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INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 41: 1030-1038, 2018 Abstract. The present study aimed to explore the roles of microRNA-21 (miR-21) and the transforming growth factor-β (TGF-β)/Smad signaling pathway in the develop- ment of peritoneal fibrosis (PF). First, dialysis effluents from 30 patients with PF were collected, and after the establishment of a mouse model of PF, hematoxylin and eosin (H&E) and Masson's staining were used to observe peritoneal tissues, inflammatory cells and blood vessels. High glucose was used to stimulate human peritoneal mesothelial cell lines and these stimulated cells were then transfected with miR‑21 inhibitor. Immunofluorescence microscopy was applied for the observa- tion of the transfected cells. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was used to detect the expression of miR‑21, and RT‑qPCR and western blot analysis were used to detect the mRNA and protein expression of Zonula occludens-1 (ZO-1), TGF-β, Smad, vimentin and connective- tissue growth factor (CTGF). The mRNA and protein expression levels TGF-β , Smad-3, vimentin and CTGF were elevated, while ZO-1 mRNA and protein expression was decreased with the prolonged duration of dialysis treatment in the patients with PF. The experiments using the mouse model of PF revealed that the peritoneal connective tissue was thickened, while the numbers of inflammatory cells and blood vessels were increased. The expression levels of miR-21, and the mRNA and protein expression levels of TGF-β , Smad-3, vimentin and CTGF were increased over time, whereas the mRNA and protein expression levels ZO-1 constantly decreased in the mice in the experimental group. Moreoever, the expression of miR‑21 positively correlated with the expression levels of TGF-β, Smad-3, vimentin and CTGF, while it negatively correlated with the expression of ZO‑1. The results of H&E and Masson's staining revealed that miR‑21 expression was associated with the degree of PF. These findings thus indicate that miR‑21 promotes the progression of PF through the activation of the TGF‑β/Smad signaling pathway. Introduction Peritoneal dialysis (PD), based on the peritoneum as a semi- permeable membrane, aims to filter out solutes for blood purification (1). PD is an economic and simple dialysis method, which can remove toxic molecules and improve the survival rate (2). However, long‑term exposure to a hypertonic and high glucose environment can induce inflammation, epithelial cell differentiation and angiogenesis, resulting in fibrosis (3). Peritoneal fibrosis (PF) is associated with abnormal histological changes in peritoneal tissues and parenchymal cells necrosis caused by fibrous connective tissue matrix deposition, which is also the main cause of ultrafiltration dialysis failure (4). The exact molecular mechansisms underlying the disease pathogenesis remain unclear. However, it has been reported that a combina- tion of mechanisms are involved in PF pathogenesis, including a high peritoneal dialysate glucose concentration, high osmotic pressure, low pH value, advanced glycation end products (AGEs) and bacterial peritonitis (5). It has been demonstrated that a number of cytokines are associated with PF, including trans- forming growth factor (TGF)-β1, connective-tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) and platelet‑derived growth factor (PDGF) (6). In addition, a previous study revealed that the activation of the TGF-β/Smad signaling pathway in patients with PF confirms the opposite role of Smad2 and Smad3 in PF and their involvement in the progres- sion of PF (7). In addition, Zheng et al reported that Astragalus membranaceus inhibited PF via the monocyte chemoattractant protein (MCP)‑1 and TGF‑β 1 pathways, exerting anti‑fibrotic and therapeutic effects (8). These studies provide insight into the treatment of PF via the targeting of TGF‑β/Smad signaling. The gene coding for microRNA (miR)‑21 is located on 17q23.2 within the TMEM49 coding region and plays an important role in DNA-dependent transcriptional regulation, which is of great significance in the development of fibrotic diseases (9). TGF‑β is a pleiotropic growth factor that regulates cell proliferation, differentiation and the immune response as well as apoptosis, and mainly exerts its role in tissue fibrosis, stimulation, mesen- MicroRNA-21 promotes the progression of peritoneal fibrosis through the activation of the TGF-β/Smad signaling pathway: An in vitro and in vivo study YA-LI MA * , FANG CHEN * , Su‑XIA YANG, BAO‑PING CHEN and JuN SHI Department of Nephrology, Huaihe Hospital of Henan university, Kaifeng, Henan 475000, P.R. China Received October 25, 2016; Accepted October 6, 2017 DOI: 10.3892/ijmm.2017.3268 Correspondence to: Dr Jun Shi, Department of Nephrology, Huaihe Hospital of Henan university, 115 Ximen Avenue, Gulou, Kaifeng, Henan 475000, P.R. China E‑mail: [email protected] * Contributed equally Key words: microRNA-21, transforming growth factor-β/Smad signaling pathway, peritoneal fibrosis, HMrSV5 human peritoneal mesothelial cells

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INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 41: 1030-1038, 20181030

Abstract. The present study aimed to explore the roles of microRNA-21 (miR-21) and the transforming growth factor-β (TGF-β)/Smad signaling pathway in the develop-ment of peritoneal fibrosis (PF). First, dialysis effluents from 30 patients with PF were collected, and after the establishment of a mouse model of PF, hematoxylin and eosin (H&E) and Masson's staining were used to observe peritoneal tissues, inflammatory cells and blood vessels. High glucose was used to stimulate human peritoneal mesothelial cell lines and these stimulated cells were then transfected with miR‑21 inhibitor. Immunofluorescence microscopy was applied for the observa-tion of the transfected cells. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was used to detect the expression of miR‑21, and RT‑qPCR and western blot analysis were used to detect the mRNA and protein expression of Zonula occludens-1 (ZO-1), TGF-β, Smad, vimentin and connective-tissue growth factor (CTGF). The mRNA and protein expression levels TGF-β, Smad-3, vimentin and CTGF were elevated, while ZO-1 mRNA and protein expression was decreased with the prolonged duration of dialysis treatment in the patients with PF. The experiments using the mouse model of PF revealed that the peritoneal connective tissue was thickened, while the numbers of inflammatory cells and blood vessels were increased. The expression levels of miR-21, and the mRNA and protein expression levels of TGF-β, Smad-3, vimentin and CTGF were increased over time, whereas the mRNA and protein expression levels ZO-1 constantly decreased in the mice in the experimental group. Moreoever, the expression of miR‑21 positively correlated with the expression levels of TGF-β, Smad-3, vimentin and CTGF, while it negatively correlated with the expression of

ZO‑1. The results of H&E and Masson's staining revealed that miR‑21 expression was associated with the degree of PF. These findings thus indicate that miR‑21 promotes the progression of PF through the activation of the TGF‑β/Smad signaling pathway.

Introduction

Peritoneal dialysis (PD), based on the peritoneum as a semi-permeable membrane, aims to filter out solutes for blood purification (1). PD is an economic and simple dialysis method, which can remove toxic molecules and improve the survival rate (2). However, long‑term exposure to a hypertonic and high glucose environment can induce inflammation, epithelial cell differentiation and angiogenesis, resulting in fibrosis (3). Peritoneal fibrosis (PF) is associated with abnormal histological changes in peritoneal tissues and parenchymal cells necrosis caused by fibrous connective tissue matrix deposition, which is also the main cause of ultrafiltration dialysis failure (4). The exact molecular mechansisms underlying the disease pathogenesis remain unclear. However, it has been reported that a combina-tion of mechanisms are involved in PF pathogenesis, including a high peritoneal dialysate glucose concentration, high osmotic pressure, low pH value, advanced glycation end products (AGEs) and bacterial peritonitis (5). It has been demonstrated that a number of cytokines are associated with PF, including trans-forming growth factor (TGF)-β1, connective-tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) and platelet‑derived growth factor (PDGF) (6). In addition, a previous study revealed that the activation of the TGF-β/Smad signaling pathway in patients with PF confirms the opposite role of Smad2 and Smad3 in PF and their involvement in the progres-sion of PF (7). In addition, Zheng et al reported that Astragalus membranaceus inhibited PF via the monocyte chemoattractant protein (MCP)‑1 and TGF‑β1 pathways, exerting anti‑fibrotic and therapeutic effects (8). These studies provide insight into the treatment of PF via the targeting of TGF‑β/Smad signaling.

The gene coding for microRNA (miR)‑21 is located on 17q23.2 within the TMEM49 coding region and plays an important role in DNA-dependent transcriptional regulation, which is of great significance in the development of fibrotic diseases (9). TGF‑β is a pleiotropic growth factor that regulates cell proliferation, differentiation and the immune response as well as apoptosis, and mainly exerts its role in tissue fibrosis, stimulation, mesen-

MicroRNA-21 promotes the progression of peritoneal fibrosis through the activation of the TGF-β/Smad

signaling pathway: An in vitro and in vivo studyYA-LI MA*, FANG CHEN*, Su‑XIA YANG, BAO‑PING CHEN and JuN SHI

Department of Nephrology, Huaihe Hospital of Henan university, Kaifeng, Henan 475000, P.R. China

Received October 25, 2016; Accepted October 6, 2017

DOI: 10.3892/ijmm.2017.3268

Correspondence to: Dr Jun Shi, Department of Nephrology, Huaihe Hospital of Henan university, 115 Ximen Avenue, Gulou, Kaifeng, Henan 475000, P.R. ChinaE‑mail: [email protected]

*Contributed equally

Key words: microRNA-21, transforming growth factor-β/Smad signaling pathway, peritoneal fibrosis, HMrSV5 human peritoneal mesothelial cells

ΜΑ et al: miR‑21 MEDIATES THE ACTIVATION OF THE TGF‑β/Smad PATHwAY IN PF 1031

chymal cell growth and collagen and fibronectin increase (10). Smad protein (Drosophila mothers against decapentaplegic protein) can be divided into 3 subfamilies: receptor‑regulated Smads (R-Smads), common-mediator Smads (Co-Smads) and inhibitory Smads (I‑Smads). There are 8 types of Smad proteins in mammals and they are TGF-β-mediated signal transduc-tion proteins, and the TGF-β/Smad signaling pathway is a key pathway in the occurrence and progression of fibrosis, involved with a variety of fibrotic diseases (11).

Certain experimental studies have detected an abnormal miR‑21 expression various organs affected by fibrosis; thus, the investigation of the function of miR‑21 in fibrosis has impli-cations for the elucidation of the pathogenesis, diagnosis and treatment of fibrosis (7‑9). Thus, this study aimed to explore the mechanisms of action of miR‑21 in PF and its association with the TGF-β/Smad pathway using dialysis effluents from patients with PF and by establishing a mouse model of PF.

Materials and methods

Study subjects. Patients with PF receiving a stable treatment for PD at the Huaihe Hospital of Henan university, Henan, China from October, 2014 to June, 2015 were selected for the study. The 30 patients included were >18 years of age and had received dialysis for >3 months. More specifically, the study subjects consisted of 23 males and 7 females with an average age of 58.5 years, whose disease duration ranged from half a month to 3 years, with an average of 15.2 months. The disease for each included subject was diagnosed from 3 days to 5.1 months from admission to the hospital with 2.1 months on average. The diagnosis was conducted and confirmed through clinical mani-festations and imaging examination. Clinical manifestations were shown as follows: back pain, leg edema, abdominal pain, lower abdominal mass, and the remaining symptoms included bloating, headaches, nausea and vomiting. The diagnostic method for the imaging examination included a B-ultrasound examination firstly, followed by a CT scan after hydronephrosis and ureterec-tasia were found. ureteral compression was also examined if the soft tissue density was found to be below the abdominal aortic bifurcation (12). According to the length of the dialysis treat-ment, there were a total of 3 groups with a 1‑year interval. For the dialysis treatment duration, there were 9 cases with <1-year duration; 14 cases with a 1‑2‑year duration and 7 cases with a 2‑3‑year duration. During peritoneal dialysis, the omentum was obtained for the collection of mesothelial cells. This study was approved by the Ethics Committee of Huaihe Hospital of Henan university. All enrolled patients had signed an informed consent. Patients suffering from a mental illness or unable to cooperate were excluded from this study.

Establishment of the mouse model of PF. Male Kunming mice at the specific pathogen‑free (SPF) level and weighing approximately 30‑40 g, were provided by the Animal Research Department of Nanchang university, Nanchang, China. All mice were reared in an SPF level environment, with a temperature of 25±1˚C and a constant humidity of 40‑60%, and free access to food and water. The feeding lasted for 7 days. A total of 20 mice were randomly divided into 2 groups with 10 mice in each group, the experimental and control groups, respectively. The mice in the experimental group were intraperitoneally injected with 0.1 mg/

kg lipopolysaccharide on day 1, 3, 5 and 7 and with 4.25% glucose dialysate (100 ml/kg) (13) each day (lasting 27 days). On day 28, the mice were sacrificed by cervical dislocation. All procedures involving animals were performed according the Guide for the Care and use of Laboratory Animals. The animal experiments were also approved by the Ethics Committee of Huaihe Hospital of Henan university, while the peritoneal mesothelial cells from 3 mice in each group were collected every 3 days during peritoneal dialysis. For the control group, 0.9% saline was intra-peritoneally injected daily, lasting for 28 days.

Peritoneal equilibrium test (PET). A PET assay was performed 28 days after modeling. The mice were substituted with 20 ml of 2.5% glucose‑based dialysate. Two hours later, the mice were sacrificed by cervical dislocation. The catheter drainage was measured, marked as S1, and the rest of glucose‑based dialy-sate was collected using a straw, marked as S2. ultrafiltration (ml) = S1 + S2 ‑20. The endpoint method was used to measure the dialysate urea concentration (D), plasma urea concentra-tion (Purea), the initial glucose concentration of peritoneal dialysis solutions (D0) and the glucose concentration of the dialysate effluent (D2), and the results of D/Purea and D2/D0 were calculated. Finally, the parietal peritoneum was fixed in 1% formaldehyde for pathological examination.

Hematoxylin and eosin (H&E) staining and Masson's staining. The visceral peritoneum specimens from the 2 groups of mice were soaked in paraformaldehyde solution and then dehydrated, embedded in paraffin, sectioned (4 µm thickness), and then baked for 12 h in an oven at 56˚C. Through H&E and Masson's staining, 10 visual fields were selected randomly (x200 magni-fication) and 5 positions were taken in each field. Image‑Pro Plus 6.0 software was used for the analysis. Peritoneal tissue thickness, the inflammatory cell number, and the number of blood vessels were also measured.

Peritoneal mesothelial cell culture and transfection. HMrSV5 human peritoneal mesothelial cells were obtained from the Institute of Nephrology in the First Affiliated Hospital of Sun Yat‑sen university. The HMrSV5 cells were cultured in DMEM‑F12 medium (Gibco, Grand Island, NY, uSA) containing 10% fetal bovine serum (FBS) in 37˚C with 5% CO2. The groups of peritoneal mesothelial cells were as follows: i) blank group (untreated peritoneal mesothelial cells); ii) high glucose group (4.5% high glucose‑induced peritoneal mesothelial cells); iii) high glucose + miR‑21 inhibitor group (4.5% high glucose‑induced peritoneal mesothelial cells were to be treated with miR‑21 inhibitors); iv) high glucose + miR‑21 inhibitor control group (4.5% high glucose‑induced peritoneal mesothelial cells were to be treated with miR‑21 inhibitor negative controls). The miR‑21 inhibitors and inhibitor negative controls were purchased from Biomics Biotechnology Corp. (Nantong, China). Twenty‑four hours before treatment, high glucose-induced peritoneal mesothelial cells were seeded in a 6‑well plate. when cell confluence reached 50%, they were incubated in a mixture of 250 µl serum-free Opti-MEM diluted miR‑21 inhibitors (4.0 µg) or miR‑21 inhibitor NC (4.0 µg) and 250 µl serum-free Opti-MEM diluted lipofectamin 2000 (10 µl; Invitrogen, Carlsbad, CA, uSA) for 6‑8 h in an atmosphere of 5% CO2 at 37˚C. when the cells were incubated for 24 h, the

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total RNA was extracted; 72 h later, total protein was extracted. The expressio levels of miR-21, Zonula occludens-1 (ZO-1), TGF-β, Smad-3, vimentin, and CTGF were detected, and some of the cells were retained for fluorescence microscopy.

Immunofluorescence. The immunofluorescence method was applied to observe he transfected cells, and fibronectin was used as a reference for fibrosis (14). Fibronectin antibody (ab2413) and biotin‑labeled goat anti‑rabbi IgG (1:1,000) were purchased from Abcam Inc. (Cambridge, MA, uSA). Primary antibodies (20 µg/ml) were diluted with 1% FBS, and secondary antibodies were diluted to 1:200. After the cells were grown on coverslips, the primary antibody was added, followed by incubation at 37˚C for 2 h; the secondary antibody was then added, followed by incubation at 37˚C for 1 h; finally 5 µg/ml DAPI were added the cells were stained for 5 min. A fluorescence microscope (Nikon, Tokyo, Japan) was used for the observation.

Reverse transcription-quantitative polymerase chain reac-tion (RT-qPCR). The recovered peritoneal mesothelial cells from patients and mice were collected every 3 days, and the extraction of total RNA from the transfected cell lines was performed using an RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. A uV Spectrophotometer (NanoDrop, walsingreen Co., Beijing, China) was used to measure the A260/A280 value and RNA concentration. RNA was reverse transcribed into cDNA using a reverse transcription kit (Promega, Madison, wI, uSA). A fluo-rescence quantitative PCR kit was purchased from Shanghai

GenePharma Technology Co., Ltd., Shanghai, China). An ABI 7500 real‑time PCR instrument (Applied Biosystem, Foster City, CA, uSA) was used for the PCR reaction. The reaction volume was 20 µl, and the reaction conditions were 40 cycles at 95˚C for 3 min, 95˚C for 12 sec and 62˚C for 50 sec. miR‑21 expression was measured quantitatively using u6 as an internal control, and the mRNA expression levels of ZO-1, TGF-β, Smad, vimentin and CTGF were measured using glyceraldehyde 3‑phosphate dehydrogenase (GAPDH) as an internal reference gene. The primers used are shown in Table I. Relative expression was calculated using the 2-ΔΔCq method.

Western blot analysis. The cells and tissues were washed with phosphate‑buffered saline (PBS), followed by the addition of 100 µl of cell lysates and incubation at 4˚C for 30 min. Centrifugation was performed at 12,000 x g for 10 min. The supernatant was collected and the protein concentration was measured using the Bradford method. The total protein (5 µg) extracted was electro-phoresed on a 12% sodium dodecyl sulfate (SDS)‑polyacrylamide gel. After being transferred onto polyvinylidene fluoride (PVDF) membranes (Jiuding High Tech. Ltd., Beijing, China), the total protein was sealed with 5% dried skimmed milk at room temper-ature for 1 h. The membranes were then incubated with mouse anti human monoclonal antibodies to ZO‑1 (ab61357; 5 µg/ml), TGF-β (ab64715, 1‑2 µg/ml), Smad (ab75512, diluted at 1:5,000), vimentin (ab8978, diluted at 1:500), CTGF (ab94939, 10 µg/ml) and GAPDH (ab8245, diluted at 1:1,000) (all from Abcam) overnight at 4˚C. After washing, the membranes were incubated with IRDye™ 800DX marked goat anti‑mouse IgG (ab6789, Abcam; diluted at 1:10,000) for 1 h. After being cultivated in the dark at room temperature for 1 h, the membranes were washed and placed into the Odyssey two-color infrared laser imaging system (LICOR Biosciences, Lincoln, NE, USA) for scanning and imaging. The integral optical density of each strip was calculated, and the ratio of the integral optical density value of the target band with the integrated optical density value of the reference GAPDH, was the relative expression value of the target protein. This experiment was repeated 3 times.

Statistical analysis. IBM SPSS statistical software (version 19.0; SPSS, Inc., Chicago, IL, uSA) was used for statistical analysis.

Table I. Primers used for RT‑qPCR.

Gene Primer sequences

miR‑21 F: 5'‑GCGGTAGCTTATCAGACTG‑3' R: 5'‑TGCGTGTCGTGGAGTC‑3'u6 F: 5'‑CTCGCTTCGGCAGCACA‑3' R: 5'‑AACGCTTCACGAATTTGCGT‑3'ZO‑1 F: 5'‑AATGAATGATGGTTGGTATGG‑3' R: 5'‑TGACAGGTAGGACAGACG‑3'TGF-β F: 5'‑GTTCTTCAATACGTCAGACATTCG‑3' R: 5'‑CATTATCTTTGCTGTCACAAGAGC‑3'Smad‑3 F: 5'‑AAATGACAGCAGCAGGGACACTA‑3' R: 5'‑TGAGGAGGTAGGACCCACAGTAGA‑3'Vimentin F: 5'‑CGTTTCCAAGCCTGACCTCACC‑3' R: 5'‑GCCATCTTTACATTGAGCAGGT‑3'CTGF F: 5'‑TGTGAAGACCTACCGGGCTA‑3' R: 5'‑TTCATGATCTCGCCATCGGG‑3'GAPDH F: 5'‑CGCTGAGTACGTCGTGGAGT‑3' R: 5'‑GTCGCTGTTGAAGTCAGAGGAG‑3'

RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction; ZO‑1, Zonula occludens‑1; CTGF, connective tissue growth factor; GAPDH, glyceraldehyde‑3‑phosphate dehydrogenase; TGF‑β, trans-forming growth factor-β; F, forward; R, reverse.

Table II. Changes in peritoneal function in mice in the experi-mental and control groups.

Control ExperimentalGroups group group P‑value

ultrafiltration 9.05±1.59 1.02±0.32 <0.001volume/mlD/Purea 0.56±0.11 0.79±0.18 0.024D2/D0 0.44±0.12 0.16±0.08 <0.001Inflammatory cells 111.15±15.32 167.51±15.28 <0.001Vascular tissues 23.40±5.93 61.26±14.77 <0.001

Purea, plasma urea concentrations; D2, glucose concentrations of dialy-sate effluent; D0, initial glucose concentrations of peritoneal dialysis solutions.

ΜΑ et al: miR‑21 MEDIATES THE ACTIVATION OF THE TGF‑β/Smad PATHwAY IN PF 1033

Count data was expressed in rate or percentage, and the Chi‑square test was used to make comparisons between 2 groups. Measurement data in groups were compared using One-way ANOVA (prior to analysis, detection of homogeneity of variance was performed), and the LSD-t-test of mean value was applied between 2 groups. Correlation analysis referred to Pearson's correlation analysis. A value of P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of miR-21 and mRNA expression levels of TGF-β, Smad-3, vimentin and CTGF in patients with PF receiving dialysis. The expression of miR-21 and the mRNA expression levels of TGF-β, Smad-3, vimentin and CTGF were increased with the increased duration of the dialysis sessions, while the expression of ZO‑1 was decreased (all P<0.05). In the patients receiving dialysis treatment for a course of 2-3 years, all expres-sion of indicators exhibited a significant difference compared to those of patients receiving dialysis treatment for 1-2 years or <1 year (all P<0.05) (Fig. 1). Correlation analysis revealed that miR-21 expression positively correlated with the mRNA expression of TGF-β, Smad‑3, vimentin and CTGF (r=0.997,

P<0.001; r=0.980, P<0.001; r=0.911, P<0.001; r=0.876, P<0.001), while miR‑21 expression was negatively correlated with ZO‑1 expression (r=‑0.826, P<0.001) (Fig. 1).

Protein expression levels of ZO-1, TGF-β, Smad-3, vimentin and CTGF in patients with PF receiving dialysis. The protein expression levels of TGF-β, Smad-3, vimentin and CTGF were increased with the increased duration of the treatment course, while ZO‑1 protein expression decreased (all P<0.05) (Fig. 2). The expression of TGF-β was found to have a significant and positive correlation with the expression levels of Smad-3, vimentin and CTGF (r=0.978, P<0.001; r=0.981, P<0.001; r=0.961, P<0.001, while TGF‑β expression was negatively correlated with ZO‑1 (r=‑0.913, P<0.001).

Peritoneal function and histological analysis of mice in the experimental and control group. The PET results revealed that the ultrafiltration volume and the D2/D0 were decreased (all P<0.01), and that the D/Purea was increased in the experi-mental group compared with the control group (P<0.05; Table II). The H&E staining results revealed that the wall of the peritoneal layer of the mice in the experimental group had thickened significantly, and the amount of fibrous tissue had

Figure 1. (A) miR‑21 expression and mRNA expression of Zonula occludens‑1 (ZO‑1), transforming growth factor‑β (TGF-β), Smad-3, vimentin and connective-tissue growth factor (CTGF) detected by RT‑qPCR. *P<0.05 compared with the treatment course for <1 year; (B) negative correlation between the miR‑21 and ZO‑1; (C) positive correlation between the miR‑21 and TGF‑β; (D) positive correlation between the miR‑21 and Smad‑3; (E) positive correlation between the miR‑21 and vimentin; (F) positive correlation between the miR‑21 and CTGF; #P<0.05 compared with the treatment course with for 1‑2 years; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction.

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significantly increased in the experimental group. From the results of Masson's staining, the cytoplasm in the experimental group had shrunk significantly and the outer wall of the fibrous

tissue was significantly thicker compared with the control group (P<0.05; Fig. 3). As observed from the two staining images, we found that the numbers of inflammatory cells and

Figure 2. Protein expression of Zonula occludens‑1 (ZO‑1), transforming growth factor‑β (TGF-β), Smad, vimentin and connective-tissue growth factor (CTGF) in patients with peritoneal fibrosis treated with dialysis as detected by western blot analysis [(A) histogram of the relative protein expression of ZO‑1, TGF‑β, Smad, vimentin and CTGF; (B) protein expression of ZO‑1, TGF‑β, Smad, vimentin and CTGF; (C) negative correlation between the TGF‑β and ZO‑1; (D) posi-tive correlation between the TGF‑β and Smad‑3; (E) positive correlation between the TGF‑β and vimentin; (F) positive correlation between the TGF‑β and CTGF]. *P<0.05 compared with the treatment course of <1 year; #P<0.05 compared with the treatment course of 1‑2 years.

Figure 3. Comparison of tissue staining and peritoneal thickness between the experimental group and the control group. *P<0.05 compared with the control group.

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vascular tissues in the experimental group were significantly higher than those of the control group (P<0.05; Table II).

Expression of miR-21 and the mRNA expression levels of ZO-1, TGF-β, Smad-3, vimentin and CTGF in the mouse model of PF.

The results revealed that the miR-21, TGF-β, Smad-3, vimentin and CTGF expression levels in the peritoneal mesothelial cells from the experimental group tended to increase over time, while ZO‑1 expression tended to decrease (Fig. 4). The expression of each biomolecule in the control group remained relatively stable.

Figure 5. Changes in the protein expressions of Zonula occludens‑1 (ZO‑1), transforming growth factor‑β (TGF-β), Smad, vimentin and connective-tissue growth factor (CTGF) at different time points. (A) Changes in the protein expression of ZO‑1, TGF‑β, Smad, vimentin and CTGF at different time points shown by a linear graph; (B) protein expression of ZO‑1, TGF‑β, Smad, vimentin and CTGF at different time points detected by western blot analysis. Differences were evident on day 10; *P<0.05 compared with the control group.

Figure 4. Changes in the expression of miR‑21 and the mRNA expression levels of Zonula occludens‑1 (ZO‑1), transforming growth factor‑β (TGF-β), Smad-3, vimentin and connective‑tissue growth factor (CTGF) at different time points. Differences were evident on day 8; *P<0.05 compared with the control group.

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From day 8, molecule expression in the experimental group differed significantly compared with the control group (all P<0.05).

Protein expression levels of ZO-1, TGF-β, Smad-3, vimentin and CTGF in the mouse model of PF. The results of western blot analysis for protein expression are shown in Fig. 5. Compared with the control group, differences in the expression levels of molecules in the experimental group were not significant on days 1‑3. Each molecule expression level in the experimental group changed over time, with the expression of TGF-β, Smad, vimentin and CTGF increasing, and that of ZO‑1 decreasing. However, no significant changes in expression were observed in the control group. From day 10, molecule protein expression in the experimental group exhibited a statistically significant difference compared with the control group (all P<0.05).

The miR-21 expression in HMrSV5 cells following transfec-tion with miR-21 inhibitor. The miR-21 expression levels in the cells in the high glucose group and the high glucose + inhibitor control group were significantly higher than those of the cells in the blank group (all P<0.05). In the high glucose + miR‑21 inhibitor group, miR‑21 expression exhibited no significant difference compared with the blank group (P>0.05; Fig. 6).

mRNA expression levels of ZO-1, TGF-β, Smad-3, vimentin and CTGF in HMrSV5 cells following transfection with miR-21 inhibitor. Compared with the blank group, the expres-sion levels of TGF-β, Smad-3, vimentin and CTGF in the high glucose group and high glucose + inhibitor control group were higher, while those of ZO‑1 were decreased. Compared with the high glucose group and high glucose + inhibitor control group, the TGF-β, Smad-3, vimentin and CTGF expression levels in the high glucose + miR‑21 inhibitor group were signif-icantly decreased, while those of ZO‑1 were increased (both P<0.05; Fig. 7). Hence, miR‑21 was proven to enhance TGF‑β, Smad-3, vimentin and CTGF expression, and to decrease ZO-1 expression, thus playing an important regulatory role in PF.

Protein expression levels of ZO-1, TGF-β, Smad-3, vimentin and CTGF in HMrSV5 cells following transfection with miR-21 inhibitor. The protein expression levels of TGF-β, Smad, vimentin and CTGF in both the high glucose group and high glucose + inhibitor control group were higher than those in the blank group, with the level of ZO‑1 significantly decreased (P<0.05). However, compared with the high glucose group and high glucose + inhibitor control group, TGF‑β, Smad, vimentin and CTGF expression levels in the high glucose + miR‑21 inhibitor group were significantly decreased, while those of ZO‑1 were significantly increased (P<0.05; Fig. 8).

Degree of PF in HMrSV5 cells in each group. As shown in Fig. 9, with high glucose as an induction source for retro-peritoneal fibrosis, compared with the blank group, the amount of fibronectin‑positive cells increased significantly, with a small intercellular distance and high density, which was evidence of an increased degree of fibrosis. By introducing miR‑21 inhibitor, in the high glucose + miR‑21 inhibitor group, the number of fibro-nectin‑positive cells significantly decreased compared to the high glucose group (P<0.05). However, there was no significant differ-ence between the high glucose + inhibitor control group and the high glucose group. Thus, these data suggested that the degree of PF correlated with miR‑21 expression. when miR‑21 expression was increased, the degree of fibrosis was also increased; however, when miR‑21 was inhibited, the degree of fibrosis decreased.

Discussion

In this study, peritoneal mesothelial cells from patients with PF receiving dialysis and from mice with PF were used to explore the correlation of miR-21 expression with the TGF-β/Smad signaling pathway and the role of miR-21 in the progression of PF. we found that miR‑21 enhanced the pathological processes of PF by regulating the TGF‑β/Smad signaling pathway.

PF is the main cause for patients abandoning PD treatment. A previous study demonstrated that innate activating fibroblast peritoneal mesothelial cytokines, inflammatory cells and growth factors play an important role in PF (15). This study found that in mesothelial cells, miR-21 expression and the corresponding retroperitoneal fibrosis index, the expression of TGF‑β, Smad-3,

Figure 6. Comparison of miR‑21 expression in HMrSV5 cells following trans-fection with miR‑21 inhibitor. *P<0.05 compared with the blank group.

Figure 7. Comparison of mRNA expression levels of Zonula occludens‑1 (ZO‑1), transforming growth factor-β (TGF-β), Smad, vimentin and connective‑tissue growth factor (CTGF) in HMrSV5 cells following transfec-tion with miR‑21 inhibitor. *P<0.05 compared with the blank group.

ΜΑ et al: miR‑21 MEDIATES THE ACTIVATION OF THE TGF‑β/Smad PATHwAY IN PF 1037

vimentin and CTGF were increased with the increasing treat-ment duration of dialysis; however, ZO‑1 expression was decreased. TGF‑β is an important factor for the promotion of PF and its gene expression is mainly regulated though the signaling protein Smad2/3, with the key molecule Smad3 mediated by TGF-β in fibrosis (16,17). A previous study demonstrated that TGF-β significantly stimulated Smad2/3 activation in peritoneal mesothelial cells and upregulated inflammatory cytokines, such as interleukin (IL)-1, tumor necrosis factor (TNF)-α and intercellular adhesion molecule 1 (ICAM-1), causing the trans-

differentiation of peritoneal mesothelial cells and the increase of extracellular matrix (18). Furthermore, high glucose dialysate can significantly upregulate CTGF and vimentin in expression peritoneal mesothelial cells, and TGF-β may be used as a down-stream target protein to promote the synthesis and degradation of the extracellular matrix, resulting in the deposition of extracel-lular matrix and mediating the occurrence of PF (19). An in vitro study also found that miR‑21 expression was downregulated by extracellular regulated protein kinase (ERK) inhibitor, Spry1, and that the expression of miR-21 had an effect on the promotion

Figure 9. Fibronectin‑positive cells in peritoneal fibrosis observed under a fluorescence microscope [(A) blank group; (B) high glucose group; (C) high glu-cose + miR‑21 inhibitor group; (D) high glucose + miR‑21 inhibitor control group].

Figure 8. Comparison of the protein expression of Zonula occludens‑1 (ZO‑1), transforming growth factor‑β (TGF-β), Smad, vimentin, and connective-tissue growth factor (CTGF) in HMrSV5 cells following transfection with miR‑21 inhibitor [(A) comparison of the protein expression of ZO‑1, TGF‑β, Smad, vimentin and CTGF in each group; (B) comparison of the protein expression of ZO‑1, TGF‑β, Smad, vimentin, and CTGF detected by western blot analysis]. *P<0.05 compared with the blank group.

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 41: 1030-1038, 20181038

of the survival of fibroblasts and the secretion of fibroblast growth factor via the ERK signaling pathway; these results indicated that miR‑21 influenced the proliferation of fibroblasts, the deposition of collagen in the extracellular matrix and fibrosis (20).

In this study, in order to further clarify the association between miR‑21 and PF, we constructed a mouse model of PF, and the expression of miR‑21, as well as that of fibrosis‑related proteins was measured. The mRNA and protein expression of miR-21, TGF-β, Smad‑3, vimentin and CTGF exhibited an upward trend over time, while ZO‑1 expression exhibited a downward trend, and it was confirmed that the degree of fibrosis increased with the unregulated expression of miR‑21. According to previous reports, TGF-β gene superfamily bound with serine/threonine kinase receptors can activate R-Smad (Smad-2 and Smad‑3) protein for phosphorylation and cause Smad‑7 to competitively bind with TGF‑β receptor through the TGF-β/Smad signaling pathway, which in turn inhibits Smad‑2/3 phos-phorylation, and regulates TGF-β signal gene transcription and expression, thereby regulating cell proliferation, differentiation, migration and apoptosis (21,22). Studies have confirmed that TGF-β is a target gene of miR-21 and TGF-β-induced endo-thelial cell transition to mesenchymal has been shown to partly occur through miR‑21‑mediated signaling pathways (23,24). In studies on renal fibrosis using humans and animals, TGF‑β binding to Smad3 was enhanced, which increased the transcrip-tion of a variety of collagen genes and miR-21 upregulation was involved in the fibrosis process. miR‑21 can regulate the expres-sion of renal extracellular matrix and smooth muscle actin via TGF-β (25,26). miR‑21 may play an important role in PF by regulating the Smad7 gene; it is an important negative regulator of proteins in the TGF-β signaling pathway (26). Based on the prediction from bioinformatics, miR‑21 can bind with the Smad7 3'uTR and inhibit Smad7 expression (27,28).

In conclusion, this study demonstrated that miR‑21 may be involved in the progression of PF via regulating the TGF‑β/Smad signaling pathway. Thus, targeting miR‑21 may prove to be an effective therapy for PF. However, as we only investigated the involvement of miR‑21 expression in PF, further studies are required to explore the potential target gene of miR‑21 in PF.

Acknowledgements

we would like to acknowledge the helpful comments for this study received from our reviewers.

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