The human glioma T98G, U87 and U251 cell lines, and the human embryonic kidney (HEK)-293 cells (CRL-1573), were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare, Life Sciences Logan, UT, USA) at 37°C in a humidified atmosphere with 5% CO₂. Human brain tissues and glioma specimens were obtained from 11 patients (5 males and 6 females; age, 35–55 years) treated at the Eye Hospital, Wenzhou Medical University (Wenzhou, China) between September 2012 and July 2013. All patients provided written informed consent. All studies and procedures involving human tissue were approved by the Wenzhou Medical University Institutional Review Board. Patient samples were used in accordance with The Declaration of Helsinki.

The T98G cells (1×10⁵) were seeded in 6-well plates and total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A 10-ng sample of total RNA was transcribed into cDNA using a TaqMan^® MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the miR-548c-3p expression level was quantified using a TaqMan^® MicroRNA Assay kit (cat. no. 479537_MIR; Ambion; Thermo Fisher Scientific, Inc.), which included primers for has-miR-548c-3p. All procedures were performed according to the manufacturer's protocols. The thermocycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 5 sec, 58°C for 10 sec and 72°C for 5 sec. Each sample was analyzed in triplicate. qPCR was performed using a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), the expression of miR-548c-3p was normalized to the expression of U6 small nuclear RNA, and relative expression levels were calculated using the 2^−ΔΔCq method (21). The expression level of miR-548c-3p in the human brain was set as the wild-type control. To determine c-Myb expression in the T98G cells (1×10⁵), the cells were transfected with miR-548c-3p using 1 µl Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cultured at 37°C for 24 h. c-Myb expression levels were then quantified by measuring cyanine dye incorporation (SYBR Green PCR Master mix; Applied Biosystems; Thermo Fisher Scientific, Inc.) using the 7500 Real-Time PCR system. The primer sequences for c-Myb were as follows: Forward, 5′-ACGAGGATGATGAGGACTTTGAG-3′; and reverse, 5′-TTTTCCCCAAGTGACGCTTT-3′.

All transfections were performed in triplicate. Cells in each well were transfected with 50 nM miR-548c-3p precursor molecule (cat. no. MC11455; Ambion; Thermo Fisher Scientific, Inc.) or a negative control (NC) precursor miRNA (cat. no. 17110; Ambion; Thermo Fisher Scientific, Inc.). Following 1–4 days of culture in a humidified atmosphere with 5% CO₂ at 37°C, cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, a colorimetric method for determining the number of viable cells, with a CellTiter 96^® AQueous One Solution Cell Proliferation Assay kit (Promega Corp., Madison, WI, USA), according to the manufacturer's protocol. MTS solution was added to each well prior to incubation at 37°C for 3 h. Cell proliferation was assessed by measuring the absorbance at 490 nm using a microtiter plate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). The T98G cells were transfected with 50 nM c-Myb-specific small interfering RNA (siRNA; 50 nM; Ambion; Thermo Fisher Scientific, Inc.) or NC siRNA (cat. no. 4392420; Ambion; Thermo Fisher Scientific, Inc.) using Lipofectamine^® 2000 reagent. The mock control group was untreated T98G cells cultured under normal conditions. The MTS assay was performed 3 days after transfection. To evaluate the colony formation ability, T98G cells transfected with miR-548c-3p or NC siRNA were seeded in 3.5-cm plates (1,000 cells/dish). The colonies were fixed with 10% formalin at room temperature for 30 min, then stained with 0.1% crystal violet (Sigma; Merck Millipore, Darmstadt, Germany) at room temperature for 30 min. Colonies with >50 cells were counted using a light microscope (Carl Zeiss, Axio Observer D1, Germany) after 6 days.

The T98G cells were plated into 60-mm dishes and cultured at 37°C until between 50 and 70% confluence for each transfection. Each cell line was transfected with 50 nM miR-548c-3p precursor molecule or NC siRNA. A total of 48 h after transfection, the cells were collected, washed with phosphate-buffered saline (PBS) and stained with propidium iodide using the Cycletest™ Plus DNA kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocol. Stained cells (1×10⁵) were analyzed for DNA content using a FACSCalibur flow cytometer with CellQuest Pro software (version 6.0) (both BD Biosciences).

TargetScan (www.targetscan.org) was used to predict the direct targets of miR-548c-3p. The 3′-untranslated region (UTR) of human c-Myb was amplified from human genomic DNA and individually cloned into a pMIR-REPORT vector (Ambion; Thermo Fisher Scientific, Inc.) using directional cloning. Seed regions were mutated to remove all complementarity to nucleotides 1 to 7 of miR-548c-3p using a QuikChange XL Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The HEK-293 cells were co-transfected with 0.4 µg firefly luciferase reporter vector and 0.02 µg control vector containing Renilla luciferase (pRL-SV40 vector) (both Promega Corp.), together with 50 nM miR-548c-3p precursor molecule or NC precursor miRNA, using Lipofectamine^® 2000 in 24-well plates. Each transfection was performed with four replicate wells. Luciferase assays were performed 24 h after transfection using the Dual-Luciferase Reporter Assay system (Promega Corp.) according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.

The T98G cells were transfected with 50 nM miR-548c-3p precursor molecule or NC. After 24 h, the cells were harvested by trypsinization and washed once with Hanks' balanced salt solution (Invitrogen; Thermo Fisher Scientific, Inc.). Transwell culture inserts (pore size, 8-µm; Costar; BD Biosciences) were placed into the wells of 24-well culture plates, separating the upper and the lower chambers. DMEM (400 µl) was added in the lower chamber and 1×10⁵ cells were added to the upper chamber. A total of 24 h after incubation at 37°C and 5% CO₂, the cells that adhered to the inserts were fixed with 70% methanol at room temperature for 30 min and then stained with 0.1% crystal violet for 30 min. The number of cells that had migrated through the pores was quantified by counting 10 independent visual fields using a light microscope and a ×20 objective.

The T98G cells (1×10⁵) were transfected with the miR-548c-3p precursor molecule or NC. A total of 24 h after transfection, the cells were washed with ice-cold PBS and subjected to lysis in a lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 20 g/l SDS, 5 mM dithiothreitol and 10 mM phenylmethylsulfonyl fluoride). Protein concentration of whole cell lysates was assessed using the Pierce™ BCA Protein Assay Kit (Pierce; Thermo Fisher Scientific, Inc.). Protein lysates (50 µg each) were separated by 10% SDS-PAGE, then electrotransferred onto nitrocellulose membranes. The membranes were blocked with a buffer containing 5% skimmed milk powder in PBS with 0.05% Tween-20 for 2 h and incubated overnight with primary antibodies (described below) at 4°C. Antibodies directed against c-Myb (1:1,000; cat. no. 12319), protein lin-28 homolog B (Lin28b; 1:1,000; cat. no. 4196), Myc proto-oncogene protein (Myc; 1:1,000; cat. no. 9402) and GAPDH (1:2,000; cat. no. 2118) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Following a second wash with PBS containing 0.05% Tween-20, the membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated anti-rabbit Immunoglobulin G secondary antibody (dilution, 1:4,000; cat. no. 7074; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and developed with an enhanced chemiluminescence detection kit (Pierce; Thermo Fisher Scientific, Inc.). GAPDH was used as a loading control.

Apoptosis in the T98G cells was determined using the Caspase-Glo^® 3/7 assay kit (Promega Corp.) according to the manufacturer's protocol. The T98G cells were plated in triplicate in 96-well plates and transfected with miR-548c-3p as aforementioned. Samples were then incubated at room temperature with the caspase substrate (provided in the kit) for 2 h followed by measurement of the optical density at 560 nm using a microtiter plate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

A total of 1.5×10⁵ T98G cells/well were cultured in a 12-well plate for 24 h prior to transfection with miR-548c-3p or NC. After 2 days, the cells were scratched with a 100-µl pipette tip, washed with Hanks' balanced salt solution three times, and cultured in serum-free DMEM at 37°C and 5% CO₂. Images were captured 0, 1 and 3 days subsequent to the wound being made.

SPSS software (version 20.0; IBM SPSS, Inc., Armonk, NY, USA) was used for statistical analysis. Student's t-test was used to determine the statistical significance of differences between groups. All results are presented as the mean ± standard error of the mean from experiments performed at least three times. P<0.05 was considered to indicate a statistically significant difference.

To determine whether miRNA was involved in the regulation of glioma tumorigenesis, the expression of miR-548c-3p was determined in glioma and wild-type brain tissues. Total RNA was extracted and RT-qPCR analysis was performed. miR-548c-3p expression was decreased significantly in all 11 glioma tissue samples compared with two wild-type brain samples (Fig. 1A). Consistent with the data from glioma tissues, miR-548c-3p expression was demonstrated to be significantly downregulated in the U87, U251 and T98G glioma cell lines compared with the brain tissue (P<0.01; Fig. 1B). These results indicated that miR-548c-3p serves certain roles in glioma development.

As miR-548c-3p expression was downregulated in glioma, its biological effects on a glioma cell line were investigated. The T98G cells were transfected with the miR-548c-3p precursor molecule or NC. An MTS assay was performed to assess growth inhibition 1, 2, 3 and 4 days after transfection. Transient transfection of miR-548c-3p into T98G cells caused a significant inhibition of proliferation at day 4 compared with that of the NC and mock cells (25±6.2% inhibition; P<0.01; Fig. 2A). To further verify miR-548c-3p-mediated inhibition of cell proliferation, a colony formation assay was used to determine visually the effect of miR-548c-3p transfection on T98G cells (Fig. 2B). Cell migration, a prerequisite for malignant transformation and metastasis, was assessed using Transwell migration and wound healing assays. In the Transwell assay, T98G cells were transfected with either the miR-548c-3p precursor or an NC precursor. The cells were seeded on culture inserts, and the ability of cells to migrate to the underside of the inserts was determined. As presented in Fig. 2C, migration of miR-548c-3p-transfected cells was significantly decreased compared with mock- and NC-transfected cells (mock, 205±45; NC, 197±49; miR-548c-3p, 137±38; both P<0.01). The results of the wound healing assay were consistent with the Transwell migration assay. Migration of miR-548c-3p-transfected T98G cells was slower than that of NC-transfected T98G cells 1 and 3 days after transfection (Fig. 2D). Overall, the introduction of miR-548c-3p resulted in reduced cell motility.

The cell cycle was investigated using flow cytometry. In T98G cells transfected with miR-548c-3p, 50% accumulated in the G₁ phase compared with 34% of cells for NC-transfected cells (Fig. 2E). To further evaluate miR-548c-3p-mediated inhibition of cell proliferation, caspase activity was investigated to determine the involvement of apoptosis. No significant difference was observed in caspase 3/7 activity between miR-548c-3p- and NC-transfected cells (Fig. 2F). Therefore, these results indicated that T98G cell growth was inhibited by miR-548c-3p expression via cell cycle G₁ arrest rather than the induction of apoptosis.

Analysis using TargetScan (www.targetscan.org) was conducted for miR-548c-3p target prediction to investigate the underlying molecular mechanisms of miR-548c-3p-mediated cell proliferation and migration. The potential targets associated with tumorigenesis were identified as c-Myb, Myc and Lin28b. The effect of miR-548c-3p on the expression of these genes was investigated, with alteration of c-Myb being the most marked (Fig. 3A). The potential binding site of miR-548c-3p was predicted in the 3′-UTR of c-Myb mRNA. Alignment between the predicted miR-548c-3p target sites and miR-548c-3p, the conserved 7-bp seed sequence for miR-548c-3p-mRNA pairing, is presented in Fig. 3B. To evaluate the specific regulation of c-Myb through the predicted binding site, the c-Myb 3′-UTR sequence was amplified and inserted downstream of the firefly luciferase coding region of a pMIR-Luc vector. Mutants of the putative binding site were also prepared.

As indicated, introduction of miR-548c-3p in HEK-293 cells with the wild-type 3′-UTR (pLuc-c-Myb 3′-UTR) construct significantly inhibited the luciferase activity compared with the NC group (P<0.01; Fig. 3C). Mutation of the binding site, using a mutant vector (pLuc-MET 3-UTR-Mut), completely eliminated the ability of miR-548c-3p to regulate luciferase expression. These results demonstrated that c-Myb was a potential target of miR-548c-3p. RT-qPCR assay results also demonstrated that miR-548c-3p was able to inhibit c-Myb mRNA expression compared with NC and mock cells (P<0.01; Fig. 3D).

To confirm that c-Myb was responsible for miR-548c-3p inhibition of T98G proliferation and migration, c-Myb was knocked down and the knockdown efficiency of c-Myb siRNA was analyzed. A western blot assay demonstrated that c-Myb siRNA was able to inhibit c-Myb expression to a marked extent (Fig. 4A). An MTS assay was performed to test the effect of c-Myb siRNA on T98G proliferation. The proliferation of T98G cells transfected with c-Myb siRNA was inhibited compared with the NC (Fig. 4B). Furthermore, cells were co-transfected with miR-548c-3p and c-Myb to evaluate the effects of c-Myb on miR-548c-3p-overexpressed cells. As presented in Fig. 4C, miR-548c-3p inhibited T98G cell proliferation; however, cells co-transfected with miR-548c-3p and c-Myb rescued proliferation markedly compared with NC. This suggested that miR-548c-3p may exert its effect through the inhibition of c-Myb. The Transwell assay demonstrated that cells co-transfected with miR-548c-3p and c-Myb rescued the inhibitory effect of miR-548c-3p on T98G migration (Fig. 4D). Therefore, these data indicated that c-Myb is responsible for the inhibition by miR-548c-3p of the proliferation and migration of T98G cells.