Molecular Medicine Reports Molecular Medicine Reports 1791-2997 1791-3004 D.A. Spandidos 10.3892/mmr.2017.7103 mmr-16-04-4293 Articles Upregulation of microRNA-25-3p inhibits proliferation, migration and invasion of osteosarcoma cells in vitro by directly targeting SOX4 WuXiaolin 1 * ZhouHongzhong 2 * YueBin 1 LiMaohua 2 LiuFengxia 1 QiuChensheng 3 ChenBohua 1 MaXuexiao 1 Department of Spine Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266000, P.R. China Department of Spine Surgery, Dezhou Municipal Hospital, Dezhou, Shandong 253000, P.R. China Department of Orthopedics, Qingdao Municipal Hospital, Qingdao, Shandong 266071, P.R. China Correspondence to: Professor Xuexiao Ma, Department of Spine Surgery, The Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Shinan, Qingdao, Shandong 266000, P.R. China, E-mail: ma_xuexiao@163.com

Contributed equally

 102017 27072017 16 4 4293 4300 16082016 18052017 Copyright © 2017, Spandidos Publications 2017

Osteosarcoma (OS) is among the most common primary tumors of bone tissue, and occurs primarily in children and young adults. Despite the development of novel therapeutic approaches, the prognosis of OS remains poor. MicroRNAs (miRNAs) are involved in the development and progression of various types of human cancer and may have potential as novel therapeutic targets for cancer treatment. The present study aimed to investigate the expression and biological functions of miRNA-25-3p in OS, and explore the molecular mechanisms underlying its actions. In the present study, miRNA-25-3p was detected in OS tissues and cell lines. The functional roles of miRNA-25-3p in OS cells were evaluated using a Cell Counting Kit 8 assay and cellular migration and invasion assays. The molecular mechanisms underlying the tumor-suppressing roles of miRNA-25-3p in OS were explored using bioinformatics analysis, luciferase reporter assay, western blotting and the reverse transcription-quantitative polymerase chain reaction. The expression of miRNA-25-3p was revealed to be downregulated in OS tissues and cell lines compared with non-tumor bone tissues and normal osteoblasts, respectively. miRNA-25-3p overexpression was demonstrated to significantly suppress the proliferation, migration and invasion of OS cells in vitro. In addition, sex-determining region-related high mobility group box (SOX) 4 was identified as a direct target gene of miRNA-25-3p, and was further investigated. Similarly to miRNA-25-3p overexpression, SOX4 knockdown was demonstrated to suppress OS cell proliferation, migration and invasion. Furthermore, SOX4 expression was revealed to be significantly upregulated in OS tissues compared with in adjacent non-tumor bone tissues, and Spearman's correlation analysis indicated a negative correlation between SOX4 mRNA and miRNA-25-3p expression levels in OS tissues. The present findings suggested that miRNA-25-3p may act as a tumor suppressor by targeting SOX4 expression in bone tissue. Therefore, miRNA-25-3p may have potential as a novel therapeutic target for the treatment of patients with OS.

 microRNA-25-3p osteosarcoma growth metastasis SOX4 Introduction

Osteosarcoma (OS) is among the most common primary tumors that develop in bone tissue, and occurs primarily in children and young adults (1). OS is mainly observed at the metaphyses of long extremity bones, with very few tumors originating from the axial skeleton and craniofacial bones (2). Currently, the primary treatments for patients with OS are surgery, radiotherapy and chemotherapy, whereas alternative approaches to OS treatment include hormonal, gene and immune therapies (3,4). Despite the development of novel therapeutic approaches, the prognosis of OS remains poor (5). The 5-year overall survival rate for patients with localized OS is ~65%; however, it falls to ~20% for patients with metastatic or recurrent disease (6). Furthermore, the molecular mechanisms of OS development and progression have yet to be fully elucidated (7). Therefore, it is imperative to elucidate the mechanisms underlying OS and identify novel therapeutic targets, in order to improve the prognosis of patients with OS.

MicroRNAs (miRNAs or miRs) are small, single-stranded endogenous non-coding RNAs containing ~21–24 nucleotides, that regulate the post-transcriptional expression of target genes through complementary base pairing with the 3′-untranslated regions (UTRs) of target mRNAs (8,9). miRNAs serve critical roles in numerous biological processes, including development, cell differentiation, proliferation and apoptosis, angiogenesis and metabolism, and their aberrant expression has been suggested to be implicated in carcinogenesis (10). Furthermore, miRNAs have been reported to suppress or promote oncogenesis, depending on whether they regulate the expression of oncogenes or tumor suppressor genes (11). Therefore, tumor-suppressing miRNAs are usually downregulated, whereas oncogenic miRNAs tend to be overexpressed in cancer (12). Previous studies have reported the deregulation of miRNA expression in OS (13–15), and miRNA expression has been correlated with tumor stage and aggressiveness (16). Therefore, it is critical to understand the roles of miRNAs in cancer development and progression, and explore their potential as novel therapeutic targets for the treatment of patients with cancer.

Previous studies have implicated miR-25-3p in several types of human cancer (17–19); however, the roles of miR-25-3p in OS have yet to be elucidated. The present study aimed to assess the expression of miR-25-3p in OS tissues and cell lines, and explore its biological functions in OS cells in vitro. In addition, it aimed to identify direct target genes of miR-25-3p in OS, and investigate the molecular mechanisms that underlie its effects in tumor development and progression.

 Materials and methods <sec> <title>Clinical specimens

The present study was approved by the Ethics Committee of The Affiliated Hospital of Qingdao University (Qingdao, China). Written informed consent was obtained from all patients. OS tissue and paired adjacent non-tumor bone tissue samples were obtained from 27 patients (male, 19; female, 8; age range, 16–53 years) with OS who received surgical treatment in The Affiliated Hospital of Qingdao University between July 2010 and September 2014. None of the patients had been treated with chemotherapy or radiotherapy prior to surgery. Tissue samples were frozen immediately upon isolation in liquid nitrogen and stored at −80°C until further use.

 Cell culture

The human MG-63, SAOS-2, HOS and U2OS OS cell lines, and the human hFOB 1.19 normal osteoblast cell line were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), and maintained at 37°C in a humidified 5% CO2 atmosphere.

 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from tissue samples (1 g) and cells (1×106) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of total RNA was measured using the NanoDrop 1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Total RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The reaction included 2 µl 5X PrimeScript Buffer, 0.5 µl PrimeScriptRT Enzyme Mix I, 0.5 µl Oligo dT Primer (50 µM), 0.5 µl Random 6 mers (100 µM) and 100 ng total RNA. The temperature protocol for reverse transcription was as follows: 37°C for 15 min and 85°C for 5 sec.

qPCR was performed on cDNA to detect SOX4 mRNA expression using SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd.) on the Applied Biosystems 7900HT Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction system contained 10 µl SYBR Premix Ex Taq, 2 µl cDNA (200 ng), 0.8 µl forward primer, 0.8 µl reverse primer, 0.4 µl ROX Reference Dye and 6 µl ddH2O. The amplification was performed with cycling conditions as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. To quantify miR-25-3p expression, cDNA was synthesized from total RNA using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction system contained 0.15 µl 100 mM dNTPs, 1 µl MultiScribe™ Reverse Transcriptase (50 U/µl), 1.5 µl 10X Reverse Transcription Buffer, 0.19 µl RNase Inhibitor, (20 U/µl), 4.16 µl Nuclease-free water and 5 µl total RNA (10 ng). The temperature protocol for reverse transcription was as follows: 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. Relative miR-485 expression was determined using a TaqMan MicroRNA PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction system contained 1 µl TaqMan® Small RNA Assay (20X), 1.33 µl cDNA, 10 µl TaqMan® Universal PCR Master Mix II (2X) and 7.67 µl Nuclease-free water. The cycling conditions for qPCR were as follows: 50°C for 2 min, 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 sec; and annealing/extension at 60°C for 60 sec. U6 small nuclear RNA and GAPDH were used as the housekeeping genes for miR-25-3p and SOX4 mRNA expression, respectively.

The primers were designed as follows: miR-25-3p, 5′-ATCCAGTGCGTGTCGTG-3′ (forward) and 5′-TGCTCATTGCACTTGTCTC-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse); SOX4, 5′-CTTGACATGATTAGCTGGCATGATT-3′ (forward) and 5′-CCTGTGCAATATGCCGTGTAGA-3′ (reverse); and GAPDH, 5′-CCAAAATCAGATGGGGCAATGCTGG-3′ (forward) and 5′-TGATGGCATGGACTGTGGTCATTCA-3′ (reverse). Relative gene expression was quantified according to the comparative Cq method (20).

 Cell transfection

Mature miR-25-3p mimics and negative control miRNAs (miR-NC) were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The miR-25-3p mimics sequence was 5′-CAUUGCACUUGUCUCGGUCUGA-3′ and the NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. SOX4-targeting small interfering (si)RNA and negative control siRNA (NC siRNA) were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The SOX4 siRNA sequence was 5′-GGGCUAGAGUUUUAACUUUTT-3′ and the NC siRNA sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. MG-63 and U2OS cells were seeded in 6-well plates at a density of 5×105 cells/well with DMEM containing 10% FBS. Cells were transfected with the miR-25-3p mimics (100 pmol), miR-NC (100 pmol), SOX4 siRNA (100 pmol) or NC siRNA (100 pmol) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as the transfection reagent, according to the manufacturer's protocol. Successful transfection was confirmed by detecting miR-25-3p or SOX4 expression after transfection using RT-qPCR, according to the aforementioned protocol.

 Cell Counting Kit-8 (CCK-8) assay

Cell proliferation was evaluated using a CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer's protocol. At 24 h post-transfection, transfected cells were seeded in 96-well plates at a density of 3×103 cells/well in DMEM containing 10% FBS and incubated at 37°C in a humidified 5% CO2 atmosphere for 24–96 h. Following incubation, the CCK-8 assay was conducted. Briefly, 10 µl CCK-8 solution was added to each well and cells were incubated at 37°C for 2 h. Subsequently, the absorbance of each sample was measured at a wavelength of 450 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

 Cell migration and invasion assay

The migratory and invasive capabilities of OS cells were assessed using Transwell chambers (8 µm pore size; Costar; Corning Incorporated, Corning, NY, USA). For the migration assay, 5×104 U2OS and MG-63 cells were seeded 48 h post-transfection in 200 µl FBS-free DMEM into the upper chambers of the inserts. A volume of 500 µl of 20% FBS-containing medium was added to the lower chambers as a chemoattractant and cells were incubated for 24 h. For the invasion assay, the Transwell inserts were coated with Matrigel (BD Biosciences, San Jose, CA, USA) and a total of 5×104 U2OS and MG-63 cells in 200 µl FBS-free medium were seeded into the upper chambers. A volume of 500 µl of 20% FBS-containing medium was added to the lower chambers as a chemoattractant and cells were incubated at 37°C for 48 h. Non-migrated and non-invaded cells on the upper surface of the membranes were scraped off with cotton swabs. Cells on the lower membranes were then fixed with 100% methanol at room temperature for 10 min, stained in 0.5% crystal violet (Beyotime Institute of Biotechnology, Haimen, China) at room temperature for 10 min, rinsed in PBS (Gibco; Thermo Fisher Scientific, Inc.) and then observed under a CKX41 inverted microscope (Olympus Corporation, Tokyo, Japan) using ×200 magnification. Cells were counted by eye in 5 random fields of view in each chamber. Each condition was assayed in triplicate and experiments were repeated at least 3 times.

 Bioinformatics analysis

In order to predict the potential target genes of miR-25-ep, TargetScan (release 6.0; November 2011; www.targetscan.org) and miRanda (release August 2010; www.microrna.org) online software were used. Human was selected as the species, and ‘hsa-miR-25-3p’ was entered.

 Western blot analysis

Cells were washed with ice-cold PBS and lysed in cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) 72 h post-transfection at 4°C for 30 min. The supernatants were collected by centrifugation at 4°C, 12,000 × g for 30 min, and protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of extracted protein samples (30 µg) were separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), which were blocked with 5% non-fat dry milk in Tris-based saline-0.5% Tween-20 (TBST) at room temperature for 2 h. Blocking was followed by an overnight incubation at 4°C with mouse anti-human monoclonal SOX4 (1:1,000 dilution; sc-130633; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse anti-human monoclonal GADPH (1:1,000 dilution; sc-137179; Santa Cruz Biotechnology, Inc.) primary antibodies, according to the manufacturer's protocol. Following 3 washes with TBS containing Tween-20, the membranes were incubated for 1 h with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (sc-2005; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.) on the FluorChem imaging system (ProteinSimple; Bio-Techne, Minneapolis, MN, USA) and analyzed with ImageJ 1.49 (National Institutes of Health, Bethesda, MD, USA).

 Luciferase reporter assay

For the luciferase reporter assay, the following plasmids were designed and synthesized by Shanghai GenePharma Co., Ltd.: The plasmid containing the wild-type (WT) 3′-UTR of the human SOX4 mRNA in a pGL3 firefly luciferase reporter vector, pGL3-SOX4-3′UTR-WT; and a plasmid containing the mutated (MUT) 3′-UTR, pGL3-SOX4-3′UTR-MUT. Human embryonic kidney (HEK) 293T cells (Shanghai Institute of Biochemistry and Cell Biology) were plated in 12-well plates at a density of 1.5×105 and co-transfected at room temperature with the luciferase reporter plasmids (1.6 µg) and miR-25-3p mimics (40 pmol) or miR-NC (40 pmol) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. Cells were harvested 48 h post-transfection and a luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA). The firefly and Renilla luciferase activities were determined using a luminometer (Tecan Group Ltd., Männedorf, Switzerland). Firefly luciferase activity was normalized to Renilla luciferase activities for each well.

 Statistical analysis

Data are presented as the mean ± standard deviation. Each assay was repeated at least three times. The statistical significance of the differences between groups was assessed using a two-tailed Student's t-test or one-way analysis of variance. Student-Newman-Keuls post hoc testing was performed to correct for multiple comparisons. Statistical analysis was performed in SPSS software version 19.0 (IBM Corp., Armonk, NY, USA). Spearman's correlation analysis was used to investigate the association between SOX4 mRNA and miR-25-3p expression levels in OS tissues. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>miR-25-3p expression is downregulated in OS

RT-qPCR was used to assess the expression levels of miR-25-3p in OS tissues and cell lines. As presented in Fig. 1A, miR-25-3p expression was significantly downregulated in OS tissue samples compared with in paired adjacent non-tumor bone tissue samples (P<0.05). In addition, the expression levels of miR-25-3p were significantly decreased in the human MG-63, SAOS-2, HOS and U2OS OS cell lines compared with in normal hFOB 1.19 cells (P<0.05; Fig. 1B). These findings suggested that downregulation of miR-25-3p may be implicated in the molecular mechanisms guiding OS development.

 Successful miR-25-3p overexpression in MG-63 and U2OS cells following transfection with miR-25-3p mimics

Since the expression of miR-25-3p was downregulated in OS tissues and cell lines, miR-25-3p may be hypothesized to act as a tumor suppressor in OS pathogenesis. MG-63 and U2OS cells were selected for further experiments, as they exhibited lower miR-25-3p expression than SAOS-2 and HOS cells (Fig. 1B). MG-63 and U2OS cells were transfected with miR-25-3p mimics or miR-NC, and RT-qPCR was performed 48 h post-transfection to assess the transfection efficiency. As demonstrated in Fig. 2, miR-25-3p was significantly upregulated in miR-25-3p mimic-transfected MG-63 and U2OS cells compared with cells transfected with miR-NC (P<0.05).

 miR-25-3p suppresses the proliferation, migration and invasion of OS cells

To analyze the roles of miR-25-3p in OS cell proliferation, migration and invasion, CCK-8 and cell migration and invasion assays were performed in MG-63 and U2OS cells. The results of the CCK-8 assays demonstrated that miR-25-3p mimic-transfected MG-63 and U2OS cells exhibited significantly suppressed proliferative capabilities at 96 h compared with miR-NC-transfected cells (P<0.05; Fig. 3A). The migration and invasion assays demonstrated similar findings: Upregulation of miR-25-3p expression significantly decreased the migratory and invasive capabilities of MG-63 and U2OS cells (P<0.05; Fig. 3B). These results suggested that miR-25-3p may serve a role in the inhibition of OS cell growth and metastasis in vitro.

 SOX4 is a direct target gene of miR-25-3p

To investigate the mechanism underlying the inhibitory effects of miR-25-3p on OS cell growth and metastasis, TargetScan and miRanda were used to predict the potential target genes of miR-25-3p. Bioinformatic analysis revealed that the SOX4 mRNA contained a miR-25-3p 7-nucleotide seed match at position 2472–2479 of the 3′-UTR (Fig. 4A). To investigate whether SOX4 may be a direct target of miR-25-3p, HEK293T cells, which are generally used for luciferase reporter assays due to the ease of exogenous gene transfection, were transfected with a pGL3-SOX4-3′UTR-WT or pGL3-SOX4-3′UTR-MUT luciferase reporter vector, along with miR-25-3p mimics or miR-NC. Following 48 h of transfection, luciferase activity was detected. miR-25-3p upregulation was demonstrated to reduce the activity of the pGL3-SOX4-3′UTR-WT reporter (P<0.05; Fig. 4B), whereas it had no effect on pGL3-SOX4-3′UTR-MUT reporter activity, thus suggesting that SOX4 may be a target of miR-25-3p. Furthermore, the effects of miR-25-3p overexpression on the expression of SOX4 were investigated in MG-63 and U2OS cells. As presented in Fig. 4C and D, respectively, miR-25-3p overexpression repressed the mRNA and protein expression of SOX4 in MG-63 and U2OS cells. The present findings suggested that SOX4 may be a direct target gene of miR-25-3p.

 SOX4 is a functional target of miR-25-3p in OS

The effects of SOX4 on the proliferation, migration and invasion of OS cells were also explored, and the results obtained were similar to those induced by miR-25-3p overexpression. Following transfection with SOX4-targeting siRNA, SOX4 was obviously downregulated in MG-63 and U2OS cells compared with NC siRNA (Fig. 5A). In addition, SOX4 knockdown was revealed to suppress the proliferation (P<0.05; Fig. 5B), migration and invasion (P<0.05; Fig. 5C) of MG-63 and U2OS cells compared with NC; these effects were similar to those following direct targeting of miR-25-3p in OS cells.

 SOX4 expression is significantly upregulated in OS tissues and is negative correlated with miR-25-3p levels

RT-qPCR was used to assess the mRNA expression of SOX4 in OS and paired adjacent non-tumor bone tissue samples, revealing that SOX4 mRNA expression was significantly upregulated in OS tissues compared with in paired adjacent non-tumor bone tissues (P<0.05; Fig. 6A). Furthermore, Spearman's correlation analysis indicated a negative correlation between SOX4 mRNA and miR-25-3p expression levels in OS tissues (R=−0.6054, P=0.0002; Fig. 6B). These findings suggested that the upregulation of SOX4 that was observed in OS tissues and cell lines may be a result of the downregulation of miR-25-3p expression in OS.

 Discussion

Aberrant miRNA expression has been reported to contribute to tumorigenesis and cancer progression in several types of human cancer (21–23). In the present study, the expression of miR-25-3p was revealed to be downregulated in OS tissues and cell lines compared with adjacent non-tumor bone tissues and normal osteoblasts, respectively. Conversely, the overexpression of miR-25-3p inhibited the proliferation, migration and invasion of OS cells. In addition, SOX4 was identified as a direct target gene of miR-25-3p, and its expression was revealed to be significantly upregulated in OS tissues and inversely correlated with miR-25-3p expression. Furthermore, the downregulation of SOX4 significantly suppressed the proliferation, migration and invasion of OS cells, similar to the effects of miR-25-3p overexpression. The present findings suggested that miR-25-3p may act as a tumor suppressor by directly modulating the expression of SOX4 in cancer cells. To the best of our knowledge, this is the first study to have investigated the expression and biological functions of miR-25-3p, and the molecular mechanisms underlying its effects in OS cells.

Previous studies have reported that miR-25-3p was dysregulated in several types of human tumors. In ovarian cancer, miR-25-3p was revealed to be upregulated in tumor tissues and cell lines (17). In addition, increased miR-25-3p expression has been positively correlated with tumor stage, histology and regional lymph node involvement, whereas Kaplan-Meier and multivariate Cox proportional hazards analyses identified the upregulated miR-25-3p expression as an indicator of poor overall survival in patients with ovarian cancer (18). In esophageal squamous cell carcinoma, the expression of miR-25-3p is increased and is significantly correlated with lymph node metastasis and TNM stage (24). Xu et al (19) reported that miR-25-3p expression is elevated in female patients with lung adenocarcinoma, and is obviously correlated with lymph node metastasis and disease stage, whereas patients with upregulated miR-25-3p expression exhibit poorer overall survival rates. Li et al (25) reported that miR-25-3p expression is enhanced in gastric cancer tumor tissue, and its increased expression is correlated with tumor node metastasis stage and lymph node metastasis. The upregulation of miR-25-3p has also been observed in hepatocellular carcinoma (26), glioblastoma (27), glioma (28), cervical cancer (29) and cholangiocarcinoma (30).

However, contradictory reports have demonstrated that the expression of miR-25-3p was downregulated in various types of human cancer. Li et al (31) reported that miR-25-3p expression is decreased in colon tumor tissues, and in prostate cancer, miR-25-3p was revealed to be downregulated and significantly negatively correlated with tumor invasiveness (32). Esposito et al (33) reported that miR-25-3p expression is decreased in anaplastic thyroid carcinoma compared with in normal thyroid tissue, and Xu et al (34) demonstrated that the expression levels of miR-25-3p are reduced in tongue squamous cell carcinoma tissues and cell lines. These contradictory studies suggested that miR-25-3p expression may be subject to tissue-specific regulatory processes in the various types of human cancer. Furthermore, miR-25-3p may have potential as a biomarker for the prognosis of several types of cancer.

Previous studies have suggested that miR-25-3p may serve critical roles in various types of human cancer: Xu et al (24) demonstrated that miR-25-3p targets cadherin-1 to enhance the migration and invasion of esophageal squamous cell carcinomas. In ovarian cancer cells, reduced expression of miR-25-3p significantly decreases cellular proliferation, migration and invasion, and promotes apoptosis, by targeting large tumor suppressor kinase 2 and B-cell lymphoma-2-like protein 11 (17,35). In gastric cancer, miR-25-3p downregulation suppresses the growth, metastasis and invasion, and enhances the apoptosis of cancer cells in vitro, whereas it also suppresses their distal pulmonary metastatic capabilities and peritoneal dissemination in vivo, by negatively regulating transducer of erbB2 1, F-box/WD repeat-containing protein 7 (FBXW7), cyclin E1 and Myc (25,36,37). In lung cancer cells, miR-25-3p overexpression was demonstrated to promote cellular proliferation and motility, and decrease apoptosis in vitro, and to enhance tumor growth in vivo; however, contradictory studies have reported that miR-25-3p overexpression enhances the sensitivity of lung cancer cells to cisplatin and promotes cell cycle arrest in the G1 phase in vitro, by modulating FBXW7, regulator of G protein signaling 3, modulator of apoptosis 1, cell division control protein 42 homolog and cyclin E2 (38–41). Peng et al (27) revealed that miR-25-3p overexpression potentiates the proliferation and invasion of glioblastoma cells in vitro, through the regulation of neurofilament light. These findings suggest that there remains controversy around the role of miR-25-3p in human cancer, and further studies are required on the matter.

miR-25-3p has been suggested to act as a tumor suppressor in several types of human cancer: In colon cancer, miR-25-3p has been reported to function as a tumor suppressor, by inhibiting the proliferation and migration of cancer cells through the regulation of mothers against decapentaplegic homolog 7 (31). In anaplastic thyroid carcinoma, the upregulation of miR-25-3p suppresses the proliferative and colony-forming capabilities of cancer cells, by targeting enhancer of zeste homolog 2 and inducing G2/M-phase cell cycle arrest (33). In tongue squamous cell carcinoma, miR-25-3p overexpression suppresses cellular proliferation and colony formation, through the regulation of cell cycle-related proteins, including cyclin D1, Akt and forkhead box protein O1 (34). These findings suggest that miR-25-3p may exert antitumor effects and may have potential as a novel therapeutic target for the development of antineoplastic agents.

Since miRNAs negatively regulate the expression of their target genes (8), the putative targets of miR-25-3p were investigated in the present study. To the best of our knowledge, this is the first study demonstrating SOX4 as a direct target gene of miR-25-3p in OS cells. SOX4 is a 47-kDa member of the SOX family, and is encoded by a single exon gene (42). Previous studies have reported that SOX4 is overexpressed in numerous types of human cancer, including breast cancer (43), hepatocellular carcinoma (44), gastric (45) and prostate cancer (46). SOX4 has been revealed to be upregulated in OS tissues, and its expression is significantly correlated with distant metastasis, pathological grade and Enneking stage. In addition, Cox multifactor regression analysis has identified SOX4 expression as an independent risk factor for patients with OS (47). Furthermore, OS patients with high SOX4 expression have lower mean overall survival time and 5-year survival rate compared with patients with low SOX4 expression (47). The present study demonstrated that following SOX4 knockdown, the proliferative, migratory and invasive capabilities of OS cells were significantly suppressed. These findings suggested that SOX4 may have potential as a novel therapeutic target for the treatment of patient with OS.

In conclusion, to the best of our knowledge, the present study is the first to have demonstrated that miR-25-3p expression was downregulated in OS tissue samples and cell lines. miR-25-3p overexpression was revealed to inhibit the proliferation, migration and invasion of OS cells, via directly targeting SOX4. The present results suggested that miR-25-3p and SOX4 may have potential as novel therapeutic targets for the development of treatments for patients with OS. However, further studies are required to fully elucidate the roles of miR-25-3p in the pathogenesis of OS and explore the molecular mechanisms that are involved in its actions.

 Acknowledgements

The present study was supported by the National Natural Science Foundation of China (grant no. H0609, H0609-81672220 and BS2011YY006).

 References MooreDDLuuHHOsteosarcomaCancer Treat Res1626592201410.1007/978-3-319-07323-1_425070231 WangBSuYYangQLvDZhangWTangKWangHZhangRLiuYOverexpression of long non-coding RNA HOTAIR promotes tumor growth and metastasis in human osteosarcomaMol Cells38432440201510.14348/molcells.2015.2327257287534443285 TanMLChoongPFDassCROsteosarcoma: Conventional treatment vs. gene therapyCancer Biol Ther8106117200910.4161/cbt.8.2.738519098456 GillJAhluwaliaMKGellerDGorlickRNew targets and approaches in osteosarcomaPharmacol Ther1378999201310.1016/j.pharmthera.2012.09.00322983152 MarinaNGebhardtMTeotLGorlickRBiology and therapeutic advances for pediatric osteosarcomaOncologist9422441200410.1634/theoncologist.9-4-42215266096 BielackSSCarrleDHardesJSchuckAPaulussenMBone tumors in adolescents and young adultsCurr Treat Options Oncol96780200810.1007/s11864-008-0057-118449804 McQueenPGhaffarSGuoYRubinEMZiXHoangBHThe Wnt signaling pathway: Implications for therapy in osteosarcomaExpert Rev Anticancer Ther1112231232201110.1586/era.11.9421916576 BartelDPMicroRNAs: Target recognition and regulatory functionsCell136215233200910.1016/j.cell.2009.01.002191673263794896 BartelDPMicroRNAs: Genomics, biogenesis, mechanism, and functionCell116281297200410.1016/S0092-8674(04)00045-514744438 RyanBMRoblesAIHarrisCCGenetic variation in microRNA networks: The implications for cancer researchNat Rev Cancer10389402201010.1038/nrc2885204955732950312 CimminoACalinGAFabbriMIorioMVFerracinMShimizuMWojcikSEAqeilanRIZupoSDonoMmiR-15 and miR-16 induce apoptosis by targeting BCL2Proc Natl Acad Sci USA1021394413949200510.1073/pnas.0506654102161662621236577 LeeYSDuttaAMicroRNAs in cancerAnnu Rev Pathol4199227200910.1146/annurev.pathol.4.110807.092222188175062769253 DongJLiuYLiaoWLiuRShiPWangLmiRNA-223 is a potential diagnostic and prognostic marker for osteosarcomaJ Bone Oncol57479201610.1016/j.jbo.2016.05.001273357754908189 LiYZengCTuMJiangWDaiZHuYDengZXiaoWMicroRNA-200b acts as a tumor suppressor in osteosarcoma via targeting ZEB1Onco Targets Ther9310131112016273077514888717 LiuQWangHSinghAShouFExpression and function of microRNA-497 in human osteosarcomaMol Med Rep14439445201610.3892/mmr.2016.525627176490 LianDWangZZLiuNSMicroRNA-1908 is a biomarker for poor prognosis in human osteosarcomaEur Rev Med Pharmacol Sci2012581262201627097944 ZhangHZuoZLuXWangLWangHZhuZMiR-25 regulates apoptosis by targeting Bim in human ovarian cancerOncol Rep27594598201222076535 WangXMengXLiHLiuWShenSGaoZMicroRNA-25 expression level is an independent prognostic factor in epithelial ovarian cancerClin Transl Oncol16954958201410.1007/s12094-014-1178-624696291 XuFXSuYLZhangHKongJYYuHQianBYPrognostic implications for high expression of MiR-25 in lung adenocarcinomas of female non-smokersAsian Pac J Cancer Prev1511971203201410.7314/APJCP.2014.15.3.119724606441 LivakKJSchmittgenTDAnalysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) methodMethods25402408200110.1006/meth.2001.126211846609 YangGZhangLLiRWangLThe role of microRNAs in gallbladder cancerMol Clin Oncol5713201610.3892/mco.2016.905273307554906925 WangZSZhongMBianYHMuYFQinSLYuMHQinJMicroRNA-187 inhibits tumor growth and invasion by directly targeting CD276 in colorectal cancerOncotarget74426644276201610.18632/oncotarget.10023273295955190094 ZhangYLiCFMaLJDingMZhangBMicroRNA-224 aggrevates tumor growth and progression by targeting mTOR in gastric cancerInt J Oncol4910681080201627315344 XuXChenZZhaoXWangJDingDWangZTanFTanXZhouFSunJMicroRNA-25 promotes cell migration and invasion in esophageal squamous cell carcinomaBiochem Biophys Res Commun421640645201210.1016/j.bbrc.2012.03.04822450326 LiBSZuoQFZhaoYLXiaoBZhuangYMaoXHWuCYangSMZengHZouQMGuoGMicroRNA-25 promotes gastric cancer migration, invasion and proliferation by directly targeting transducer of ERBB2, 1 and correlates with poor survivalOncogene3425562565201510.1038/onc.2014.21425043310 SadeghianYKamyabi-MoghaddamZNodushanSMKhoshbakhtSPedramBYahaghiEMokarizadehAMohebbiMRETRACTED ARTICLE: Profiles of tissue microRNAs; miR-148b and miR-25 serve as potential prognostic biomarkers for hepatocellular carcinomaTumour BiolJul252015(Epub ahead of print)26209296 PengGYuanXYuanJLiuQDaiMShenCMaJLiaoYJiangWmiR-25 promotes glioblastoma cell proliferation and invasion by directly targeting NEFLMol Cell Biochem409103111201510.1007/s11010-015-2516-x26209061 ZhangJGongXTianKChenDSunJWangGGuoMmiR-25 promotes glioma cell proliferation by targeting CDKN1CBiomed Pharmacother71714201510.1016/j.biopha.2015.02.00525960208 QiuGFangBXinGWeiQYuanXWuDmiR-25 promotes cell proliferation by targeting RECK in human cervical carcinoma HeLa cellsXi Bao Yu Fen Zi Mian Yi Xue Za Zhi3140432015(In Chinese)25575057 RazumilavaNBronkSFSmootRLFingasCDWerneburgNWRobertsLRMottJLmiR-25 targets TNF-related apoptosis inducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinomaHepatology55465475201210.1002/hep.2469821953056 LiQZouCZouCHanZXiaoHWeiHWangWZhangLZhangXTangQMicroRNA-25 functions as a potential tumor suppressor in colon cancer by targeting Smad7Cancer Lett335168174201310.1016/j.canlet.2013.02.02923435373 ZoniEvan der HorstGvan de MerbelAFChenLRaneJKPelgerRCCollinsATVisakorpiTSnaar-JagalskaBEMaitlandNJvan der PluijmGmiR-25 modulates invasiveness and dissemination of human prostate cancer cells via regulation of αv- and α6-integrin expressionCancer Res7523262336201510.1158/0008-5472.CAN-14-215525858144 EspositoFTornincasaMPallantePFedericoABorboneEPierantoniGMFuscoADown-regulation of the miR-25 and miR-30d contributes to the development of anaplastic thyroid carcinoma targeting the polycomb protein EZH2J Clin Endocrinol Metab97E710E718201210.1210/jc.2011-306822399519 XuJYYangLLMaCHuangYLZhuGXChenQLMiR-25-3p attenuates the proliferation of tongue squamous cell carcinoma cell line Tca8113Asian Pac J Trop Med6743747201310.1016/S1995-7645(13)60130-323827155 FengSPanWJinYZhengJMiR-25 promotes ovarian cancer proliferation and motility by targeting LATS2Tumour Biol351233912344201410.1007/s13277-014-2546-025179841 GongJCuiZLiLMaQWangQGaoYSunHMicroRNA-25 promotes gastric cancer proliferation, invasion, and migration by directly targeting F-box and WD-40 Domain Protein 7, FBXW7Tumour Biol3678317840201510.1007/s13277-015-3510-325944166 ZhangYPengZZhaoYChenLmicroRNA-25 inhibits cell apoptosis of human gastric adenocarcinoma cell line AGS via regulating CCNE1 and MYCMed Sci Monit2214151420201610.12659/MSM.896118271207284913832 XiangJHangJBCheJMLiHCMiR-25 is up-regulated in non-small cell lung cancer and promotes cell proliferation and motility by targeting FBXW7Int J Clin Exp Pathol8914791532015264646594583891 ChenZWuYMengQXiaZElevated microRNA-25 inhibits cell apoptosis in lung cancer by targeting RGS3In Vitro Cell Dev Biol Anim526267201610.1007/s11626-015-9947-226416661 WuTChenWKongDLiXLuHLiuSWangJDuLKongQHuangXLuZmiR-25 targets the modulator of apoptosis 1 gene in lung cancerCarcinogenesis36925935201510.1093/carcin/bgv06825998847 ZhaoZLiuJWangCWangYJiangYGuoMMicroRNA-25 regulates small cell lung cancer cell development and cell cycle through cyclin E2Int J Clin Exp Pathol7772677342014255508094270611 LiYZuLWangYWangMChenPZhouQmiR-132 inhibits lung cancer cell migration and invasion by targeting SOX4J Thorac Dis7156315692015265436034598519 ZhangJLiangQLeiYYaoMLiLGaoXFengJZhangYGaoHLiuDXSOX4 induces epithelial-mesenchymal transition and contributes to breast cancer progressionCancer Res7245974608201210.1158/0008-5472.CAN-12-104522787120 ZhengJHJianZXJinHSChenSCWangGYExpression of SOX4 gene and early recurrence of hepatocellular carcinoma: Their relationship and the clinical significanceNan Fang Yi Ke Da Xue Xue Bao308188192010(In Chinese)20423819 FangCLHseuYCLinYFHungSTTaiCUenYHLinKYClinical and prognostic association of transcription factor SOX4 in gastric cancerPLoS One7e52804201210.1371/journal.pone.0052804232851873527618 WangLLiYYangXYuanHLiXQiMChangYWWangCFuWYangMERG-SOX4 interaction promotes epithelial-mesenchymal transition in prostate cancer cellsProstate74647658201410.1002/pros.2278324435928 BaoZQZhangCCXiaoYZZhouJSTaoYSChaiDMOver-expression of Sox4 and β-catenin is associated with a less favorable prognosis of osteosarcomaJ Huazhong Univ Sci Technolog Med Sci36193199201610.1007/s11596-016-1565-z27072961

miR-25-3p expression in OS tissue samples and cell lines assessed using reverse transcription-quantitative polymerase chain reaction. (A) miR-25-3p expression was downregulated in OS tissues (n=27) compared with paired adjacent non-tumor bone tissues (Normal; n=27). Data are expressed as the mean ± standard deviation. *P<0.05 vs. Normal. (B) miR-25-3p expression was downregulated in the human MG-63, SAOS-2, HOS and U2OS OS cell lines compared with in hFOB 1.19 normal osteoblasts. Data are expressed as the mean ± standard deviation. *P<0.05 vs. hFOB 1.19. miR, microRNA; OS, osteosarcoma.

Successful overexpression of miR-25-3p in OS cells confirmed using reverse transcription-quantitative polymerase chain reaction. Human MG-63 and U2OS OS cell lines were transfected with miR-25-3p mimics or miR-NC. miR-25-3p expression was significantly upregulated in MG-63 and U2OS cells transfected with miR-25-3p mimics compared with in miR-NC-transfected cells. Data are expressed as the mean ± standard deviation. *P<0.05 vs. miR-NC. miR, microRNA; OS, osteosarcoma; NC, negative control.

miR-25-3p overexpression inhibits the proliferation, migration and invasion of human MG-63 and U2OS OS cells in vitro. (A) A Cell Counting Kit-8 assay revealed that transfection with miR-25-3p mimics significantly suppressed the proliferation of MG-63 and U2OS cells at 96 h compared with miR-NC-transfected cells. (B) Transwell migration and invasion assays revealed that transfection with miR-25-3p mimics significantly impaired the migratory and invasive capabilities of MG-63 and U2OS cells compared with miR-NC-transfected cells. Representative photomicrographs are included. Original magnification, ×200. Data are expressed as the mean ± standard deviation. *P<0.05 vs. miR-NC. miR, microRNA; OS, osteosarcoma; NC, negative control.

SOX4 is a direct target gene of miR-25-3p. (A) Schematic representation of the WT and MUT miR-25-3p-targeted sequences within the 3′-UTR of SOX4 mRNA. (B) Luciferase reporter assays were performed in human embryonic kidney 293T cells 48 h following co-transfection with pGL3-SOX4-3′UTR-WT or pGL3-SOX4-3′UTR-MUT and miR-25-3p mimic or miR-NC, confirming SOX4 as a direct target gene of miR-25-3p. (C) Reverse transcription-quantitative polymerase chain reaction revealed that SOX4 mRNA expression was significantly downregulated in human MG-63 and U2OS OS cells transfected with miR-25-3p mimics compared with in miR-NC-transfected cells. (D) Representative blots demonstrating that SOX4 protein expression appeared to be downregulated in MG-63 and U2OS cells transfected with miR-25-3p mimics compared with miR-NC-transfected cells. Data are expressed as the mean ± standard deviation. *P<0.05 vs. miR-NC. SOX, sex-determining region-related high mobility group box; miR, microRNA; WT, wild-type; MUT, mutated; UTR, untranslated region; NC, negative control; OS, osteosarcoma.

SOX4 knockdown inhibits the proliferation, migration and invasion of human MG-63 and U2OS OS cells in vitro. (A) Western blot analysis indicated that SOX4 protein expression was downregulated in MG-63 and U2OS cells following transfection with SOX4-targeting siRNA. (B) Cell Counting Kit-8 assays revealed that transfection with SOX4-siRNA significantly suppressed the proliferation of MG-63 and U2OS cells at 96 h compared with NC siRNA-transfected cells. (C) Transwell migration and invasion assays revealed that transfection with SOX4-siRNA significantly impaired the migratory and invasive capabilities of MG-63 and U2OS cells compared with NC siRNA-transfected cells. Representative photomicrographs are included. Original magnification, ×200. Data are expressed as the mean ± standard deviation. *P<0.05 vs. NC siRNA. SOX, sex-determining region-related high mobility group box; OS, osteosarcoma; si, small interfering; NC, negative control.

SOX4 mRNA expression is inversely correlated with miR-25-3p levels in OS tissues. (A) Reverse transcription-quantitative polymerase chain reaction revealed that SOX4 mRNA expression was significantly upregulated in OS tissues (n=27) compared with paired adjacent non-tumor bone tissues (Normal; n=27). Data are expressed as the mean ± standard deviation. *P<0.05 vs. Normal. (B) Spearman's correlation analysis detected a negative correlation between SOX4 mRNA and miR-25-3p expression levels in OS tissues. SOX, sex-determining region-related high mobility group box; miR, microRNA; OS, osteosarcoma.