the SF-SIS scaffolds and stabilized the mitochondrial metabolism through miR-330-5p-PDK2 signaling, which contributes to endometrial repair progression. Conclusion In this study, we demonstrated that circPTP4A2 was elevated in the HuMSCs seeded on the SF-SIS scaffolds and stabilized the mitochondrial metabolism through miR-330-5p-PDK2 signaling, which contributes to endometrial repair progression. These findings demonstrate that HuMSC-seeded SF-SIS scaffolds are an encouraging method for the treatment of lUA. Stem Cell & Developmental Cell Biology Intrauterine adhesions Human umbilical cord MSCs (HuMSC) Silk fibroin surface modified small-intestinal submucosa (SF-SIS) CircPTP4A2 Mitochondrial metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Embryo implantation failure caused by female endometrial damage is the main factor affecting the success rate of assisted reproduction. Cells in the functional layer of the endometrium in a normal cycle are apoptotic and shed, and release signals to activate endometrial stem cells (ESCs), promoting angiogenesis and tissue repair. However, after the damage, the endometrium is deficient in blood vessels and has few glands leading to thinning of the uterine lining (atrophy)[ 1 , 2 ]. Intrauterine adhesions (IUA), also called Asherman's syndrome, is a common uterine disease caused by damage to the basal lining of the endometrium caused by mechanical injury or infection, resulting in endometrial fibrosis, uterine obstruction, menstrual abnormalities, infertility and pregnancy [ 3 , 4 ]. Presently, hysteroscopic adhesiolysis is performed clinically, and anti-adhesion drugs and IUD are administered simultaneously [ 5 ]. Several therapeutic agents have been used to improve endometrial regrowth [ 6 ]. However, due to the loss and functional destruction of endometrial basal stem cells, the therapeutic effect is not ideal, and a normally functional endometrial environment cannot be provided. Mesenchymal stem cells can differentiate into endometrial epithelial and mesenchymal cells after induction, suggesting that mesenchymal stem cells have biological functions similar to those of endometrial basement ESCs and may be involved in the repair of the endometrial structure and improve its function [ 7 ]. However, multiple challenges persist in the treatment of endometrial damage by exogenous mesenchymal stem cells, such as differences in the cell carrier and the intrauterine environment [ 8 ]. Generally, human umbilical cord MSCs (HuMSCs) were superior to bone marrow MSCs (BM-MSCs) in terms of cell content and proliferative capacity, and have lower immunogenicity than BM-MSCs [ 9 , 10 ]. In addition, (HuMSCs) are convenient to obtain materials and are free from ethical controversies, and other advantages have attracted increasing attention [ 11 ]. Studies using in vivo HuMSC transplantation experiments to observe endometrial reconstruction, two months after the detection of transplantation, showed a thickening of the damaged endometrial tissue, a reduction in the area affected by fibrosis, and a similar repair period. It is speculated that HuMSCs secreted by paracrine factors have anti-inflammatory effects, facilitate the repair of the endometrium, and maintain cell function and angiogenesis of the microenvironment [ 12 ]. However, the molecular mechanism underlying the effect of HuMSCs on the repair and regeneration of the endometrium remains unclear. Recently, a significant focus has been expressed on the application of tissue-engineered artificial reconstruction materials for tissue repair and regeneration. Small-intestinal submucosa (SIS) is a cell-free, allogeneic, and collagen-matrix material with good histocompatibility and biomechanical properties and no immunogenicity and toxicity [ 13 ]. It is a natural biodegradable biological material [ 14 – 16 ]. The submucosa of the small intestine is mainly composed of type I collagen isotype, and is rich in fibronectin and growth factors, among which aplasia collagen and fibronectin can specifically bind to the cell membrane and activate the cell conduction pathway to facilitate cell adhesion[ 17 ]. As a tissue-engineered material, SIS has been widely used in the research of cartilage, tendon, bladder, cardiovascular regeneration, and other fields, however a few studies have been reported on the repair of injuries of the endometrium and the molecular mechanism [ 18 , 19 ]. Silk cellulose (SF) has the three advantages of good mechanical properties, controllable biodegradation and excellent biocompatibility, and has been widely used in the field of biomedicine [ 20 , 21 ]. The SIS membrane is a material coated with SF layers, which can adjust the diffusion rate of drugs or biomolecules and improve the mechanical and structural stability of its properties. In this study, we constructed HuMSC-seeded SIS scaffolds and evaluated the impact of the repair of damaged endometrium in an lUA mouse model. Moreover, We also studied the potential mechanism of HuMSCs in repairing the endometrium. 2. Materials And Method 2.1 Preparation and characterization of SF-SIS scaffolds The SF-SIS scaffolds used in this study was kindly donated from Dr. Li (The Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China). The SF-SIS scaffolds were prepared and characterized as previously described [ 20 ]. 2.2 Characterization and culture of HuMSCs HuMSCs is purchased from Promocell (Miaotong (Shanghai) Biological Science & Technology Co., Ltd. Shanghai, China). Frozen HuMSCs between P2 and P10 were freshly inoculated in 100 mm culture dishes (1×10 6 cells per dish) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, USA), penicillin (100 U/ml, Gibco) and Streptomyces (100 mg/ml, Gibco). Briefly, the phenotype of HuMSCs was identified by FACS specificity (CD34, CD45, CD29, CD90, CD105 and HLA-DR). The osteogenic and adipogenic abilities of mesenchymal stem cells were determined by the Cyagen Osteogenic stimulation kit (Cyage, Guangzhou, China) and the Cyagen Adipogenic Differentiation Kit (Cyage, Guangzhou, China). 2.3 Osteogenic and adipogenic differentiation of HuMSCs According to the manufacturer's instructions (RASMX-90021 and RASMX-90031, Cyagen, Guangzhou, China), the ability of cells to differentiate into osteoblasts or adipocytes was characterized using commercial osteogenic and adipogenic induction kits. Osteoblasts were stained with Alizarin Red S and adipocytes with oil red O. 2.4 Animal IUA model All animal procedures are approved by the the first affiliated hospital of Zhengzhou University, Institutional Animal Care and Use Committee and are carried out in accordance with the National Research Council Laboratory Animal Care and Use Guidelines. 6-week-old BALB /c mice were obtained from Shanghai SLAC Animal Center and allowed to acclimatize to the new environment for a week. To establish an IUA model, the uterus was mechanically damaged during pregnancy. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital. The uterus is excised at the midline below the abdomen. Insert a size 7 needle into the left and right uterine junction and cut it carefully back and forth until the uterine congestion is visible to the naked eye. Then, the abdominal cavity was closed. The operation was performed under aseptic conditions. The control group did not undergo surgery. 2.5 Histological analysis Uterine specimens were collected 8 days after surgery. The sample was fixed with 4% paraformaldehyde, dehydrated, removed with xylene, and finally embedding with paraffin. The embedded tissue sections were 5-µm thick. The uterine morphology and structure were evaluated by hematoxylin - eosin (H&E) staining. Masson staining was performed to evaluate the fibrosis according to manufacturer's instructions (Yeasen, Shanghai, China). Fibrosis areas (light blue) were evaluated using ImageJ software (MD, USA). 2.6 circRNA sequencing Total RNAs were extracted with Trizol (Invitrogen, Carlsbad, CA, USA). Sequencing libraries were generated and sequenced by Sango Bio Technology (Shanghai, China). A total amount of 5 µg RNA per sample was used. The libraries were subjected to paired-end sequencing with pair end 150 bp reading length on an Illumina HiSeq sequencer (Illumina, San Diego, CA, USA). 2.7 Real-time PCR Total RNA was extracted with Trizol reagent (TAKARA), and 500 ng total RNA was transcribed into cDNA using PrimeScript RT Master Mix (TAKARA, Dalian, China). Subsequently, SYBR Premix Ex Taq II Kit (TAKARA) was used to detect the expression of circRNAs and mRNA by RT-qPCR, which was normalized to endogenous control GAPDH expression. Folding changes were calculated using 2 −ΔΔCT method. 2.8 RNA fluorescence in situ hybridization RNA fluorescence in situ hybridization was performed using a fluorescence in situ hybridization kit (RiboBio, Guangzhou, China) in accordance with the manufacturer's guidelines. Cy3-labeled circPTP4A2 probe (RiboBio, Guangzhou, China) was detected with fluorescence in situ hybridization kit and then observed with LSM800 confocal microscopy (Zeiss, Germany). 2.9 Measurement of mitochondrial metabolism ATP content and synthase activity was performed using the quantification kits (Sango Biotech, Shanghai, China) in accordance with the manufacturer's guidelines. Real time integrated cellular oxygen consumption rate (OCR) was measured using the Seahorse XF24 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA, USA) as previous described. In brief, HuMSCs cells were treated with 10 µg/mL curcumin for 12 h and 10 4 cells were plated into the seahorse customized cell plates. After the probes were calibrated, the OCR was detected with sequential injection of the following compounds which regulate mitochondrial respiration: oligomycin (ATP synthase inhibitor; 1 µM), FCCP (uncoupler; 1 µM), rotenone (complex I inhibitor; 1 µM), and antimycin A (complex III inhibitor; 1 µM). 2.10 Dual-luciferase reporter assays The 293T cells were incubated in 24-well plates with 2×10 4 cells per well. These cells were then transfected with psiCheck2-circPTP4A2-WT or psiCheck2-circPTP4A2-Mut, miR-330-5p mimics, or miR-NC. After 48 h, the cells were lysed with a passive lysis buffer (Promega, Madison, WI, USA) and the dual luciferase reporting assay (Promega) was used to calculate the relative luciferase activity by normalized firefly luminescence to Renilla luminescence. 2.11 Western blotting The HuMSCs were lysed with RIPA lysis buffer. The same amount of protein was then broken down by SDS-PAGE analysis and electrically transferred to PVDF membranes (Milliore, Schwalbach, Germany), which were then sealed with 5% skimmed milk powder and incubated overnight with primary antibody at 4°C. The primary antibodies used were anti-PDK2 (Cell Signaling Technology) and anti-GAPDH (Kanchen Biotech). The membrane was then incubated with HRB-conjugated secondary antibodies at room temperature for 1 h, and the imprinting was observed using an enhanced chemiluminescence kit (Pierce, Waltham, MA, USA). 2.12 Statistical Analysis Data were expressed as the Means ± SEM. For two groups, 2-tailed t-test (unpaired) was used for comparisons. For multiple comparisons, ANOVA followed by the post hoc Bonferroni test was taken with GraphPad Prism® version 9.0 software (GraphPad Software, Inc., La Jolla, CA, USA). 3. Results 3.1 Culture and characterization of HuMSCs BMSCs are adsorbed on plastic discs. The typical morphology of HuMSCs is similar to that of spindle-shaped fibroblasts, which grow in a tightly packed vortex pattern, is shown in Fig. 1 A. The HuMSCs were successfully differentiated into osteoblasts and adipocytes in vitro (Fig. 1 A), this indicates that this cell population was a pluripotent mesenchymal stromal cell. Furthermore, FACS results showed that most cells were negative for hematopoietic markers after 7 days in culture for CD34+ (5.12%), CD45+ (2.05%), HLA-DR (0.61%) and highly positive for CD29 (61.9%), CD90 (94.9%) and CD105 (94.9)%.expression (Fig. 1 B). 3.2 HuMSCs-SF-SIS reduces fibrotic area and increases the number of glands in the lUA model To identify the functional effect of HuMSCs-SF-SIS scaffolds on the repair of damaged endometrium, a mouse lUA model was established.The HuMSCs-SF-SIS scaffolds were transplanted after the damage to the endometrium was reduced. After 4 weeks, uterine tissue was collected up for modeling. According to the HE staining results, the shape of the uterine cavity in the pseudopod group was irregular. Columnar epithelial cells cover the uterus and gland cavities. Epithelial cell structure was complete, stromal glands were abundant, and oval. Connective tissue fragments were found in the uterine cavity in the IUA model group, the number of glandular blood vessels were significantly reduced, and the connective tissue was congested one week after the damage. The number of endometrial glands in the HuMSCs-SF-SIS transplanted group increased, and the lumen of neonates was not completely covered by the monolayer columnar epithelium (Fig. 2 A,P < 0.05). According to the results of statistical analysis, for the number of endometrial glands, the model group and HuMSCs-SF-SIS transplantation group were lower than the sham-operated group (Fig. 2 B, P < 0.05), and the model group was significantly lower than the sham-operated group (P < 0.05). Differently, the group of HuMSCs-SF-SIS transplantation group was significantly higher than the IUA model group (Fig. 2 B, P < 0.05). Endometrial adhesions are characterized by fibrosis. In the study, we used Masson staining (Fig. 2 C) to assess the degree of fibrosis. A significant increase in the area of endometrial fibrosis was observed in lUA model mice compared with the sham group (Fig. 2 D,P < 0.05), but HuMSCs-SF-SIS transplantation resulted in a significant decrease in fibrosis (Fig. 2 D, P < 0.05). In conclusion, transplantation of HuMSCs-SF-SIS not only increased the number of endometrial glands, but also repaired the injured endometrium. 3.3 The expression level of circPTP4A2 was significantly elevated in the HuMSCs cultured on the SF-SIS scaffolds To explore whether circRNA is involved in endometrial repair, we first performed RNA-seq analysis of total RNA from ribosomal RNA of normal HuMSCs and HuMSCs cultured on SF-SIS scaffolds (Fig. 3 A). A total of 54 circRNAs were significantly downregulated, and 27 circRNAs were upregulated in HuMSCs cultured on the SF-SIS scaffolds (filtered by FC(fold change) > 2 and P < 0.05). Variability expression of circRNAs was directly displayed by volcano clustering analysis. (Fig. 3 B). We then chose several significantly upregulated circRNAs (circUXSI, circPTP4A2, circCNTRL, circEPSTII, circSFMBT2, circZNF680, and circEMB) to verify their existence via real-time PCR (Fig. 3 C). We concluded that circPTP4A2 was upregulated in HuMSCs cultured on the SF-SIS scaffolds (Fig. 3 C). In a follow-up study, we found resistance between circPTP4A2 and RNase R and also confirmed that PTP4A2 mRNA showed a significant reduction after RNase R treatment (Fig. 3 D). Moreover,(sub-fractional real-time PCR (Fig. 3 E) and fluorescence in situ hybridization (FISH) assays (Fig. 3 F) data indicated that circPTP4A2 was mainly present in the cytoplasm. 3.4 CircPTP4A2 facilitates the mitochondrial metabolism of HuMSCs under hypoxia condition Mitochondria are essential in the cellular biochemistry of most eukaryotic cells, producing nearly 95% of cellular ATP through oxidative phosphorylation of mitochondria, thereby controlling cell death or survival under hypoxic conditions, such as the transplanted SF-SIS scaffolds. Considering the critical role of mitochondrial metabolism, we tested whether circPTP4A2 facilitates the biological functions of HuMSCs via the regulation of mitochondrial metabolism. For the purpose of hypothesis testing, we first determined the effects of circPTP4A2 on ATP content (Fig. 4 A) and ATP synthase activity (Fig. 4 B).We found that the ATP content and ATP synthase activity were dramatically decreased in hypoxic HuMSCs, but significantly increased with the overexpression of circPTP4A2. Using the seahorse XF24 extracellular flux analyzer, we also analyzed the cell oxygen consumption rate (OCR) (Fig. 4 C), indicating reduced OXPHOS in HuMSCs cells. We then assessed the mitochondrial functions, in particular, basal respiration (Fig. 4 D), maximal respiration (Fig. 4 F), ATP production (Fig. 4 E), spin respiratory capacity (Fig. 4 G), proton leak (Fig. 4 H), and non-mitochondrial respiration (Fig. 4 I). Hypoxia treatment dramatically impaired the OCR value of basal respiration, spare respiratory capacity, maximal respiration, and ATP production in HuMSCs. In contrast, overexpression of circPTP4A2 significantly attenuated the inhibitory effect of hypoxia on ATP content, ATP synthase activity, and cell oxygen consumption rate (OCR). These data indicate that circPTP4A2 could facilitate the mitochondrial metabolism of HuMSCs under hypoxic conditions. 3.5 CircPTP4A2 is targeted by miR-330-5p in HuMSCs cells To explore whether circPTP4A2 can function as “miRNA sponge” in HuMSCs cells, we selected several potential miRNAs (miR-326, miR-487a, miR-335, miR-532-3p, miR-421, miR-502-5p, miR-330-5p, miR-1290 and miR-1305) through Starbase 2.0 database. After circPTP4A2 knockout, we found an increased level of miR-330-5p (Fig. 5 A) in HuMSCs cells. Enrichment of miR-330-5p with circPTP4A2 was derived from the Ago2 co-immunoprecipitation assay (Fig. 5 B) and miR-330-5p RNA pull-down assay (Fig. 5 C). To further verify the miR-330-5p target circPTP4A2, we added a luciferase reporter gene test. The results showed that miR-330-5p mimics significantly reduced the luciferase activity of HuMSCs transfected with wild-type circPTP4A2. But the miR-330-5p mimic failed to reduce the luciferase activity of the mutant circPTP4A2 transfected HuMSCs cells (Fig. 5 D). These results suggest that circPTP4A2 is targeted by miR-330-5p in HuMSCs. 3.6 MiR − 330- 5p over-expression impaired the circPTP4A2 enhanced mitochondrial metabolism in hypoxia - treated HuMSCs. To identify whether miR − 330–5p is critical to circPTP4A2 enhanced mitochondrial metabolism in hypoxia – treated HuMSCs. We transfected the miR − 330–5p mi mic in hypoxia - treated HuMSCs with or without circPTP4A2 overexpression. The miR − 330–5p mimic dramatically reduced ATP content (Fig. 6 A ) and ATP synthase activity (Fig. 6 B). Moreover, miR-330-5p mimics the Reduced basal respiration, spare respiratory capacity, ATP produ ction, and maximal respiration in HuMSCs cells (Fig. 6 C). Notably, circPTP4A2 overexpression failed to increase basal respiration (Fig. 6 D), ATP production (Fig. 6 E), maximal respiration (Fig. 6 F) ,and spare respiratory capacity(Fig. 6 G) in HuMSC cells transfected with miR-330-5p mimic. Through the above results, it is shown that miR-330-5p overexpression impaired circPTP4A2 enhanced mitochondrial metabolism in hypoxia - treated HuMSCs. 3.7 MiR − 330–5p inhibits PDK2 expression through the 3' UTR target region To further explore the underlying mechanism of the circPTP4A2/miR-330-5p axis on mitochondrial metabolism in hypoxia-treated HuMSCs, we then screened the potential targets of the miR-330-5p Target Scan and Star Base databases. Further by dual luciferase assay, we found that the 3’UTR mRNA of the mitochondrial metabolism regulator PDK2 was directly targeted by miR-330-5p (Fig. 7 A- 7 D). In addition, PDK2 mRNA (Fig. 7 B) and protein (Fig. 7 C) levels were significantly reduced by miR-330-5p mimics in mouse umbilical cord MSCs and HuMSC. Taken together, it can be concluded that miR-330-5p may regulate mitochondrial metabolism by suppressing the expression of PDK2. 3.8CircPTP4A2-miR-330-5p-PDK2 signaling is critical to HuMSCs-SF-SIS decreasing the fibrosis area and increasing the number of glands in IUA model To confirm the critical role of circPTP4A2-miR-330-5p-PDK2 signaling in the progress of the repair of the endometrium by HuMSCs-SF-SIS in IUA model,we altered the levels of miR-330-5p and PDK2 in HuMSCs-SF-SIS in the IUA model. As shown in Fig. 8 A and 8 B, the number of endometrial glands in the HuMSCs-SF-SIS transplanted group was significantly impaired by miR-330-5p and PDK2 knockdown (Fig. 8 A and 8 B, P < 0.05). However, PDK2 overexpression significantly enhanced the endometrial glands in the miR-330-5p mimic-transfected HuMSCs-SF-SIS transplanted group (Fig. 8 A and 8 B, P < 0.05). Consistently, Masson staining (Fig. 8 C and 8 D) results showed that the area of fibrosis in the endometrium of IUA model mice was significantly increased in the miR-330-5p or PDK2 knockdown group, but PDK2 led to a remarkable reduction in fibrosis (Fig. 8 D, P < 0.05) in miR-330-5p mimic-transfected HuMSCs-SF-SIS transplanted group. From these results it follows that circPTP4A2-miR-330-5p-PDK2 signaling is critical to HuMSCs-SF-SIS increasing the number of glands and decreasing the area of fibrosis in the IUA Model. 4. Discussion Presently, according to reports, 2.8%-45.5% of women with IUA have impaired fertility, notably occurring after pregnancy-related dilatation and curettage in more than 90% of cases [ 5 , 6 ]. Up to now, lUA has been treated mainly with both surgical and estrogenic modalities, and unfortunately, the recurrence rate is high, ranging from 20–63% [ 3 ]. Additionally, there is also a high risk for placental implantation. Therefore, it is particularly important to develop safe and feasible treatment options for patients with lUA at this time. Mesenchymal stem cells have now been widely used for tissue repair.Gargett et al. proposed that LUA patients could reconstruct endometrial tissue with endometrial mesenchymal stem cells [ 1 ]. Nagori et al. showed that bone marrow mesenchymal stem cell transplantation was effective in promoting repair of damaged endometrium in vivo [ 22 ], a view shared by Phermthai et al. Mesenchymal stem cell transplantation has been reported to be effective in repairing endometrial defects such as infertility and endometrial hyperplasia [ 23 , 24 ]. In fibrotic diseases, mesenchymal stem cells play an anti-fibrotic role, for example, pulmonary fibrosis, renal fibrosis and hepatic fibrosis can be treated with mesenchymal stem cells [ 25 – 29 ]. One of the typical representatives of ECM is the submucosa of the small intestine (SIS), such as skin, bone, bladder, ligaments, and abdominal wall, which has been widely used for tissue repair and clinical trials [ 13 ].After SF coating by a single-component LbL assembly, the SIS membrane exhibited good cell compatibility and are not only well resistant to rapid degradation, but also maintain structural integrity [ 20 ]. In this study, We seeded HuMSCs on the surface of the SF-SIS scaffold and were surprised to find an increase in the number of HuMSCs-seeded SF-SIS scaffold glands, as well as a reduction in the fibrotic area of the lUA model. Based on the study data, we conclude that HuMSC-seeded SF-SIS scaffolds may be used for IUA treatment. CircRNAs are circular non-coding RNAs that are resistant to the digestive action of RNase R. CircRNAs are mainly made by selective splicing (post splicing) of information exchange between upstream splice acceptors and downstream splice donors [ 30 , 31 ]. Our comparison of circRNAs with long-stranded noncoding RNAs (IncRNAs) and microRNAs (miRNAs) in mammalian cells revealed that circRNAs are better in terms of stability and conservation[ 32 ]. Meanwhile, circRNA can interfere with the expression of related genes, transcribe and interfere with RNA responses, and also act as a scaffold or template to assemble or synthesize protein complexes through circRNA sponge action [ 33 ].Recent reports have confirmed the role of several functional circRNAs in regulating tissue regeneration of MSCs. For example, the pluripotency of human embryonic stem cells (hESCs) is maintained by sponge transfection of circBIRC6 with miR-34a and miR-145 [ 34 ]. CircHIPK3 has been reported to promote a variety of cancers by absorbing multiple miRNAs through sponge uptake [ 35 ]. CircSMARCA5 inhibits glioblastoma pleomorphic cell migration but facilitates prostate cancer cell proliferation [ 36 ]. In this study, we found that circPTP4A2 is critical to HuMSCs-SF-SIS increasing the number of glands and decreasing the fibrosis area in the lUA model by targeting miR-330-5p-PDK2 signaling. A distinctive feature of eukaryotic cells is the presence of intracellular mitochondria, which play an important role in energy metabolism and apoptosis and are essential in biological longevity[ 37 ]. In cells, mitochondria carry out complex biological reactions and are one of the most complex reactive sensing systems [ 38 ]. Recent studies have shown that balancing mitochondrial dynamics, which regulate the fate of stem cells, and morphology is crucial for maintaining tissue homeostasis[ 39 ]. Recent studies have found that mitochondrial metabolism can be significantly altered by environmental stimuli [ 40 ]. An important feature of the MSC niche is hypoxia, which has been shown over the last decade to have a key role in maintaining three aspects of stem cell survival, self-replication and pluripotency [ 41 ]. It was found that for the glycolytic pathway, transcription and synthesis of enzymes increased in hypoxic cells, but synthesis of proteins involved in mitochondrial catabolism decreased [ 42 ].The proliferation, differentiation and survival of BMSCs has been shown to be affected by culture under hypoxic pressure[ 43 ]. Cytochrome oxidase, an enzyme located at the end of the mitochondrial respiratory chain, is involved in aerobic synthesis in mammalian cells, mainly using oxygen as a substrate [ 27 ]. Under hypoxic conditions, mitochondrial size and average velocity were significantly reduced [ 44 ]. In this study, we found that circPTP4A2-miR-330-5p-PDK2 signaling is critical for the stability of mitochondrial metabolism in HuMSCs under hypoxic conditions. Collectively, we constructed HuMSC-seeded SF-SIS scaffolds and evaluated the impact of repairing damaged endometrium in an lUA mouse model. Interestingly, we performed an in-depth study of the underlying mechanisms of endometrial repair progression in HuMSCs, in which circPTP4A2 is elevated in the HuMSCs seeded on the SF-SIS scaffolds and stabilizes mitochondrial metabolism via miR-330-5p-PDK2 signaling. Furthermore, these findings demonstrated that HuMSC-seeded SF-SIS scaffolds signify future clinical applications in the treatment of lUA. Conclusion In this study, we demonstrated that circPTP4A2 was elevated in the HuMSCs seeded on the SF-SIS scaffolds and stabilized the mitochondrial metabolism through miR-330-5p-PDK2 signaling, which contributes to endometrial repair progression. These findings demonstrate that HuMSC-seeded SF-SIS scaffolds are an encouraging method for the treatment of lUA. Declarations Ethics approval and consent to participate The study was ethically approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University. The study was performed according to the international, national and institutional rules considering animal experiments and biodiversity rights. The ethics review number is 2019-KY-72. Consent for publication Not applicable. Availability of data and material The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by National Natural Science Foundation of China (31400823 to R.-Y.X, the Chinese Medical Association of Clinical Medicine Research Special Fund Project (17020600729 to R.-Y.X. ),The Youth Innovation Fund of First Affiliated Hospital of Zhengzhou University (to R.-Y.X). Authors' contributions YYZ and RYX performed the research, analyzed data, and participated in writing the manuscript. LHL collected and analyzed the data. XWB contributed to the study performance. All authors read and approved the final manuscript. RYX conceived this study and gave final approval of this manuscript. Acknowledgements We thank for Jinlong Liu for the technique support and discussion. References Gargett CE, Schwab KE, Deane JA. Endometrial stem/progenitor cells: the first 10 years. Hum Reprod Update. 2016;22(2):137–63. Tempest N, Maclean A, Hapangama DK. Endometrial Stem Cell Markers: Current Concepts and Unresolved Questions . Int J Mol Sci, 2018. 19(10). Hooker AB, et al. 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Hypoxic preconditioning of human urine-derived stem cell-laden small intestinal submucosa enhances wound healing potential. Stem Cell Res Ther. 2020;11(1):150. Zhang XZ, et al. Procyanidins-crosslinked small intestine submucosa: A bladder patch promotes smooth muscle regeneration and bladder function restoration in a rabbit model. Bioact Mater. 2021;6(6):1827–38. Bi X, et al. The effects of silk layer-by-layer surface modification on the mechanical and structural retention of extracellular matrix scaffolds. Biomater Sci. 2020;8(14):4026–38. Algarrahi K, et al. Acellular bi-layer silk fibroin scaffolds support functional tissue regeneration in a rat model of onlay esophagoplasty. Biomaterials. 2015;53:149–59. Nagori CB, Panchal SY, Patel H. Endometrial regeneration using autologous adult stem cells followed by conception by in vitro fertilization in a patient of severe Asherman's syndrome. J Hum Reprod Sci. 2011;4(1):43–8. Cao Y, et al. Allogeneic cell therapy using umbilical cord MSCs on collagen scaffolds for patients with recurrent uterine adhesion: a phase I clinical trial. Stem Cell Res Ther. 2018;9(1):192. Phermthai T, et al. Successful derivation of xeno-free mesenchymal stem cell lines from endometrium of infertile women. Reprod Biol. 2016;16(4):261–8. El Agha E, et al. Mesenchymal Stem Cells in Fibrotic Disease. Cell Stem Cell. 2017;21(2):166–77. Eom YW, Shim KY, Baik SK. Mesenchymal stem cell therapy for liver fibrosis. Korean J Intern Med. 2015;30(5):580–9. Ishiuchi N, et al. Hypoxia-preconditioned mesenchymal stem cells prevent renal fibrosis and inflammation in ischemia-reperfusion rats. Stem Cell Res Ther. 2020;11(1):130. Shojaati G, et al. Mesenchymal Stem Cells Reduce Corneal Fibrosis and Inflammation via Extracellular Vesicle-Mediated Delivery of miRNA. Stem Cells Transl Med. 2019;8(11):1192–201. Watanabe Y, et al. Mesenchymal Stem Cells and Induced Bone Marrow-Derived Macrophages Synergistically Improve Liver Fibrosis in Mice. Stem Cells Transl Med. 2019;8(3):271–84. Han B, Chao J, Yao H. Circular RNA and its mechanisms in disease: From the bench to the clinic. Pharmacol Ther. 2018;187:31–44. Salzman J. Circular RNA Expression: Its Potential Regulation and Function. Trends Genet. 2016;32(5):309–16. Xu X, et al. CircRNA inhibits DNA damage repair by interacting with host gene. Mol Cancer. 2020;19(1):128. Chen J, et al. circPTN sponges miR-145-5p/miR-330-5p to promote proliferation and stemness in glioma. J Exp Clin Cancer Res. 2019;38(1):398. Yang H, et al. CircRNA BIRC6 promotes non-small cell lung cancer cell progression by sponging microRNA-145. Cell Oncol (Dordr). 2020;43(3):477–88. Xu Q, et al. CircHIPK3 regulates pulmonary fibrosis by facilitating glycolysis in miR-30a-3p/FOXK2-dependent manner. Int J Biol Sci. 2021;17(9):2294–307. Stella M, et al., Serum Extracellular Vesicle-Derived circHIPK3 and circSMARCA5 Are Two Novel Diagnostic Biomarkers for Glioblastoma Multiforme . Pharmaceuticals (Basel), 2021. 14(7). Hsu YC, et al. Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer. Semin Cell Dev Biol. 2016;52:119–31. Li Q, et al. The role of mitochondria in osteogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells. Protein Cell. 2017;8(6):439–45. Mahrouf-Yorgov M, et al. Mesenchymal stem cells sense mitochondria released from damaged cells as danger signals to activate their rescue properties. Cell Death Differ. 2017;24(7):1224–38. Phinney DG, et al. Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. Nat Commun. 2015;6:8472. Wanet A, et al. Connecting Mitochondria, Metabolism, and Stem Cell Fate. Stem Cells Dev. 2015;24(17):1957–71. Wang J, et al. Cell adhesion-mediated mitochondria transfer contributes to mesenchymal stem cell-induced chemoresistance on T cell acute lymphoblastic leukemia cells. J Hematol Oncol. 2018;11(1):11. Chen J, et al. Mesenchymal stem cell-derived exosomes protect beta cells against hypoxia-induced apoptosis via miR-21 by alleviating ER stress and inhibiting p38 MAPK phosphorylation. Stem Cell Res Ther. 2020;11(1):97. Wobma HM, et al. The influence of hypoxia and IFN-gamma on the proteome and metabolome of therapeutic mesenchymal stem cells. Biomaterials. 2018;167:226–34. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-895708","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":54806456,"identity":"c4c59d2a-eea9-42ce-a288-53f45837cd49","order_by":0,"name":"Yuanyuan Zheng","email":"","orcid":"","institution":"The First Affiliated Hospital of ZhengzhouUniversity","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Zheng","suffix":""},{"id":54806457,"identity":"f9f7c4f5-5f66-41fb-a7a8-a0df73ecf279","order_by":1,"name":"Linhao Li","email":"","orcid":"","institution":"BeiHang University School of Biological Science and Medical Engineering","correspondingAuthor":false,"prefix":"","firstName":"Linhao","middleName":"","lastName":"Li","suffix":""},{"id":54806458,"identity":"bac9dec8-21bc-4814-ba8e-1ff81dbb142e","order_by":2,"name":"Xuewei Bi","email":"","orcid":"","institution":"Beihang University","correspondingAuthor":false,"prefix":"","firstName":"Xuewei","middleName":"","lastName":"Bi","suffix":""},{"id":54806459,"identity":"7a4d3c75-2b21-4e9b-8a6a-0616cbd5a53e","order_by":3,"name":"Ruyue Xue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBAC9gYogx9CMRPWwnMAypBsIFmLwQGitbAffibNU3EvcfP5488kGCqsExvYzx7Ar4UnzdiY50xx4rYDB9IkGM6kJzbw5CXg1WIvwcP4mLctIXHbwYZjEoxthxMbJHgM8NsiwcNwmPdfQuLmZsY2CcZ/xGkB2tKQkLiBjZlNgrGBGC1AvxjOOZZgPOMMG7NFwrF04zaeHAJagCEm8aYmQba///jDGx9qrGX72c/g1wIDjg0gMgGI2YhSDwT2xCocBaNgFIyCEQgAou4+pTTz1MUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9028-7794","institution":"Zhenzhou University","correspondingAuthor":true,"prefix":"","firstName":"Ruyue","middleName":"","lastName":"Xue","suffix":""}],"badges":[],"createdAt":"2021-09-11 10:49:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-895708/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-895708/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":14161892,"identity":"df4ad288-01e3-4ff4-b936-f99167b51fe8","added_by":"auto","created_at":"2021-09-30 17:26:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":150647,"visible":true,"origin":"","legend":"Culture and characterization of HuMSCs. (A) The cell morphology of the cultured HuMSCs. (B) The cell markers were analyzed by FACs. N=3.","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-895708/v1/b57a2a070fe9bc517dc08e72.jpg"},{"id":14161505,"identity":"ace81418-70c9-4f11-ae26-9cc069b66920","added_by":"auto","created_at":"2021-09-30 17:23:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":187510,"visible":true,"origin":"","legend":"HuMSCs-SF-SIS increased the number of glands and reduced the area of fibrosis in the lUA model (A) The number of endometrial glands and the lumen of neonates were analyzed by HE staining. (B) Statistical analysis were performed to evaluate the number of endometrial glands. (C) The extent of fibrosis was assessed with Masson staining. (D) The fibrotic area of endometrium was carried out using Image J software. N=3. *P\u003c0.05 indicated group.","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-895708/v1/a0d7f6d01ea037d06cbb362d.jpg"},{"id":14161507,"identity":"e474450f-ce91-48f3-83e6-509a81556b93","added_by":"auto","created_at":"2021-09-30 17:23:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":117000,"visible":true,"origin":"","legend":"The expression level of circPTP4A2 is significantly elevated in the HuMSCs cultured on the SF-SIS scaffolds. (A)The profile of circRNA from normal HuMSCs and HuMSCs cultured on the SF-SIS scaffolds were analyzed by performing RNA-seq on ribosomal RNA-depleted total RNA. (B)The differential expression of total circRNAs was directly revealed by volcanic eruption-type clustering analysis. (C) Several significant up-regulated circRNAs (circUXSI , circPTP4A2, circCNTRL, circEPSTM, circSFMBT2, circZNF680 and circEMB) were verified via real-time PCR. (D) CircPTP4A2 was identified by real-time PCR, indicating a resistance between circPTP4A2 and RNase R.And the RNase R-treated PTP4A2 mRNA was significantly reduced. (E) Sub-fractional real-time PCR and (F) fluorescence in situ hybridization (FISH) assay was used to analyze the sub-cellular location of circPTP4A2 in HuMSCs. N=3, P\u003c 0.05 indicated group.","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-895708/v1/dbde05998d7cc721c0132bbd.jpg"},{"id":14162201,"identity":"266eaae2-12da-4f02-9cfe-ef8a4d9acbc8","added_by":"auto","created_at":"2021-09-30 17:29:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141708,"visible":true,"origin":"","legend":"CircPTP4A2 facilitates the mitochondrial metabolism of HuMSCs under hypoxia condition. (A) The effects of circPTP4A2 on the ATP content and (B) ATP synthase activity was analyzed by kits. (C) The cell oxygen consumption rate (OCR) of HuMSCs was analyzed by the seahorse XF24 Extracellular Flux Analyser (D) The Basal Respiration, (E) ATP production (F) Maximal Respiration, (G) Spare Respiratory Capacity, (H)Proton leak and (I) non-mitochondrial Respiration were analyzed. N=3. *P\u003c 0.05 indicated group.","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-895708/v1/9f0dbdc8ad7f8b4dbba770c8.jpg"},{"id":14161893,"identity":"604768ff-ebc5-47e4-86f8-66da4c6f88c6","added_by":"auto","created_at":"2021-09-30 17:26:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98893,"visible":true,"origin":"","legend":"CircPTP4A2 is targeted by miR-330-5p in HuMSCs cells. \n(A) The level of miRNAs (miR-487a, miR-330-5p, miR-1290, miR-326,miR421, miR-502-5p, miR-532-3p, miR-335 and miR-1305) in HuMSCs cells with or without circPTP4A2 knockout were analyzed by real-time PCR. (B) Ago2 Co-lmmunoprecipitation assay and (C) miR-330-5p RNA pull down were used to evaluate the interaction between miR-330-5p and circPTP4A2. (D) Dual-luciferase reporter assay was used to evaluate the control of miR-30-5p on circPTP4A2 in HuMSCs cells. N=3. *P\u003c 0.05 indicated group.","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-895708/v1/5401655b95c46af27674a063.jpg"},{"id":14161508,"identity":"0b33c964-19d9-4472-b492-7f357845efb3","added_by":"auto","created_at":"2021-09-30 17:23:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":145968,"visible":true,"origin":"","legend":"MiR-330-5p overexpression impairs the circPTP4A2 enhanced mit ochondrial metabolism in hypoxia treated HuMSCs. (A) The rescuer effects of miR-330-5p on the circPTP4A2 enhanced ATP content and (B) ATP synthase activity was analyzed by kits. (C) The rescuer effects of miR-330-5p on the circPTP4A2 enhanced cell oxygen consumption rate (OCR) of HuMSCs was analyzed by the seahorse XF24 Extracellular Flux Analyser. (D) The Basal Respiration, (E) ATP production (F) Maximal Respiration, (G) Spare Respiratory Capacity, (H) Proton leak and (I) non-mitochondrial Respiration were analyzed. N=3. *P\u003c 0.05 indicated group.","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-895708/v1/53cbc8cef4e86b3d0b512368.jpg"},{"id":14161894,"identity":"2b31e7c2-6837-4715-89e7-e79a5cfaef8b","added_by":"auto","created_at":"2021-09-30 17:26:20","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":110294,"visible":true,"origin":"","legend":"MiR-330-5p suppresses the expression of PDK2 via the 3’UTR target region. (A-C) Dual-luciferase reporter assay was used to evaluate the control of miR-30-5p on PDK2 mRNA 3’UTR in HuMSCs cells. (D)The mRNA and (E) protein levels of PDK2 in mouse umbilical MSCs and HuMSCs was analyzed by real-time PCR and Western Blot. N=3. *P\u003c0.05 indicated group.","description":"","filename":"fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-895708/v1/672c40b45bbfd7024b8740d0.jpg"},{"id":14161509,"identity":"174456ba-d1e9-43e6-82dd-d1b6891a14a9","added_by":"auto","created_at":"2021-09-30 17:23:20","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":259367,"visible":true,"origin":"","legend":"CircPTP4A2-miR-330-5p-PDK2 signaling is critical to the HuMSCs-SF-SIS increased number of glands and decreased fibrosis area in lUA model. (A) The number of endometrial glands and the lumen of neonates was analyzed by HE staining. (B) Statistical analysis was performed to evaluate the number of endometrial glands. (C) The degree of fibrosis was assessed using Masson staining. (D) The fibrotic area of endometrium was carried out using Image J software. N=3. *P\u003c 0.05 indicated group.","description":"","filename":"fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-895708/v1/0e0cc92876909f62a172e910.jpg"},{"id":15231171,"identity":"8defc198-45c3-47c3-810d-7b6cffd73aba","added_by":"auto","created_at":"2021-11-04 19:24:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1346834,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-895708/v1/17d2f435-d794-4a6f-978b-d6e45721d2cf.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003ecircPTP4A2-miR-330-5p-PDK2 Signaling Facilitates In Vivo Survival of HuMSCs on SF-SIS Scaffolds and Improves The Repair of Damaged Endometrium\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEmbryo implantation failure caused by female endometrial damage is the main factor affecting the success rate of assisted reproduction. Cells in the functional layer of the endometrium in a normal cycle are apoptotic and shed, and release signals to activate endometrial stem cells (ESCs), promoting angiogenesis and tissue repair. However, after the damage, the endometrium is deficient in blood vessels and has few glands leading to thinning of the uterine lining (atrophy)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Intrauterine adhesions (IUA), also called Asherman's syndrome, is a common uterine disease caused by damage to the basal lining of the endometrium caused by mechanical injury or infection, resulting in endometrial fibrosis, uterine obstruction, menstrual abnormalities, infertility and pregnancy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Presently, hysteroscopic adhesiolysis is performed clinically, and anti-adhesion drugs and IUD are administered simultaneously [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Several therapeutic agents have been used to improve endometrial regrowth [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, due to the loss and functional destruction of endometrial basal stem cells, the therapeutic effect is not ideal, and a normally functional endometrial environment cannot be provided.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells can differentiate into endometrial epithelial and mesenchymal cells after induction, suggesting that mesenchymal stem cells have biological functions similar to those of endometrial basement ESCs and may be involved in the repair of the endometrial structure and improve its function [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, multiple challenges persist in the treatment of endometrial damage by exogenous mesenchymal stem cells, such as differences in the cell carrier and the intrauterine environment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Generally, human umbilical cord MSCs (HuMSCs) were superior to bone marrow MSCs (BM-MSCs) in terms of cell content and proliferative capacity, and have lower immunogenicity than BM-MSCs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition, (HuMSCs) are convenient to obtain materials and are free from ethical controversies, and other advantages have attracted increasing attention [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Studies using in vivo HuMSC transplantation experiments to observe endometrial reconstruction, two months after the detection of transplantation, showed a thickening of the damaged endometrial tissue, a reduction in the area affected by fibrosis, and a similar repair period. It is speculated that HuMSCs secreted by paracrine factors have anti-inflammatory effects, facilitate the repair of the endometrium, and maintain cell function and angiogenesis of the microenvironment [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the molecular mechanism underlying the effect of HuMSCs on the repair and regeneration of the endometrium remains unclear.\u003c/p\u003e \u003cp\u003eRecently, a significant focus has been expressed on the application of tissue-engineered artificial reconstruction materials for tissue repair and regeneration. Small-intestinal submucosa (SIS) is a cell-free, allogeneic, and collagen-matrix material with good histocompatibility and biomechanical properties and no immunogenicity and toxicity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It is a natural biodegradable biological material [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The submucosa of the small intestine is mainly composed of type I collagen isotype, and is rich in fibronectin and growth factors, among which aplasia collagen and fibronectin can specifically bind to the cell membrane and activate the cell conduction pathway to facilitate cell adhesion[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As a tissue-engineered material, SIS has been widely used in the research of cartilage, tendon, bladder, cardiovascular regeneration, and other fields, however a few studies have been reported on the repair of injuries of the endometrium and the molecular mechanism [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Silk cellulose (SF) has the three advantages of good mechanical properties, controllable biodegradation and excellent biocompatibility, and has been widely used in the field of biomedicine [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The SIS membrane is a material coated with SF layers, which can adjust the diffusion rate of drugs or biomolecules and improve the mechanical and structural stability of its properties.\u003c/p\u003e \u003cp\u003eIn this study, we constructed HuMSC-seeded SIS scaffolds and evaluated the impact of the repair of damaged endometrium in an lUA mouse model. Moreover, We also studied the potential mechanism of HuMSCs in repairing the endometrium.\u003c/p\u003e"},{"header":"2. Materials And Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation and characterization of SF-SIS scaffolds\u003c/h2\u003e \u003cp\u003eThe SF-SIS scaffolds used in this study was kindly donated from Dr. Li (The Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China). The SF-SIS scaffolds were prepared and characterized as previously described [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Characterization and culture of HuMSCs\u003c/h2\u003e \u003cp\u003eHuMSCs is purchased from Promocell (Miaotong (Shanghai) Biological Science \u0026amp; Technology Co., Ltd. Shanghai, China). Frozen HuMSCs between P2 and P10 were freshly inoculated in 100 mm culture dishes (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells per dish) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, USA), penicillin (100 U/ml, Gibco) and Streptomyces (100 mg/ml, Gibco). Briefly, the phenotype of HuMSCs was identified by FACS specificity (CD34, CD45, CD29, CD90, CD105 and HLA-DR). The osteogenic and adipogenic abilities of mesenchymal stem cells were determined by the Cyagen Osteogenic stimulation kit (Cyage, Guangzhou, China) and the Cyagen Adipogenic Differentiation Kit (Cyage, Guangzhou, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Osteogenic and adipogenic differentiation of HuMSCs\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer's instructions (RASMX-90021 and RASMX-90031, Cyagen, Guangzhou, China), the ability of cells to differentiate into osteoblasts or adipocytes was characterized using commercial osteogenic and adipogenic induction kits. Osteoblasts were stained with Alizarin Red S and adipocytes with oil red O.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Animal IUA model\u003c/h2\u003e \u003cp\u003e All animal procedures are approved by the the first affiliated hospital of Zhengzhou University, Institutional Animal Care and Use Committee and are carried out in accordance with the National Research Council Laboratory Animal Care and Use Guidelines. 6-week-old BALB /c mice were obtained from Shanghai SLAC Animal Center and allowed to acclimatize to the new environment for a week. To establish an IUA model, the uterus was mechanically damaged during pregnancy. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital. The uterus is excised at the midline below the abdomen. Insert a size 7 needle into the left and right uterine junction and cut it carefully back and forth until the uterine congestion is visible to the naked eye. Then, the abdominal cavity was closed. The operation was performed under aseptic conditions. The control group did not undergo surgery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Histological analysis\u003c/h2\u003e \u003cp\u003eUterine specimens were collected 8 days after surgery. The sample was fixed with 4% paraformaldehyde, dehydrated, removed with xylene, and finally embedding with paraffin. The embedded tissue sections were 5-\u0026micro;m thick. The uterine morphology and structure were evaluated by hematoxylin - eosin (H\u0026amp;E) staining. Masson staining was performed to evaluate the fibrosis according to manufacturer's instructions (Yeasen, Shanghai, China). Fibrosis areas (light blue) were evaluated using ImageJ software (MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 circRNA sequencing\u003c/h2\u003e \u003cp\u003eTotal RNAs were extracted with Trizol (Invitrogen, Carlsbad, CA, USA). Sequencing libraries were generated and sequenced by Sango Bio Technology (Shanghai, China). A total amount of 5 \u0026micro;g RNA per sample was used. The libraries were subjected to paired-end sequencing with pair end 150 bp reading length on an Illumina HiSeq sequencer (Illumina, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted with Trizol reagent (TAKARA), and 500 ng total RNA was transcribed into cDNA using PrimeScript RT Master Mix (TAKARA, Dalian, China). Subsequently, SYBR Premix Ex Taq II Kit (TAKARA) was used to detect the expression of circRNAs and mRNA by RT-qPCR, which was normalized to endogenous control GAPDH expression. Folding changes were calculated using 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 RNA fluorescence in situ hybridization\u003c/h2\u003e \u003cp\u003eRNA fluorescence in situ hybridization was performed using a fluorescence in situ hybridization kit (RiboBio, Guangzhou, China) in accordance with the manufacturer's guidelines. Cy3-labeled circPTP4A2 probe (RiboBio, Guangzhou, China) was detected with fluorescence in situ hybridization kit and then observed with LSM800 confocal microscopy (Zeiss, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Measurement of mitochondrial metabolism\u003c/h2\u003e \u003cp\u003eATP content and synthase activity was performed using the quantification kits (Sango Biotech, Shanghai, China) in accordance with the manufacturer's guidelines. Real time integrated cellular oxygen consumption rate (OCR) was measured using the Seahorse XF24 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA, USA) as previous described. In brief, HuMSCs cells were treated with 10 \u0026micro;g/mL curcumin for 12 h and 10\u003csup\u003e4\u003c/sup\u003e cells were plated into the seahorse customized cell plates. After the probes were calibrated, the OCR was detected with sequential injection of the following compounds which regulate mitochondrial respiration: oligomycin (ATP synthase inhibitor; 1 \u0026micro;M), FCCP (uncoupler; 1 \u0026micro;M), rotenone (complex I inhibitor; 1 \u0026micro;M), and antimycin A (complex III inhibitor; 1 \u0026micro;M).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Dual-luciferase reporter assays\u003c/h2\u003e \u003cp\u003eThe 293T cells were incubated in 24-well plates with 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well. These cells were then transfected with psiCheck2-circPTP4A2-WT or psiCheck2-circPTP4A2-Mut, miR-330-5p mimics, or miR-NC. After 48 h, the cells were lysed with a passive lysis buffer (Promega, Madison, WI, USA) and the dual luciferase reporting assay (Promega) was used to calculate the relative luciferase activity by normalized firefly luminescence to Renilla luminescence.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.11 Western blotting\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe HuMSCs were lysed with RIPA lysis buffer. The same amount of protein was then broken down by SDS-PAGE analysis and electrically transferred to PVDF membranes (Milliore, Schwalbach, Germany), which were then sealed with 5% skimmed milk powder and incubated overnight with primary antibody at 4\u0026deg;C. The primary antibodies used were anti-PDK2 (Cell Signaling Technology) and anti-GAPDH (Kanchen Biotech). The membrane was then incubated with HRB-conjugated secondary antibodies at room temperature for 1 h, and the imprinting was observed using an enhanced chemiluminescence kit (Pierce, Waltham, MA, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.12 Statistical Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eData were expressed as the Means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. For two groups, 2-tailed t-test (unpaired) was used for comparisons. For multiple comparisons, ANOVA followed by the post hoc Bonferroni test was taken with GraphPad Prism\u0026reg; version 9.0 software (GraphPad Software, Inc., La Jolla, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Culture and characterization of HuMSCs\u003c/h2\u003e \u003cp\u003eBMSCs are adsorbed on plastic discs. The typical morphology of HuMSCs is similar to that of spindle-shaped fibroblasts, which grow in a tightly packed vortex pattern, is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The HuMSCs were successfully differentiated into osteoblasts and adipocytes in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), this indicates that this cell population was a pluripotent mesenchymal stromal cell. Furthermore, FACS results showed that most cells were negative for hematopoietic markers after 7 days in culture for CD34+ (5.12%), CD45+ (2.05%), HLA-DR (0.61%) and highly positive for CD29 (61.9%), CD90 (94.9%) and CD105 (94.9)%.expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 HuMSCs-SF-SIS reduces fibrotic area and increases the number of glands in the lUA model\u003c/h2\u003e \u003cp\u003eTo identify the functional effect of HuMSCs-SF-SIS scaffolds on the repair of damaged endometrium, a mouse lUA model was established.The HuMSCs-SF-SIS scaffolds were transplanted after the damage to the endometrium was reduced. After 4 weeks, uterine tissue was collected up for modeling. According to the HE staining results, the shape of the uterine cavity in the pseudopod group was irregular. Columnar epithelial cells cover the uterus and gland cavities. Epithelial cell structure was complete, stromal glands were abundant, and oval. Connective tissue fragments were found in the uterine cavity in the IUA model group, the number of glandular blood vessels were significantly reduced, and the connective tissue was congested one week after the damage. The number of endometrial glands in the HuMSCs-SF-SIS transplanted group increased, and the lumen of neonates was not completely covered by the monolayer columnar epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). According to the results of statistical analysis, for the number of endometrial glands, the model group and HuMSCs-SF-SIS transplantation group were lower than the sham-operated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the model group was significantly lower than the sham-operated group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Differently, the group of HuMSCs-SF-SIS transplantation group was significantly higher than the IUA model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Endometrial adhesions are characterized by fibrosis. In the study, we used Masson staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) to assess the degree of fibrosis. A significant increase in the area of endometrial fibrosis was observed in lUA model mice compared with the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but HuMSCs-SF-SIS transplantation resulted in a significant decrease in fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In conclusion, transplantation of HuMSCs-SF-SIS not only increased the number of endometrial glands, but also repaired the injured endometrium.\u003c/p\u003e\u003cp\u003e \u003cb\u003e3.3 The expression level of circPTP4A2 was significantly elevated in the HuMSCs cultured on the SF-SIS scaffolds\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore whether circRNA is involved in endometrial repair, we first performed RNA-seq analysis of total RNA from ribosomal RNA of normal HuMSCs and HuMSCs cultured on SF-SIS scaffolds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A total of 54 circRNAs were significantly downregulated, and 27 circRNAs were upregulated in HuMSCs cultured on the SF-SIS scaffolds (filtered by FC(fold change)\u0026thinsp;\u0026gt;\u0026thinsp;2 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Variability expression of circRNAs was directly displayed by volcano clustering analysis. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We then chose several significantly upregulated circRNAs (circUXSI, circPTP4A2, circCNTRL, circEPSTII, circSFMBT2, circZNF680, and circEMB) to verify their existence via real-time PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We concluded that circPTP4A2 was upregulated in HuMSCs cultured on the SF-SIS scaffolds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In a follow-up study, we found resistance between circPTP4A2 and RNase R and also confirmed that PTP4A2 mRNA showed a significant reduction after RNase R treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Moreover,(sub-fractional real-time PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) and fluorescence in situ hybridization (FISH) assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) data indicated that circPTP4A2 was mainly present in the cytoplasm.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 CircPTP4A2 facilitates the mitochondrial metabolism of HuMSCs under hypoxia condition\u003c/h2\u003e \u003cp\u003eMitochondria are essential in the cellular biochemistry of most eukaryotic cells, producing nearly 95% of cellular ATP through oxidative phosphorylation of mitochondria, thereby controlling cell death or survival under hypoxic conditions, such as the transplanted SF-SIS scaffolds. Considering the critical role of mitochondrial metabolism, we tested whether circPTP4A2 facilitates the biological functions of HuMSCs via the regulation of mitochondrial metabolism. For the purpose of hypothesis testing, we first determined the effects of circPTP4A2 on ATP content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and ATP synthase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).We found that the ATP content and ATP synthase activity were dramatically decreased in hypoxic HuMSCs, but significantly increased with the overexpression of circPTP4A2. Using the seahorse XF24 extracellular flux analyzer, we also analyzed the cell oxygen consumption rate (OCR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), indicating reduced OXPHOS in HuMSCs cells. We then assessed the mitochondrial functions, in particular, basal respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), maximal respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), spin respiratory capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), proton leak (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), and non-mitochondrial respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Hypoxia treatment dramatically impaired the OCR value of basal respiration, spare respiratory capacity, maximal respiration, and ATP production in HuMSCs. In contrast, overexpression of circPTP4A2 significantly attenuated the inhibitory effect of hypoxia on ATP content, ATP synthase activity, and cell oxygen consumption rate (OCR). These data indicate that circPTP4A2 could facilitate the mitochondrial metabolism of HuMSCs under hypoxic conditions.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 CircPTP4A2 is targeted by miR-330-5p in HuMSCs cells\u003c/h2\u003e \u003cp\u003eTo explore whether circPTP4A2 can function as \u0026ldquo;miRNA sponge\u0026rdquo; in HuMSCs cells, we selected several potential miRNAs (miR-326, miR-487a, miR-335, miR-532-3p, miR-421, miR-502-5p, miR-330-5p, miR-1290 and miR-1305) through Starbase 2.0 database. After circPTP4A2 knockout, we found an increased level of miR-330-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) in HuMSCs cells. Enrichment of miR-330-5p with circPTP4A2 was derived from the Ago2 co-immunoprecipitation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and miR-330-5p RNA pull-down assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). To further verify the miR-330-5p target circPTP4A2, we added a luciferase reporter gene test. The results showed that miR-330-5p mimics significantly reduced the luciferase activity of HuMSCs transfected with wild-type circPTP4A2. But the miR-330-5p mimic failed to reduce the luciferase activity of the mutant circPTP4A2 transfected HuMSCs cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results suggest that circPTP4A2 is targeted by miR-330-5p in HuMSCs.\u003c/p\u003e\u003cp\u003e \u003cb\u003e3.6 MiR \u0026minus;\u0026thinsp;330- 5p over-expression impaired the circPTP4A2 enhanced mitochondrial metabolism in hypoxia - treated HuMSCs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo identify whether miR \u0026minus;\u0026thinsp;330\u0026ndash;5p is critical to circPTP4A2 enhanced mitochondrial metabolism in hypoxia \u0026ndash; treated HuMSCs. We transfected the miR \u0026minus;\u0026thinsp;330\u0026ndash;5p mi mic in hypoxia - treated HuMSCs with or without circPTP4A2 overexpression. The miR \u0026minus;\u0026thinsp;330\u0026ndash;5p mimic dramatically reduced ATP content (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA ) and ATP synthase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Moreover, miR-330-5p mimics the Reduced basal respiration, spare respiratory capacity, ATP produ ction, and maximal respiration in HuMSCs cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Notably, circPTP4A2 overexpression failed to increase basal respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), maximal respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) ,and spare respiratory capacity(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) in HuMSC cells transfected with miR-330-5p mimic. Through the above results, it is shown that miR-330-5p overexpression impaired circPTP4A2 enhanced mitochondrial metabolism in hypoxia - treated HuMSCs.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 MiR \u0026minus;\u0026thinsp;330\u0026ndash;5p inhibits PDK2 expression through the 3' UTR target region\u003c/h2\u003e \u003cp\u003eTo further explore the underlying mechanism of the circPTP4A2/miR-330-5p axis on mitochondrial metabolism in hypoxia-treated HuMSCs, we then screened the potential targets of the miR-330-5p Target Scan and Star Base databases. Further by dual luciferase assay, we found that the 3\u0026rsquo;UTR mRNA of the mitochondrial metabolism regulator PDK2 was directly targeted by miR-330-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). In addition, PDK2 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) levels were significantly reduced by miR-330-5p mimics in mouse umbilical cord MSCs and HuMSC. Taken together, it can be concluded that miR-330-5p may regulate mitochondrial metabolism by suppressing the expression of PDK2.\u003c/p\u003e\u003cp\u003e \u003cb\u003e3.8CircPTP4A2-miR-330-5p-PDK2 signaling is critical to HuMSCs-SF-SIS decreasing the fibrosis area and increasing the number of glands in IUA model\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo confirm the critical role of circPTP4A2-miR-330-5p-PDK2 signaling in the progress of the repair of the endometrium by HuMSCs-SF-SIS in IUA model,we altered the levels of miR-330-5p and PDK2 in HuMSCs-SF-SIS in the IUA model. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, the number of endometrial glands in the HuMSCs-SF-SIS transplanted group was significantly impaired by miR-330-5p and PDK2 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, PDK2 overexpression significantly enhanced the endometrial glands in the miR-330-5p mimic-transfected HuMSCs-SF-SIS transplanted group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Consistently, Masson staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD) results showed that the area of fibrosis in the endometrium of IUA model mice was significantly increased in the miR-330-5p or PDK2 knockdown group, but PDK2 led to a remarkable reduction in fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in miR-330-5p mimic-transfected HuMSCs-SF-SIS transplanted group. From these results it follows that circPTP4A2-miR-330-5p-PDK2 signaling is critical to HuMSCs-SF-SIS increasing the number of glands and decreasing the area of fibrosis in the IUA Model.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePresently, according to reports, 2.8%-45.5% of women with IUA have impaired fertility, notably occurring after pregnancy-related dilatation and curettage in more than 90% of cases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Up to now, lUA has been treated mainly with both surgical and estrogenic modalities, and unfortunately, the recurrence rate is high, ranging from 20\u0026ndash;63% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, there is also a high risk for placental implantation. Therefore, it is particularly important to develop safe and feasible treatment options for patients with lUA at this time.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells have now been widely used for tissue repair.Gargett et al. proposed that LUA patients could reconstruct endometrial tissue with endometrial mesenchymal stem cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nagori et al. showed that bone marrow mesenchymal stem cell transplantation was effective in promoting repair of damaged endometrium in vivo [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], a view shared by Phermthai et al. Mesenchymal stem cell transplantation has been reported to be effective in repairing endometrial defects such as infertility and endometrial hyperplasia [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In fibrotic diseases, mesenchymal stem cells play an anti-fibrotic role, for example, pulmonary fibrosis, renal fibrosis and hepatic fibrosis can be treated with mesenchymal stem cells [\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. One of the typical representatives of ECM is the submucosa of the small intestine (SIS), such as skin, bone, bladder, ligaments, and abdominal wall, which has been widely used for tissue repair and clinical trials [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].After SF coating by a single-component LbL assembly, the SIS membrane exhibited good cell compatibility and are not only well resistant to rapid degradation, but also maintain structural integrity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, We seeded HuMSCs on the surface of the SF-SIS scaffold and were surprised to find an increase in the number of HuMSCs-seeded SF-SIS scaffold glands, as well as a reduction in the fibrotic area of the lUA model. Based on the study data, we conclude that HuMSC-seeded SF-SIS scaffolds may be used for IUA treatment.\u003c/p\u003e \u003cp\u003eCircRNAs are circular non-coding RNAs that are resistant to the digestive action of RNase R. CircRNAs are mainly made by selective splicing (post splicing) of information exchange between upstream splice acceptors and downstream splice donors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our comparison of circRNAs with long-stranded noncoding RNAs (IncRNAs) and microRNAs (miRNAs) in mammalian cells revealed that circRNAs are better in terms of stability and conservation[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Meanwhile, circRNA can interfere with the expression of related genes, transcribe and interfere with RNA responses, and also act as a scaffold or template to assemble or synthesize protein complexes through circRNA sponge action [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].Recent reports have confirmed the role of several functional circRNAs in regulating tissue regeneration of MSCs. For example, the pluripotency of human embryonic stem cells (hESCs) is maintained by sponge transfection of circBIRC6 with miR-34a and miR-145 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. CircHIPK3 has been reported to promote a variety of cancers by absorbing multiple miRNAs through sponge uptake [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. CircSMARCA5 inhibits glioblastoma pleomorphic cell migration but facilitates prostate cancer cell proliferation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, we found that circPTP4A2 is critical to HuMSCs-SF-SIS increasing the number of glands and decreasing the fibrosis area in the lUA model by targeting miR-330-5p-PDK2 signaling.\u003c/p\u003e \u003cp\u003eA distinctive feature of eukaryotic cells is the presence of intracellular mitochondria, which play an important role in energy metabolism and apoptosis and are essential in biological longevity[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In cells, mitochondria carry out complex biological reactions and are one of the most complex reactive sensing systems [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Recent studies have shown that balancing mitochondrial dynamics, which regulate the fate of stem cells, and morphology is crucial for maintaining tissue homeostasis[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Recent studies have found that mitochondrial metabolism can be significantly altered by environmental stimuli [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. An important feature of the MSC niche is hypoxia, which has been shown over the last decade to have a key role in maintaining three aspects of stem cell survival, self-replication and pluripotency [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. It was found that for the glycolytic pathway, transcription and synthesis of enzymes increased in hypoxic cells, but synthesis of proteins involved in mitochondrial catabolism decreased [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].The proliferation, differentiation and survival of BMSCs has been shown to be affected by culture under hypoxic pressure[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Cytochrome oxidase, an enzyme located at the end of the mitochondrial respiratory chain, is involved in aerobic synthesis in mammalian cells, mainly using oxygen as a substrate [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Under hypoxic conditions, mitochondrial size and average velocity were significantly reduced [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In this study, we found that circPTP4A2-miR-330-5p-PDK2 signaling is critical for the stability of mitochondrial metabolism in HuMSCs under hypoxic conditions. Collectively, we constructed HuMSC-seeded SF-SIS scaffolds and evaluated the impact of repairing damaged endometrium in an lUA mouse model. Interestingly, we performed an in-depth study of the underlying mechanisms of endometrial repair progression in HuMSCs, in which circPTP4A2 is elevated in the HuMSCs seeded on the SF-SIS scaffolds and stabilizes mitochondrial metabolism via miR-330-5p-PDK2 signaling. Furthermore, these findings demonstrated that HuMSC-seeded SF-SIS scaffolds signify future clinical applications in the treatment of lUA.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we demonstrated that circPTP4A2 was elevated in the HuMSCs seeded on the SF-SIS scaffolds and stabilized the mitochondrial metabolism through miR-330-5p-PDK2 signaling, which contributes to endometrial repair progression. These findings demonstrate that HuMSC-seeded SF-SIS scaffolds are an encouraging method for the treatment of lUA.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was ethically approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University. The study was performed according to the international, national and institutional rules considering animal experiments and biodiversity rights. The ethics review number is 2019-KY-72.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (31400823 to R.-Y.X, the Chinese Medical Association of Clinical Medicine Research Special Fund Project (17020600729 to R.-Y.X. ),The Youth Innovation Fund of First Affiliated Hospital of Zhengzhou University (to R.-Y.X).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYYZ and RYX\u0026nbsp;performed the research, analyzed data, and participated in writing the manuscript. LHL collected and analyzed the data. XWB contributed to the study performance. All authors read and approved the final manuscript. RYX conceived this study and gave final approval of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank for Jinlong Liu for the technique support and discussion.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGargett CE, Schwab KE, Deane JA. Endometrial stem/progenitor cells: the first 10 years. Hum Reprod Update. 2016;22(2):137\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTempest N, Maclean A, Hapangama DK. \u003cem\u003eEndometrial Stem Cell Markers: Current Concepts and Unresolved Questions\u003c/em\u003e. Int J Mol Sci, 2018. 19(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHooker AB, et al. 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Biomaterials. 2018;167:226\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Intrauterine adhesions, Human umbilical cord MSCs (HuMSC), Silk fibroin surface modified small-intestinal submucosa (SF-SIS), CircPTP4A2, Mitochondrial metabolism","lastPublishedDoi":"10.21203/rs.3.rs-895708/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-895708/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHuman umbilical cord MSCs (HuMSC)-based therapy has shown promising results in the treatment of intrauterine adhesions (lUA). In this study, our aim was to construct a HuMSC-seeded silk fibroin small-intestinal submucosa (SF-SIS) scaffold and evaluate the impact of repairing the damaged endometrium in an lUA mouse model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo identify the functional effect of HuMSCs-silk cellulose (SF)- small-intestinal submucosa (SIS) scaffolds on the repair of damaged endometrium, a mouse lUA model was established in this study. The uterine morphology and fibrosis were evaluated by hematoxylin - eosin (H\u0026amp;E) staining and Masson staining. CircRNA sequencing, real-time PCR and RNA fluorescence in situ hybridization were used to screen and verify the potential circRNAs that involved in the repair of damaged endometrium by HuMSCs. Real time integrated cellular oxygen consumption rate (OCR) was measured using the Seahorse XF24 Extracellular Flux Analyser. The potential down-stream miRNAs and proteins of circRNAs were analyzed dual-luciferase report and Western Blot.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that HuMSCs-SF-SIS not only increased the number of glands, but also reduced the ulcer area in the IUA model. Furthermore, we demonstrated that circPTP4A2 was elevated in the HuMSCs seeded on the SF-SIS scaffolds and stabilized the mitochondrial metabolism through miR-330-5p-PDK2 signaling, which contributes to endometrial repair progression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn this study, we demonstrated that circPTP4A2 was elevated in the HuMSCs seeded on the SF-SIS scaffolds and stabilized the mitochondrial metabolism through miR-330-5p-PDK2 signaling, which contributes to endometrial repair progression. These findings demonstrate that HuMSC-seeded SF-SIS scaffolds are an encouraging method for the treatment of lUA.\u003c/p\u003e","manuscriptTitle":"circPTP4A2-miR-330-5p-PDK2 Signaling Facilitates In Vivo Survival of HuMSCs on SF-SIS Scaffolds and Improves The Repair of Damaged Endometrium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-09-30 17:23:18","doi":"10.21203/rs.3.rs-895708/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a9d9cb53-f6a3-48c1-ab12-66686ba9253f","owner":[],"postedDate":"September 30th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":7548054,"name":"Stem Cell \u0026 Developmental Cell Biology"}],"tags":[],"updatedAt":"2021-11-04T19:24:22+00:00","versionOfRecord":[],"versionCreatedAt":"2021-09-30 17:23:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-895708","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-895708","identity":"rs-895708","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}