MIR503HG silencing promotes endometrial stromal cell progression and metastasis and suppresses apoptosis in adenomyosis by activating the Wnt/β‑catenin pathway via targeting miR‑191
- Authors:
- Published online on: January 30, 2023 https://doi.org/10.3892/etm.2023.11816
- Article Number: 117
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Copyright: © Xu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Adenomyosis (AD) is defined as invasion of endometrial glands and stroma into the myometrium (1). Following a limited or diffuse growth pattern, AD develops into endometrial glands in the myometrium (2) and can coexist with endometriosis (3). Manifesting as pelvic pain and abnormal uterine bleeding, AD has a close association with female infertility, dysmenorrhea and dyspareunia (2,4,5). AD is defined as a benign gynecological disease, however, according to some reports, patients with AD may have a risk of malignant transformation (6,7), during which the endometrium invaded by AD can progress into endometrioid adenocarcinoma followed by serous and clear cell carcinoma and poorly differentiated adenocarcinoma, severely threatening health (8). Therefore, early diagnosis and timely treatment are key for suppressing the progression and the malignant transformation of AD. Understanding of the AD condition remains poor despite the upgrade in diagnostic tools (6) and current management of AD still lacks international guidelines (9).
It may be a novel approach to develop the treatment of AD from the perspective of genetics, as long non-coding RNAs (lncRNAs) are aberrantly expressed in both eutopic and ectopic endometria of patients with AD (10,11). Classified as a subgroup of ncRNAs, lncRNAs are >200 nucleotides in length and possess regulatory effects on AD progression (12-14). Placenta-enriched lncRNA MIR503 host gene (MIR503HG), located on chromosome Xq26.3 where genes associated with human reproduction are enriched (15), is highly expressed and associated with unfavorable outcomes in endometrial cancer (16) and preeclampsia (17), but is lowly expressed and suppresses tumor growth by inhibiting cell migration and invasion in triple-negative breast cancer (18). However, the specific expression and mechanism of MIR503HG in AD remain unclear.
In addition, the conventional view is that AD results from the abnormal down-growth and invagination of the endometrium into the myometrium (19). Studies have reported that lncRNA-mediated inhibition of proliferation and enhancement of apoptosis in endometrial stromal cells (ESCs) ameliorates AD (12-14). Meanwhile, at only ~22 nucleotides in length and also classified as ncRNAs, microRNAs (miRNAs or miRs) have been discovered to act as a downstream mechanism of lncRNA-mediated changes in processes of ESCs during AD progression (13). Among them, miR-191 is notably dysregulated in the endometrium tissues of patients with AD (20). Therefore, identifying downstream miRNAs of lncRNAs that regulate AD is key.
In the present study, the expression of MIR503HG in AD was characterized and a potential mechanism by which MIR503HG regulates AD by modulating its downstream miRNAs was investigated. Meanwhile, since MIR503HG has been reported to target miR-191 to promote tumor inhibition in cervical cancer (21), further experiments were performed to determine their roles in AD.
Materials and methods
Ethics approval
The study was ratified by the Ethics Committee of People's Hospital of Deyang City (approval no. GD202000524; Deyang, China) and written informed consent from all participants was obtained for experimental work involving tissues obtained from humans.
Clinical samples
AD tissue (n=30) was collected from the endometrium of patients with AD (mean age, 42.75±5.62 years; range, 31-58 years) who had been diagnosed clinically and pathologically at People's Hospital of Deyang City in July 2020 to November 2020. None of the patients had received preoperative chemotherapy, radiotherapy or hormone therapy or had a history of chronic disease such as coronary heart disease and hypertension. During the same period, specimens taken from the endometrium (n=30) of patients with cervical lesions or uterine fibroids after hysterectomy but without AD (mean age, 40.24±3.73 years; range, 32-49 years) were used as the control group. In the control group, the patients had no history of hormone therapy or chronic diseases. All tissues were immediately snap-frozen in liquid nitrogen at -80˚C for 40 min for further use.
Isolation, identification and culture of ESCs
ESCs were isolated from the endometrium of patients with AD (n=3) as follows: The endometrium was cut into 0.5-1.0 mm3 sections and digested with 0.25% trypsin (cat. no. 9002-07-7; Sigma-Aldrich; Merck KGaA) that was diluted to 1.25 mg/l with Dulbecco's Modified Eagle Medium (DMEM)/F-12 (cat. no. 21041025; Thermo Fisher Scientific, Inc.). The sections were incubated at 37˚C with 5% CO2 for 80 min, then a cell suspension was obtained and filtered twice through a nylon mesh (140- and 37-µm mesh in sequence). Subsequently, the filtered suspension was centrifugated at 1,000 x g for 5 min at 4˚C and ESCs were obtained as previously described (22). When the confluence of ESCs was 90% under an optical microscope (BX50; Olympus Corporation; magnification, x200), ESCs were maintained in DMEM/F12 (50 ml), supplemented with 10% fetal bovine serum (FBS; cat. no. F2442), 2 mmol glutamine (cat. no. 1294808) and 1% penicillin-streptomycin (cat. no. P4333; all Sigma-Aldrich; Merck KGaA) at 37˚C with 5% CO2.
Immunocytochemistry assay
After being fixed in 4% paraformaldehyde (cat. no. P6148; Sigma-Aldrich; Merck KGaA) for 15 min at room temperature, ESCs (2x104 cells/ml) were washed with PBS (cat. no. P5493, Sigma-Aldrich; Merck KGaA) three times and incubated with 0.1% Triton X-100 (cat. no. X100; Sigma-Aldrich; Merck KGaA) for improvement of permeability. The cells were washed with PBS three times and blocked in 5% bovine serum albumin (cat. no. A7030; Sigma-Aldrich; Merck KGaA) for 20 min at room temperature. Subsequently, anti-vimentin (cat. no. PA5-27231; 1:1,000; Thermo Fisher Scientific, Inc.) and anti-cytokeratin antibody (cat. no. PA5-32465; 1:100; Thermo Fisher Scientific, Inc.) were used to incubate the cells at 4˚C overnight. After being washed with PBS, cells were incubated with goat anti-Rabbit IgG HRP (cat. no. 31466; 1:1,000; Thermo Fisher Scientific, Inc.) at 4˚C for 60 min in the dark. Following washing with PBS, cells were color-developed using diaminobenzidine (cat. no. D8001; Sigma-Aldrich; Merck KGaA) for 5 min at room temperature and counterstained with hematoxylin (cat. no. H3136, Sigma-Aldrich; Merck KGaA) for 1 min at room temperature. ESCs were observed by a light microscope (IX71; Olympus Corporation; magnification, x200).
Cell transfection
MIR503HG overexpression plasmids were constructed with pcDNA3.1 vector (cat. no. V79520; Thermo Fisher Scientific, Inc.). Short hairpin (sh)-MIR503HG (5'-CATCCAGCATCTCCAGTTA-3') was constructed using MISSION pLKO.1-puro Empty Vectors (cat. no. SHC001; Sigma-Aldrich; Merck KGaA). Empty vector and scrambled sequence (5'-TTCTCCGAACGTGTCACGT-3') were used as negative controls (NCs). miR-191 inhibitor/inhibitor control (IC; miR20000440-1-5, 5'-CAGCUGCUUUUGGGAUUCCGUUG-3'; miR2N0000001-1-5, 5'-UCUACUCUUUCUAGGAGGUUGUGA-3') and mimic/mimic control (miR10000440-1-5, 5'-CAACGGAAUCCCAAAAGCAGCUG-3'; miR1N0000001-1-5, 5'-UUCUCCGAACGUGUCACGU-3') were purchased from Guangzhou RiboBio Co., Ltd. ESCs were transfected with MIR503HG overexpression plasmids, sh-MIR503HG, miR-191 inhibitor/mimic or a combination of sh-MIR503HG and miR-191 inhibitor using Lipofectamine 3000® (cat. no. L3000015; Thermo Fisher Scientific, Inc.). Briefly, ESCs were plated in 96-well plates at a density of 1x104 cells/well and cultured to 80% confluence. Opti-MEM (10 µl; cat. no. 31985062; Thermo Fisher Scientific, Inc.) and P3000 reagents (0.4 µl; Thermo Fisher Scientific, Inc.) were used in combination to dilute the plasmids (0.2 µg) and Lipofectamine 3000 (0.15 µl), and the diluted Lipofectamine 3000 reagent and diluted plasmid was mixed, followed by incubation at 37˚C for 10 min. Finally, RNA-lipid complex was added to 96-well plates to incubate the cells at 37˚C for 24 or 48 h before further use.
Dual-luciferase reporter assay
Starbase v.2.0 (https://starbase.sysu.edu.cn/) was used to predict the targeting association between MIR503HG and miR-191. Wild-type (WT; 5'-ATGCTGCTTTT-GATTTCCGTTA-3') and mutant (MUT; 5'-ATGCTGACTTT-GATTTCCGTTA-3') MIR503HG 3'-untranslated region were cloned into pmirGLO vectors (cat. no. E1330, Promega Corporation) to synthesize pMirGLO-MIR503HG-WT and pMirGLO-MIR503HG-MUT, respectively. ESCs were plated into 12-well plates at a density of 1x107 cells/well and cultured to 70% confluence at 37˚C. The cells were co-transfected with miR-191 mimic (100 ng) and pmirGLO vectors (100 ng) inserted with fragments of MIR503HG WT or MUT sequence using Lipofectamine 3000® (Thermo Fisher Scientific, Inc.) at 37˚C for 48 h. Dual-luciferase reporter assay was immediately performed with Dual-Luciferase Reporter Assay System (cat. no. E1980, Promega Corporation). Firefly luciferase activity, normalized to Renilla luciferase activity and measured using a luminometer (GloMax® 20/20; Promega Corporation), was used to express the binding specificity.
RNA immunoprecipitation (RIP) assay
RIP assay was performed using a RIP kit (cat. no. RIP-12RXN; Sigma-Aldrich; Merck KGaA). ESCs were digested with trypsin (Sigma-Aldrich; Merck KGaA) and collected the cells, and then cells were ransferred into a mixed solution containing PBS, nuclear separation buffer and double-distilled H2O. The solution was stirred on ice and centrifuged at 2,500 x g for 15 min at 4˚C. The precipitated nucleus of ESCs was resuspended in RIP buffer. The solution was uniformly divided into IgG and the Ago2 group. The supernatant in the IgG group was added to 10 µg anti-IgG antibody (cat. no. ab171870; 1:1,000; Abcam) while that in the Ago2 group was added to 10 µg Ago2 antibody (cat. no. ab186733; 1:30; Abcam), gently shaken and then incubated at 4˚C overnight. The supernatant was added to 40 µl protein A/G magnetic beads (cat. no. M2400; Beijing Solarbio Science & Technology Co., Ltd.) and cultured at 4˚C for 1 h. Unconjugated protein was removed by RIP buffer washing. The untreated cell lysate was used as an input group. The RNAs in the input group, IgG and Ago2 group were extracted and reverse-transcribed into cDNA. Subsequently, the expression of miR-191 and MIR503HG were measured via quantitative polymerase chain reaction (qPCR). Expression in the input group was considered to be positive control.
Cell Counting Kit (CCK)-8 assay
Following transfection, ESCs were plated into 96-well plates at a density of 5x103 cells/well. CCK-8 reagent (cat. no. C0037; Beyotime Institute of Biotechnology) was added into the cells at a ratio of 1:10, after which cell incubation was performed at 37˚C for 1 h. Cell viability was measured based on the optical density using a microplate reader (Synergy Neo2; BioTek Instruments, Inc.; Agilent Technologies, Inc.) at a wavelength of 450 nm.
Transwell assay
Cell migration and invasion of transfected ESCs were evaluated by Transwell chambers (cat. no. 428; Corning, Inc.). For invasion assays, Transwell upper chambers were precoated with Matrigel (cat. no. 356234; Corning, Inc.) diluted at a ratio of 1:3 and incubated at 37˚C for 2 h. Following transfection, ESCs were suspended in serum-free DMEM/F12 to a concentration of 2x105 cells/ml and cell suspension (100 µl) was poured into the upper chamber. The lower chamber was filled with DMEM/F12 (600 µl) supplemented with 10% FBS (cat. no. F2442; Sigma-Aldrich; Merck KGaA). The whole Transwell chamber was incubated at 37˚C for 24 h, after which non-migratory or non-invading cells in the upper chamber were removed. The remaining cells were washed twice with PBS, fixed in 4% paraformaldehyde (cat. no. P6148; Sigma-Aldrich; Merck KGaA) at room temperature for 20 min and stained with Giemsa (800 µl; cat. no. 10092013; Thermo Fisher Scientific, Inc.) at room temperature for 15 min. Finally, stained cells were observed under an inverted light microscope (IX71; Olympus Corporation) and counted with ImageJ v.1.47 (National Institutes of Health, Bethesda, MD, USA) from eight randomly selected fields. The cell migration or invasion rate was set to 100% in control groups, while that in other groups was calculated by comparing the mean cell number with that in the control group.
Flow cytometry
The apoptosis of transfected ESCs was measured using Annexin V-FITC/PI apoptosis detection kit (cat. no. 40302ES20; Shanghai Yeasen Biotechnology Co., Ltd.). After transfection, ESCs were digested in EDTA-free trypsin (cat. no. T2600000; Sigma-Aldrich; Merck KGaA) at room temperature for 2 min and centrifugated at 3,000 x g for 5 min at 4˚C, followed by washing with PBS. Subsequently, the cells were resuspended with 1X Binding Buffer to reach the concentration of 1x106 cells/ml. Annexin V-FITC solution (5 µl) and PI solution (10 µl) were used to incubate the cells in the dark for 10 min at room temperature. Apoptotic cells (early and late apoptosis) were examined using a flow cytometer (CytoFLEX; Beckman Coulter, Inc.) and analyzed with CytExpert software (Version 2.2.0.97; Beckman Coulter, Inc.).
Reverse transcription-(RT)qPCR
AD tissues were homogenized using a UH-05 homogenizer (Union-Biotech). Total RNA and miRNA were extracted from ESCs, with or without transfection and AD and control tissues using TRIzol® (cat. no. 15596026; Thermo Fisher Scientific, Inc.) and PureLink miRNA Isolation kit (cat. no. K157001; Thermo Fisher Scientific, Inc.), respectively. The extracted total RNA and miRNA were reverse-transcribed to cDNA using SuperScript IV reverse transcriptase (cat. no. 18090010; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) using PowerUp SYBR Green Master Mix (cat. no. A25742; Thermo Fisher Scientific, Inc.). The primer pairs used for qPCR are listed in Table I. The thermocycling conditions were as follows: Initial denaturation at 95˚C for 10 min; 40 cycles of annealing at 95˚C for 15 sec and elongation at 60˚C for 60 sec. Quantification was performed using the 2-ΔΔCq method (23) and the expression levels of MIR503HG were normalized to GAPDH while miR-191 was normalized to U6.
Western blotting
Total protein from transfected ESCs was extracted using RIPA Buffer (cat. no. 89900; Thermo Fisher Scientific, Inc.) and quantified using a BCA assay (cat. no. A53227; Thermo Fisher Scientific, Inc.). The extracted protein (40 µg/lane) and marker (4 µl; cat. no. PR1910; Beijing Solarbio Science & Technology Co., Ltd.) were loaded and separated by SDS-PAGE on a 10 or 12% gel (cat. nos. P0670 and P0672, Beyotime Institute of Biotechnology) and then transferred onto PVDF membranes (cat. no. P2438; Sigma-Aldrich; Merck KGaA). The membranes were blocked with 5% non-fat milk in TBS with 1% Tween-20 (TBST; cat. no. T9039, Sigma-Aldrich; Merck KGaA) for 1 h at 37˚C. Subsequently, membranes were incubated with primary antibodies against E-cadherin (cat. no. ab40772; 97 kDa; 1:10,000; Abcam), N-cadherin (cat. no. ab18203; 130 kDa; 1:1,000; Abcam), β-catenin (cat. no. 9562; 92 kDa, 1:1,000; Cell Signaling Technology, Inc.), cleaved caspase-3 (cat. no. ab32042; 17 kDa; 1:500; Abcam) and GAPDH (cat. no. 5174; 37 kDa; 1:1,000; Cell Signaling Technology, Inc.) at 4˚C overnight. Following washing with TBST, membranes were incubated with a secondary Goat anti-Rabbit IgG Alexa Fluor™ Plus 488 (cat. no. A32731; 1:10,000; Thermo Fisher Scientific, Inc.) at 37˚C for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagent kit (cat. no. WP20005; Thermo Fisher Scientific, Inc.) on an imaging device (iBright CL750; Thermo Fisher Scientific, Inc.) and analyzed using ImageJ Software (1.52s version; National Institutes of Health).
Statistical analysis
All statistical analyses were conducted with GraphPad Prism (version 8.0; GraphPad Software Inc.). Data were obtained from independent experiments performed in triplicate and are presented as mean ± standard deviation. Differences between two groups were analyzed using the unpaired Student's t test, while those between multiple groups were analyzed using one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
MIR503HG is lowly expressed in AD tissue and regulates viability, migration, invasion and apoptosis in ESCs derived from the endometrium of patients with AD
RT-qPCR demonstrated that the MIR503HG expression in AD tissues was much lower than that in tissues collected from patients with cervical lesions or uterine fibroids (P<0.001; Fig. 1A). The physiological and pathological characteristics of ESCs isolated from the endometrium of patients with AD were investigated. Morphological examination via an optical microscope of isolated ESCs revealed that ESCs derived from patients with AD were polygons with an elliptical nucleus that was primarily located at the center of the cell and had noticeable nucleoli (Fig. 1B). In addition, immunocytochemistry assay illustrated that the cytoplasm of the ESCs was positive for vimentin with brownish yellow, but was negative for cytokeratin with purplish red nuclei (Fig. 1C). To investigate the role of MIR503HG in AD, isolated ESCs were subjected to transfection with MIR503HG overexpression plasmids or sh-MIR503HG; MIR503HG was successfully overexpressed and knocked down (P<0.001; Fig. 1D), respectively. CCK-8 and Transwell assays were performed to detect the viability, migration and invasion rates of ESCs derived from patients with AD; these were confirmed to be significantly decreased by MIR503HG overexpression (P<0.01 for viability and P<0.001 for both migration and invasion vs. NC; Fig. 1E-I) and increased by MIR503HG knockdown (P<0.05, P<0.01 and P<0.001 vs. NC, respectively; Fig. 1F-I). Moreover, flow cytometry analysis showed that apoptosis of ESCs was promoted by MIR503HG overexpression but inhibited by MIR503HG knockdown (P<0.001; Fig. 1J and K).
MIR503HG is a sponge of miR-191 and inhibits miR-191 expression in ESCs derived from patients with AD while miR-191 is highly expressed in AD tissues
Starbase bioinformatics analysis demonstrated that miR-191 had binding sites with MIR503HG (Fig. 2A). Dual-luciferase reporter assay was performed, the result of which showed that with transfection of miR-191 mimic, luciferase activity of ESCs derived from patients with AD and transfected with pMirGLO-MIR503HG-WT was suppressed (P<0.001; Fig. 2B), while that of cells transfected with pMirGLO-MIR503HG-MUT remained unchanged (Fig. 2B). RIP assay demonstrated that the expression levels of both miR-191 and MIR503HG were increased in the anti-Ago2 group, indicating binding of miR-191 and MIR503HG (P<0.001 vs. IgG; Fig. 2C). RT-qPCR showed that miR-191 expression was downregulated by MIR503HG overexpression but upregulated by MIR503HG knockdown in ESCs derived from patients with AD (P<0.001; Fig. 2D), while high expression of miR-191 was observed in AD tissue (P<0.001; Fig. 2E).
miR-191 inhibition partially reversed the effect of MIR503HG knockdown on the AD-derived ESC viability and migration/invasion and apoptosis
miR-191 inhibitor was introduced to determine the role of miR-191 in the MIR503HG-associated effects on ESCs derived from patients with AD. According to RT-qPCR results, expression of miR-191 was significantly decreased after ESCs were transfected with miR-191 inhibitor (P<0.001; Fig. 3A) and upregulation of miR-191 was observed following MIR503HG knockdown (P<0.001; Fig. 3B), and miR-191 inhibitor reversed MIR503HG knockdown-induced upregulation of miR-191 (P<0.001; Fig. 3B). CCK-8, Transwell and flow cytometry assay results demonstrated that miR-191 inhibition decreased the viability and the migration and invasion rates of ESCs derived from patients with AD and augmented the apoptosis rate (P<0.001 vs. NC + IC; Fig. 3C-I). MIR503HG knockdown promoted the viability and the migration and invasion of ESCs derived from patients with AD and inhibited the apoptosis (P<0.01, P<0.001 vs. NC + IC; Fig. 3C-I). In addition, miR-191 inhibition attenuated the effect of MIR503HG knockdown on viability and migration, invasion and apoptosis of ESCs derived from patients with AD (P<0.001; Fig. 3C-3I). Moreover, cleaved caspase-3 was used as the indicator of apoptosis and its expression was decreased by MIR503HG knockdown (P<0.001; Fig. 3J and K) but increased by miR-191 inhibitor in ESCs (P<0.001; Fig. 3J and K). Moreover, miR-191 inhibitor attenuated the effect of MIR503HG knockdown on the expression of cleaved caspase-3 (P<0.001; Fig. 3J and K).
MIR503HG knockdown facilitates epithelial-mesenchymal transition (EMT) and activates the Wnt/β-catenin pathway by regulating miR-191 expression in ESCs derived from patients with AD
A previous study demonstrated that EMT is a cellular process key for miR-191 upregulation-promoted cancer progression (24). In addition, the Wnt/β-catenin pathway is one of the underlying mechanisms of EMT in AD (25). Western blotting showed that MIR503HG knockdown significantly downregulated the levels of E-Cadherin and upregulated those of N-cadherin and β-catenin (P<0.01, P<0.001 and P<0.01 vs. NC + IC, respectively; Fig. 4A-D), while miR-191 inhibition significantly upregulated the levels of E-Cadherin and downregulated those of N-cadherin and β-catenin (P<0.001, P<0.05 and P<0.001 vs. NC + IC, respectively; Fig. 4A-D). Moreover, miR-191 inhibition attenuated the effect of MIR503HG knockdown on levels of E-cadherin, N-cadherin and β-catenin (P<0.05, P<0.01 and P<0.001, vs. sh-MIR503HG + IC, respectively; Fig. 4A-D).
Discussion
Although no direct evidence has indicated that AD triggers infertility, AD interferes with the success of in vitro fertilization (26). Prolonged treatment time of AD can result not only in placental dislocation, preeclampsia and premature delivery, but also cause endometrial adenocarcinoma (7,27,28). Currently, AD, as a common benign pathology, is asymptomatic in one-third of cases (29) and diagnostic indices for AD are still lacking (6). There is no early classification of the extent of the disease (30) and biomarkers need to be discovered for the management of AD.
Aberrantly expressed lncRNAs, which are been proposed to serve as diagnostic and therapeutic markers in assorted diseases, such as cancer, cardiovascular disease and osteoarthritis, have been detected in patients with AD (10,11). MIR503HG has been previously identified as key in sustaining endothelial cell biology and angiogenic processes (31) and is highly expressed in the placenta and other reproductive tissues (32). As AD refers to invasion of the endometrium into the myometrium, it is reasonable to hypothesize that MIR503HG is lowly expressed in patients with AD; this was confirmed in AD tissue in the present study.
ESCs are closely associated with the pathogenesis of AD, and inhibited ESC proliferation contributes to AD progression (13). Previous studies have demonstrated that transition of ESCs to epithelial cells suppresses AD progression (33) and resistance to the apoptosis of ESCs may account for the pathogenesis of AD (22). Moreover, inhibition of proliferation, migration and enhancement of apoptosis are considered a radically and efficiently conservative method for the treatment of AD (22). Overexpression of MIR503HG in preeclampsia-derived trophoblast cells inhibits cell proliferation, migration and invasion, which underly the pathogenesis of preeclampsia (17). MIR503HG overexpression leads to enhanced apoptosis of high glucose-cultured human proximal tubular (HK-2) cells (34). Contrary to the role of MIR503HG in preeclampsia but consistent with that in high glucose-induced HK-2 cells, the present study demonstrated that overexpression of MIR503HG impeded the viability, migration and invasion and enhanced the apoptosis of ESCs isolated successfully from the endometrial tissue of patients with AD. In addition, the pathogenesis of AD is affected by EMT (33). EMT is characterized by epithelial cells obtaining mesenchymal phenotypes through undergoing loss of cell polarity, remodeling of the cytoskeleton, acquired invasiveness and resistance to apoptosis (35,36). During this process, E-cadherin (an epithelial marker) expression is downregulated and N-cadherin (a mesenchymal marker) expression is upregulated (37). The present study demonstrated that the knockdown of MIR503HG led to downregulated E-cadherin and upregulated N-cadherin, indicating that MIR503HG overexpression may reverse EMT during AD progression. Overall, the present findings demonstrated that restoration of MIR503HG expression may help suppress AD progression.
Eutopic endometrium of patients with AD exhibits reciprocally dysregulated miRNAs such as upregulated miR-191, the analysis of which may be a promising low-invasive method for efficient diagnosis of AD (20). In addition, miRNAs participate in lncRNA-mediated AD progression by serving as the downstream targets of lncRNAs (13). The present study identified that miR-191 was directly targeted by MIR503HG and enriched in AD tissue, which is in line with its expression in adipose-derived mesenchymal stem cells that have stromal features (38). In addition, inhibition of miR-191 reversed the MIR503HG knockdown-induced the viability, migration, invasion increased and apoptosis decreased in ESCs derived from patients with AD to suppress AD progression, which signified that inhibition of AD progression induced by restoration of MIR503HG expression was miR-191 inhibition-dependent.
Additionally, a previous study discovered that the activation of the Wnt/β-catenin signaling pathway contributes to the pathogenesis of AD via EMT (39). In the nucleus of epithelial cells, functioning as the main effector of the canonical Wnt signaling which initiates EMT, β-catenin is key for tissue differentiation during embryonic development (39) and elevated β-catenin levels indicate activation of Wnt/β-catenin signaling (25). By analyzing β-catenin expression, the present study demonstrated that the Wnt/β-catenin pathway was activated by MIR503HG knockdown but suppressed by miR-191 inhibition, which also reversed MIR503HG knockdown-induced Wnt/β-catenin activation, denoting that the MIR503HG/miR-191 axis-mediated amelioration of AD resulted from inhibited Wnt/β-catenin signaling.
In conclusion, the present findings demonstrated that MIR503HG was lowly expressed in AD, and MIR503HG exerted a suppressive impact on AD progression by targeting miR-191, which was enriched in ESCs derived from patients with AD, to decrease viability, migration, invasion and EMT while enhancing apoptosis. This suppression may involve inhibition of the Wnt/β-catenin pathway. The present study provided new insights into the therapeutic strategies for AD. However, examinations in a clinical situation or peripheral blood samples were not performed to elucidate the role of MIR503HG or miR-191 in survival or prognosis of patients with AD. Also, the size of samples was small. In addition, the analysis of Wnt/β-catenin signaling pathway in future studies requires MIR503HG overexpression and rescue experiments.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported by the Deyang Science and Technology Bureau (grant no. 2020SZZ078).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
XX and BC provided substantial contributions to conception and design and wrote and revised the manuscript. YL, RL and JL performed data acquisition, analysis and interpretation. All authors have read and approved the final manuscript. XX and BC confirm the authenticity of all the raw data.
Ethics approval and consent to participate
The present study was approved by the ethics committee of People's Hospital of Deyang City (approval no. GD202000524) and written informed consent from all participants was obtained for experiments involving tissue.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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