The present study was approved by the Ethics Committee of the Brain Hospital of Hunan Province (Changsha, China). Glioma tissues (n=66) were collected from patients with glioma (39 male, 27 female; age, 24–64 years; median age, 49.3 years) by surgical resection at the Brain Hospital of Hunan Province between March 2013 and October 2016. Patients were excluded from the current study if they received chemotherapy or radiotherapy prior to surgical resection. A total of 8 patients with glioma were diagnosed at World Health Organization stage II, 24 at stage III and 34 at stage IV. Furthermore, 15 normal brain tissues were also collected from healthy patients (9 male, 6 female; age, 33–57 years; median age, 46.7 years) via surgical resection at the same hospital between June 2013 and September 2016. Written informed consent was obtained from all patients. The clinicopathologic characteristics of the patients are summarized in Table I. Fresh tissues were stored at −80°C prior to further use.

Several common human glioma cell lines (U251-MG, T98G, U-87MG Uppsala and U-373MG Uppsala) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Normal human astrocyte (NHA) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37°C, 5% CO₂ and 95% O₂.

Lipofectamine^™ 2000 (Thermo Fisher Scientific, Inc.) was used to perform cell transfection according to the manufacturer's protocol. T98G and U251 cells were transfected with 100 nM miR-200a mimic (cat. no. HmiR0002-MR04; Guangzhou Fulengen Co., Ltd., Guangzhou, China), 100 nM miR-200a inhibitor (cat. no. HmiR-AN0298-SN-10; Guangzhou Fulengen Co., Ltd.) and 100 nM miR negative control (miR-NC; cat. no. CmiR0001-MR04; Guangzhou Fulengen Co., Ltd.). Cells were also co-transfected with 100 nM miR-200a mimic and 100 nM blank pcDNA3.1 vector (cat. no. ZL00008; Yearthbio, Changsha, China) or with 100 nM miR-200a mimic and 100 nM FOXA1 plasmid (cat. no. ZL00371; Yearthbio). Non-transfected cells were used as control. Further experimentation was performed 48 h following transfection.

Total RNA was extracted from tissues or U251, T98G, U-87MG Uppsala, U-373MG Uppsala and NHA cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. Total RNA (1 µg) was then reverse transcribed into cDNA using the PrimeScript Reverse Transcription kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. qPCR reactions were performed using the SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.) with a 7300 plus ABI system (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. For the assessment of mRNA expression, GAPDH was used as internal reference. For the expression of miR-200a, U6 was used as internal reference. The thermocycling conditions were as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 15 sec and 60°C for 30 sec. Relative expression was analyzed using the 2^−ΔΔCq method (26). The primers for FOXA1 (cat. no. HQP055418), GAPDH (cat. no. HQP006940), miR-200a (cat. no. HmiRQP0298) and U6 (cat. no. HmiRQP9001) were purchased from Guangzhou Fulengen Co., Ltd.

TargetScan 7.1 software (www.targetscan.org) was used to assess the putative target genes of miR-200a. The mutant type (MT) or wild type (WT) 3′UTR of FOXA1 was amplified and cloned into the psiCHECK-2 vector (Promega Corporation, Madison, WI, USA) to construct luciferase reporter plasmids, which were obtained from Yearthbio. U251 and T98G cells were co-transfected with the WT FOXA1 plasmid or the MT FOXA1plasmid and miR-200a mimic or miR-NC, respectively, using Lipofectamine^™ 2000 (Thermo Fisher Scientific, Inc.). Following transfection for 48 h, a dual-luciferase reporter assay system (Promega Corporation) was used to examine the luciferase activity, according to the manufacturer's protocol. Renilla luciferase activity was normalized to Firefly luciferase activity.

T98G and U251 Cells were lysed using radio immunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.). Protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Protein (50 µg per lane) was separated using 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). Membranes were then blocked with 5% evaporated milk at room temperature for 1 h and subsequently incubated with rabbit anti-human FOXA1 (1:200; cat. no. ab170933; Abcam, Cambridge, UK)or rabbit anti-human GAPDH (1:200; cat. no. ab9485; Abcam) antibodies overnight at 4°C. Membranes were further incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000; cat. no. ab6721; Abcam) for 40 min at room temperature. Chemiluminescence was detected using the SuperSignal West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific, Inc.). The quantity of protein was analyzed using Image J 1.45 software (National Institutes of Health, Bethesda, MD, USA).

U251 and T98G cells (5×10³ cells/well) were plated into 96-well plates and cultured for different durations (0, 24, 48 or 72 h) at 37°C in a humidified atmosphere containing 5% CO₂. A total of 10 µl MTT solution (5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added at different time points (0, 24, 48 or 72 h). Cells were then incubated at 37°C for 4 h. The supernatant was then removed and 100 µl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) was added. The absorbance value (optical density) was measured at 570 nm using a microplate reader.

U251 and T98G cells (5×10³ cells/well) were seeded into 96-well plates and incubated at 37°C for 24 h. A total of 50 µM EdU (Guangzhou RiboBio Co., Ltd., Guangzhou, China) was then added. Following this, cells were incubated at 37°C for 2 h. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then treated with 0.5% Triton-X-100 (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. Subsequently, 100 µl Apollo^® Reaction Cocktail (Guangzhou RiboBio Co., Ltd.) was added and cells were incubated at room temperature for 30 min. Following staining with DAPI (5 µg/ml; Sigma-Aldrich; Merck KGaA) at room temperature for 30 min, cells were visualized under a fluorescent microscope (IX71; Olympus Corporation, Tokyo, Japan).

Matrigel pre-coated Transwell chambers (BD Biosciences, Franklin Lakes, NJ, USA) were utilized to examine cell invasion. T98G and U251 cell suspensions (3×10⁵ cells/ml) were prepared using DMEM. Subsequently, 300 µl cell suspension was added into the upper chamber and 300 µl DMEM supplemented with 10% FBS was added into the lower chamber. Cells were incubated at 37°C for 24 h and the cells that did not invade through the membrane were carefully removed. The transmigrated cells were then fixed using 4% polyformaldehyde at room temperature for 30 min, stained with crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature for 10 min and counted in five randomly selected microscopic fields using a light microscope (magnification, ×400). All experiments were performed in triplicate.

Data are presented as the mean ± standard deviation. GraphPad Prism version 5.01 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical analysis. Differences between two groups were analyzed using a Student's t-test and differences among more than two groups were analyzed using one-way analysis of variance followed by a Tukey's post-hoc test. Glioma patients were divided into high and low miR-200a expression groups, based on its mean expression value, and a χ² test was used to analyze the results presented in Table I. A Pearson correlation test was used to determine the relationship between miR-200a and FOXA1expression. P<0.05 was considered to indicate a statistically significant result.

RT-qPCR was used to assess the expression of miR-200a in glioma and normal brain tissues. As presented in Fig. 1A, the levels of miR-200a were significantly lower in glioma tissues compared with normal brain tissues. Furthermore, grade IV glioma tissues demonstrated significantly lower miR-200a levels when compared with grade II–III glioma tissues (Fig. 1B). Subsequently, glioma patients were divided into high and low miR-200a expression groups, based on its mean expression value. As presented in Table I, the low expression of miR-200a was significantly associated with advanced clinical stage in glioma. In addition, miR-200a levels were significantly reduced in several common glioma cell lines compared with those in NHA cells (Fig. 1C). As T98G and U251 exhibited the lowest expression of miR-200a, they were used for the following experiments.

T98G and U251 cells were transfected with miR-200a mimics to upregulate the expression of miR200a. The results indicated that miR-200a levels were significantly increased in the miR-200a group compared with the miR-NC group. However, transfection with miR-NC did not significantly affect the expression of miR-200a (Fig. 2A). The effects of miR-200a upregulation on glioma cells proliferation, survival and invasion were then assessed. As presented in Fig. 2B-D, the overexpression of miR-200a markedly reduced T98G and U251 cell proliferation (at 48 and 72 h), viability and invasion. These findings suggest that miR-200a demonstrated suppressive effects on the proliferation, survival and invasion of glioma cells.

The potential downstream target genes of miR-200a in glioma were then assessed. TargetScan software data indicated that the 3′UTR of FOXA1 mRNA contained the binding sequences of miR-200a (Fig. 3A). To confirm this prediction, the present study constructed the WT and MT FOXA1 3′UTR luciferase reporter plasmids (Fig. 3B). Dual luciferase reporter gene assay data indicated that the luciferase activity was significantly reduced in glioma cells co-transfected with miR-200a mimics and WT reporter plasmids, when compared with cells transfected with WT reporter plasmids. However, this result was reversed following transfection with the MT reporter plasmid (Fig. 3C and D). These data indicate that FOXA1 is a target gene of miR-200a in glioma cells.

The expression of FOXA1 in glioma was further assessed. The results demonstrated that FOXA1 was significantly upregulated in glioma tissues compared with normal brain tissues (Fig. 4A). A negative correlation between FOXA1 and miR-200a expression in glioma tissues was also identified (Fig. 4B). Therefore, FOXA1was demonstrated to be upregulated in glioma, which may be due to the downregulation of miR-200a.

The effect of miR-200a on the expression of FOXA1in glioma cells was assessed. The results demonstrated that FOXA1 mRNA and protein expression was significantly reduced following miR-200a overexpression (Fig. 5A and B). miRNAs not only affect mRNA expression, but also function to inhibit protein translation (5–7). Due to this double effect, transfection with miR-200a mimics demonstrated a greater reduction in FOXA1 protein expression when compared with mRNA expression. T98G and U251 cells were transfected with amiR-200a inhibitor to knockdown its expression. RT-qPCR data revealed that miR-200a expression was significantly reduced in the miR-200a inhibitor group compared with the control group. Furthermore, no marked difference in miR-200a expression between the NC inhibitor and control group was identified (Fig. 5C). As presented in Fig. 5D and E, the expression of FOXA1 mRNA and protein was significantly increased in the miR-200a inhibitor group compared with the NC inhibitor group. These findings suggest that the expression of FOXA1 was negatively regulated by miR-200a in T98G and U251 cells.

Rescue experiments were performed to assess whether FOXA1 acted as a downstream effecter of miR-200a in glioma cells. miR-200a-overexpressing T98G and U251 cells were transfected with FOXA1 expression plasmid or blank vectors (acting as the negative control group). Following transfection, FOXA1 protein levels were markedly higher in the miR-200a+FOXA1 group compared with those in the miR-200a+blank group. It was also demonstrated that the proliferation (at 48 and 72 h), viability and invasion of glioma cells were significantly increased in the miR-200a+FOXA1 group compared with those in the miR-200a+blank group (Fig. 6B-D). These results indicated that FOXA1 rescued the inhibitory effects of miR-200a by affecting the proliferation, viability and invasion of glioma cells.