GBM and adjacent normal tissues were collected from 26 patients (15 males and 11 females; age range: 37–65 years old) who were newly diagnosed as GBM and treated with surgical resection in China-Japan Union Hospital of Jilin University (Changchun, China) between July 2014 and May 2017. None of these patients received radiotherapy or chemotherapy prior to surgery. Tissue samples were collected immediately after surgical resection, frozen in liquid nitrogen and stored at −80°C until further use. The present study was approved by the Ethics Committee of China-Japan Union Hospital of Jilin University. Written informed consent was obtained from all enrolled patients prior to surgery.

A total of four human GBM cell lines, including U138, U251, T98, and LN229, were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Normal human astrocytes (NHAs) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA), and were cultured in astrocyte medium (Sciencell Research Laboratories) supplemented with 10% FBS. All cells were grown at 37°C under normoxic conditions of 95% air and 5% CO₂.

Cells were plated into 6-well plates one day prior to transfection. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used in cell transfection according to the manufacturer's protocol. miR-876-5p mimics, miRNA mimics negative control (miR-NC), small interfering RNA (siRNA) targeting FOXM1 (FOXM1 siRNA) and negative control siRNA (NC siRNA) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The miR-876-5p mimics sequence was 5′-UGGAUUUCUUUGUGAAUCACCA-3′ and the miR-NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. The FOXM1 siRNA sequence was 5′-GGACCACUUUCCCUACUUUTT-3′ and the NC siRNA sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. The FOXM1 overexpression plasmid pCMV-FOXM1 and empty pCMV plasmid were constructed by the Chinese Academy of Sciences (Changchun, China). Cells were transfected with miRNA mimics (100 pmol), siRNA (100 pmol) or plasmids (4 µg), and transfected cells were incubated at 37°C with 5% CO₂ and then subjected to the evaluation of transfection efficiency. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was used to assess the transfection efficiency of miR-876-5p mimics. The efficiencies of siRNA and plasmid transfection were determined through western blot analysis. Forty-eight hours after transfection, RT-qPCR analysis was performed. Cell Counting Kit-8 (CCK-8) assay and flow cytometry assay were carried out at 24 and 48 h post-transfection, respectively. In vitro migration and cell invasion assays were conducted 48 h following transfection. After 72 h incubation, western blot analysis was performed to determine the FOXM1 protein expression.

Total RNA was isolated from tissue specimens or cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For the detection of miR-876-5p expression, total RNA was used for complementary DNA (cDNA) synthesis using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). 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. Quantitative polymerase chain reaction (qPCR) was conducted using a TaqMan MicroRNA PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). 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. For FOXM1 mRNA expression determination, reverse transcription was performed using a Prime-Script RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The temperature protocol for reverse transcription was as follows: 37°C for 15 min and 85°C for 5 sec. The synthesized cDNA was subjected to qPCR using a SYBR Premix Ex Taq™ Kit (Takara Biotechnology Co., Ltd.). The cycling conditions for qPCR were as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. miR-876-5p and FOXM1 expression levels were normalized with reference to U6 snRNA and GAPDH, respectively. The primers were designed as follows: Forward, 5′-AGGACUUCUCCCUCCUCCCAG-3′ and reverse, 5′-UCCUCUUCUCCCUCCAGGGAG-3′ for miR-876-5p; forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′ for U6; forward, 5′-GAAGAACTCCATCCGCCACA-3′ and reverse, 5′-GCCTTAAACACCTGGTCCAATGTC-3′ for FOXM1 and forward and 5′-TGGATTTGGACGCATTGGTC-3′ and reverse 5′-TTTGCACTGGTACGTGTTGATA-3′ for GAPDH. Relative gene expression levels were analysed using the 2^−ΔΔCq method (29).

Cells were transfected and cultured at 37°C with 5% CO₂ for 24 h. Following the culture period, transfected cells were collected and plated into 96-well plates at an initial density of 3×10³ cells/well. A CCK-8 assay was performed to detect cell proliferation at different time points (0, 1, 2 and 3 days). A tostal of 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added into each well of the plate, and then the cells were incubated at 37°C for a further 2 h. The absorbance of each well was detected at a wavelength of 450 nm (A450) by a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

An Annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit (Biolegend, San Diego, CA, USA) was utilized to assess cell apoptosis rate. In brief, transfected cells were incubated at 37°C under 5% CO₂ for 48 h. Subsequently, the transfected cells were harvested, washed twice with phosphate-buffered saline (PBS) and suspended in 100 µl binding buffer. Following this, the transfected cells were incubated with Annexin V-FITC (5 µl) and propidium iodide (5 µl) at room temperature in the dark. Following incubation for 15 min, a flow cytometer (FACScan; BD Biosciences, Franklin Lakes, NJ, USA) was used to acquire data on cell apoptosis rate. The data was analysed with CellQuest version 5.1 (BD Biosciences).

In vitro migration and cell invasion assays were performed to detect cell migration and invasion using 24-well Transwell chambers coated without or with Matrigel (both from BD Biosciences), respectively. Cells were collected 48 h after transfection and suspended in FBS-free DMEM. The upper compartments of the Transwell chambers were loaded with 200 µl of cell suspension containing 1×10⁵ transfected cells. DMEM (500 µl) supplemented with 20% FBS was placed in the lower compartments. After 24 h of incubation, cells remaining on the upper surface were removed using a cotton swab, whereas the migrated and invaded cells were fixed in 100% methanol at room temperature for 15 min, stained with 0.1% crystal violet at room temperature for 15 min, washed with PBS and air-dried. Migration and invasion capacities were quantified by counting the number of migrated and invaded cells in five randomly selected microscopic fields seen under an inverted microscope (×200 magnification; IX83; Olympus Corp., Tokyo, Japan).

The putative targets of miR-876-5p were predicted using TargetScan (www.targetscan.org), microRNA.org (www.microrna.org) and miRDB (www.mirdb.org). FOXM1 was predicted as a major target of miR-876-5p, and was selected for further verification experiments.

The 3′-UTR fragments of FOXM1 containing the wild-type (Wt) or mutant (Mut) miR-876-5p targeting sequences were chemically produced by Shanghai GenePharma Co., Ltd., and inserted into the pmirGLO luciferase reporter vector (Promega Corp., Madison, WI, USA). The constructed luciferase plasmids were defined as pmirGLO-Wt-FOXM1-3′-UTR and pmirGLO-Mut-FOXM1-3′-UTR, respectively. For reporter assays, cells were inoculated into 24-well plates at a density of 1×10⁵ cells/well. After overnight incubation, miR-876-5p mimics or miR-NC were transfected into cells containing pmirGLO-Wt-FOXM1-3′-UTR or pmirGLO-Mut-FOXM1-3′-UTR with Lipofectamine 2000, according to the manufacturer's protocol. Subsequent to 48 h incubation, transfected cells were harvested and the luciferase activity was analysed using the Dual-Luciferase Reporter Assay System (Promega Corp.). Renilla luciferase activity was used for normalization.

Cells or homogenized tissues were lysed using a Total Protein Extraction Kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Protein concentration was measured by a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). The same amounts of proteins (30 µg) were subjected to 10% SDS-PAGE gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Inc.). The membranes were subsequently blocked with 5% fat-free milk diluted in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room temperature and incubated overnight at 4°C with primary antibodies. Following washing with TBST three times, the membranes were further immersed in goat anti-mouse horseradish peroxidase-conjugated IgG secondary antibody (dilution 1:5,000; cat. no. ab205719; Abcam, Cambridge, UK) at room temperature for 2 h. Subsequent to three washes with TBST, chemiluminescence detection was conducted using an ECL Protein Detection Kit (Pierce; Thermo Fisher Scientific, Inc.). Primary antibodies used in the present study included mouse anti-human monoclonal FOXM1 (dilution 1:500; cat. no. sc-271746; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and mouse anti-human monoclonal GAPDH (dilution 1:500; cat. no. ab8245; Abcam). GAPDH was used as endogenous control. Protein expression was quantified using Quantity One software version 4.62 (BioRad Laboratories, Inc., Hercules, CA, USA).

Data are presented as the mean ± standard deviation of three independent experiments. Differences between groups were determined using two-tailed Student's t-test or one-way analysis of variance (ANOVA) for multiple comparisons. The Student-Newman-Keuls method was used as a post hoc test following ANOVA. The association between miR-876-5p and FOXM1 mRNA levels in GBM tissues was assessed using Spearman's correlation analysis. SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

To illustrate the potential relevance of miR-876-5p in GBM, the present study first detected miR-876-5p expression in GBM and adjacent normal tissues obtained from 26 patients. RT-qPCR data demonstrated that miR-876-5p expression was significantly downregulated in GBM tissues compared with adjacent normal tissues (Fig. 1A; P<0.05). In addition, the expression levels of miR-876-5p in four GBM cell lines (U138, U251, T98 and LN229) and normal human astrocytes (NHAs) were examined. The miR-876-5p expression in all four tested GBM cell lines was decreased compared with in NHAs (Fig. 1B; P<0.05). U251 and T98 cell lines exhibited relatively lower miR-876-5p expression compared with the two other GBM cell lines; hence, U251 and T98 cell lines were selected for subsequent experiments. These observations suggested that decreased expression of miR-876-5p was associated with the development and progression of GBM.

To investigate the functional role of miR-876-5p in GBM, the present study transfected U251 and T98 cells with miR-876-5p mimics that resulted in increased endogenous miR-876-5p expression (Fig. 2A; P<0.05). The impact of miR-876-5p overexpression on GBM cell proliferation was determined using a CCK-8 assay. The results indicated that transfection of miR-876-5p mimics significantly reduced the proliferation of U251 and T98 cells (Fig. 2B; P<0.05). A flow cytometry assay was performed to measure the apoptosis rate of U251 and T98 cells treated with miR-876-5p mimics or miR-NC. miR-876-5p overexpression significantly increased the apoptosis rate of U251 and T98 cells compared with miR-NC groups (Fig. 2C; P<0.05). These results suggested that miR-876-5p served a tumour suppressive role in GBM cell proliferation and apoptosis.

In vitro migration and invasion assays were performed to further examine the potential role of miR-876-5p in metastasis of GBM cells. As presented in Fig. 3A, the migratory ability of the miR-876-5p mimics-transfected U251 and T98 cells was significantly suppressed compared with miR-NC-transfected cells (P<0.05). Additionally, overexpression of miR-876-5p expression resulted in the reduced number of invaded U251 and T98 cells compared with miR-NC groups (Fig. 3B; P<0.05). Taken together, miR-876-5p overexpression inhibited the metastasis of GBM.

To elucidate the mechanism of miR-876-5p activity in GBM, the present study employed bioinformatics analysis to predict the putative targets of miR-876-5p. It was demonstrated that the 3′-UTR of FOXM1 matched the seed sequences of miR-876-5p (Fig. 4A). FOXM1 has been well documented to play crucial roles in GBM progression (30–34) and thus was selected for further verification. To determine whether miR-876-5p could directly target the 3′-UTR of FOXM1, luciferase reporter plasmids were constructed and were transfected into U251 and T98 cells containing miR-876-5p mimics or miR-NC. The results of the luciferase reporter assay indicated that upregulation of miR-876-5p significantly reduced the luciferase activity of the plasmid carrying the wild type (Wt) 3′-UTR of FOXM1 in U251 and T98 cells (P<0.05). Conversely, the luciferase activity was unaltered in cells transfected with plasmid harbouring the mutant (Mut) 3′-UTR of FOXM1 (Fig. 4B).

To further explore the association between miR-876-5p and FOXM1 in GBM, the present study measured FOXM1 expression levels in 26 pairs of GBM tissues and adjacent normal tissues. RT-qPCR analysis revealed that FOXM1 expression in GBM tissues was significantly upregulated compared with adjacent normal tissues (Fig. 4C; P<0.05). In addition, a negative correlation between miR-876-5p and FOXM1 mRNA levels was identified in GBM specimens (Fig. 4D; r=−0.5589, P=0.0030). Furthermore, enforced miR-876-5p expression suppressed FOXM1 expression in U251 and T98 cells at the mRNA (Fig. 4E; P<0.05) and protein (Fig. 4F; P<0.05) levels. The results collectively suggested that FOXM1 was a direct target gene of miR-876-5p in GBM cells.

FOXM1 was confirmed to be a direct target of miR-876-5p in GBM cells; hence, the authors hypothesized that inhibition of FOXM1 could imitate the suppressive roles of miR-876-5p in GBM cells. To confirm this hypothesis, U251 and T98 cells were transfected with NC siRNA or FOXM1 siRNA, and western blot analysis was performed in order to validate the transfection efficiency. As expected, FOXM1 protein was efficiently knocked down in U251 and T98 cells treated with FOXM1 siRNA (Fig. 5A; P<0.05). Functional experiments revealed that siRNA-mediated knockdown of FOXM1 inhibited the proliferation (Fig. 5B; P<0.05) and promoted the apoptosis (Fig. 5C; P<0.05) of U251 and T98 cells. In vitro migration and invasion assays revealed that FOXM1 suppression remarkably restricted both the migration (Fig. 5D; P<0.05) and invasion (Fig. 5E; P<0.05) capabilities of U251 and T98 cells. These results demonstrated that FOXM1 downregulation could mimic the tumour suppressive role of miR-876-5p in GBM cells, further suggesting that FOXM1 is a functional downstream target of miR-876-5p in GBM.

Rescue experiments were conducted to determine whether FOXM1 mediates the tumour suppressive role of miR-876-5p in GBM cells. The present study co-transfected miR-876-5p mimics with empty pCMV or pCMV-FOXM1 plasmid lacking a 3′-UTR into U251 and T98 cells; subsequently, western blot analysis was performed to detect FOXM1 protein expression. As presented in Fig. 6A, the downregulated FOXM1 protein expression caused by miR-876-5p overexpression was restored in U251 and T98 cells following co-transfection with pCMV-FOXM1 (P<0.05). Furthermore, CCK-8 assays revealed that restoration of FOXM1 expression partially rescued the miR-876-5p-mediated suppression of cell proliferation (Fig. 6B; P<0.05). Similarly, the flow cytometry assay revealed that while miR-876-5p promoted apoptosis of U251 and T98 cells, this effect was abolished by forced FOXM1 expression (Fig. 6C; P<0.05). Furthermore, FOXM1 upregulation partially abrogated the suppressive effects of miR-876-5p overexpression on migration (Fig. 6D; P<0.05) and invasion (Fig. 6E; P<0.05) of U251 and T98 cells. These results collectively demonstrated that miR-876-5p may inhibit the progression of GBM, at least partly through downregulation of FOXM1 expression.