A total of 10 GBM tissues and 10 corresponding adjacent normal tissues were collected from the Department of Neurosurgery, The People's Hospital of Leshan City (Leshan, China). The use of human tissues in the present study was evaluated and approved by the Ethic Committee of The People's Hospital of Leshan City. All patients enrolled in the present study provided written informed consent, and all specimens were handled and made anonymous according to the ethical and legal standards of China. All GBM tissues were confirmed and evaluated by a pathologist, and were frozen at −80°C immediately following surgery.

The normal human astrocytes (NHA; cat. no. 43923), and human glioblastoma cell lines U373 and U118, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. The U373 cell line is known to be contaminated with the U251 astrocytoma cell line and the U118 cell line is known to be contaminated with the U138 astrocytoma cell line (22).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The level of miR-194-5p was evaluated using a TaqMan MiRNA Assay (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. U6 small nuclear RNA was used as an internal loading control. Reverse transcription-quantitative polymerase chain reaction was performed on an ABI 7900HT instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers were synthesized and tested by Sangon Biotech Co., Ltd. (Shanghai, China). The primer sequences were as follows: U6 forward, GTG GAC CGC ACA AGC TCG CT, and reverse, TTG TTG AAC GGC ACT GTG TAT AGC A; miR-194-5p forward, AGT GTG ACG TTG ACA TCC GT, and reverse, GCA GCT CAG TAA CAG TCC GC. Relative miR-194-5p expression was calculated according to the comparative Cq method (23) and normalized to the expression of U6.

miR-194-5p was overexpressed and inhibited in vitro by transfection with miR-194-5p mimics and an miR-194-5p antisense oligonucleotide (ASO), respectively. The miR-194-5p mimics and miR-194-5p ASO were purchased from Sangon Biotech Co., Ltd. The miRNA sequences were as follows: miR-194-5p mimic, 5′-AAGGCAGGGCCCCCGCUCCCC-3′; miR-194-5p ASO, 5′-GGAGCGGGGGCCCUGCCUUUU-3; scrambled miRNA control, 5′-CAGUACUUUUGUGUAGUACAA-3. The cells were seeded (1×10⁶ cells/well) and cultured overnight. Transfection was performed for 30 min using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

After 24, 48 and 72 h of transfection, cellular proliferation was assessed by the MTT assay. Cells were seeded into 96-well plates at a density of 5×10⁵ cells/well. MTT reagent was added into the medium at a final concentration of 0.1 mg/ml. A total of 100 µl DMSO was added. Optical density was measured by a microplate reader at 570 nm.

A cell suspension (5×10⁵ cells/ml) was prepared in annexin V-fluorescein isothiocyanate (FITC; Abcam, Shanghai, China) binding buffer and Annexin V-FITC was added and incubated for 15 min at room temperature. Subsequently, propidium iodide (PI; Abcam, Shanghai, China) was added, samples were analyzed by flow cytometry using an LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) at an excitation wavelength of 488 nm (argon-ion laser or solid state laser), and an emission wavelength of 530 nm for FITC and 590 nm for PI (24). Data analysis was performed using the BD FACSDiva software version 8.0.1 (BD Biosciences).

Targetscan software (www.targetscan.org) was used to predict the potential targets of miR-149-5p.

Cells were seeded at 1×10⁵ cells/well and were serum starved for 6 h prior to transfection. The wild-type (WT) 3′UTR of IGF1R and mutated controls were cloned and inserted into the dual-luciferase-reporter plasmid (500 ng; Shengong Biotechnology Company) and the pGL3-control (100 ng; Promega Corporation, Madison, WI, USA). miR-194-5p mimics and controls were subsequently transfected into U373 cells containing wild-type or mutant 3′UTR plasmids with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were harvested and luciferase activities were analyzed after 24 h using the Dual-Luciferase Reporter assay system (Promega Corporation). The mutant IGF1R 3′UTRs were generated using a Site-Directed Mutagenesis kit (Promega Corporation). The primers used to perform mutagenesis were as follows: IGF1R forward, 5′-ctgtccctgatgctgaaggcaggcagaatgacttc-3′, and reverse, 5′-gaagtcattctgctctgccttcagctcagggacag-3′. The kit was used according to the manufacturer's protocol.

Whole-cell lysates were prepared as previously described (25). The cell lysates were centrifuged at 4°C, 12,000 × g for 10 min, extracted and the proteins were quantified using a bicinchoninic acid protein quantification kit (cat. no. ab102536; Abcam, Cambridge, UK). Proteins (30 µg) were separated via 4–10% SDS-PAGE. Membranes were electrotransferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk overnight at 4°C and incubated with the anti-IGF1R antibody (cat. no. 3027; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. β-actin (cat. no. 4967; 1:1,000; Cell Signaling Technology, Inc.) was used as the endogenous control, incubated overnight at 4°C. Then the membranes were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat. no. 7074; 1:1,000; Cell Signaling Technology, Inc.) for 2 h at room temperature. The bands were visualized using enhanced chemiluminescence (GE Healthcare Life Sciences, Little Chalfont, UK).

Data are presented as the mean ± standard deviation of 3 independent experiments. Statistical analysis was performed using SPSS software (version 10.0; SPSS, Inc., Chicago, IL, USA). The difference between two groups was analyzed using a two-tailed Student's t-test. One-way analysis of variance was used to analyze the difference between multiple groups, followed by the Student-Newman-Keuls method. P<0.05 was considered to indicate a statistically significant difference.

IGF1R is an independent prognostic factor associated with decreased survival of patients with GBM (21). Therefore, miR-194-5p was selected for investigation, as it was identified to target IGF1R. A total of three miR-194-5p binding sites were identified in the 3′UTR of the IGF1R gene and three mutants were subsequently generated (Fig. 1A). The three mutated versions of the IGF1R 3′UTRs were cloned into luciferase reporter plasmids. miR-194-5p mimics and the mutant 3′UTRs were cotransfected into U373 cells, and it was observed that the miR-194-5p mimics reduced the luciferase activity of the WT 3′UTR, while the mutated 3′UTRs partially reduced the inhibitory function of miR-194-5p, scrambled miRNA was used as a vehicle (Fig. 1B). miR-194-5p mimics were subsequently transfected into U373 cells and the IGF-1R protein level was observed to be reduced 48 h following transfection (Fig. 1C). The results indicated that miR-194-5p may serve a role in GBM via IGF1R, thus miR-194-5p was selected for further study.

miR-194-5p levels were evaluated in 10 GBM tissues and compared with the miR-194-5p levels in the 10 corresponding adjacent normal tissues. All the GBM tissues were observed to express decreased miR-194-5p levels compared with the corresponding adjacent normal tissues (Fig. 2A). The mean levels of the 10 GBM tissues also exhibited a decreased level of miR-194-5p compared with the normal tissues (Fig. 2B).

miR-194-5p was overexpressed in GBM cells to test the function of miR-194-5p. The miR-194-5p levels in U373, U118 and NHA cells were analyzed by RT-qPCR, and it was observed that U373 and U118 cells expressed decreased levels of miR-194-5p compared with NHA cells (Fig. 3A). miR-194-5p was successfully overexpressed in U373 and U118 cells by miR-194-5p mimic transfection (Fig. 3B). MTT analysis revealed that the miR-194-5p mimics inhibited the growth of U373 and U118 cells (Fig. 3C). The cellular apoptotic rate was analyzed 48 h following miR-194-5p mimic transfection, and it was observed that miR-194-5p upregulated the apoptotic rate of U373 and U118 cells (Fig. 3D).

miR-194-5p was successfully downregulated in U373 and U118 cells by miR-194-5p ASO transfection. After 48 h, the miR-194-5p levels in cells were analyzed by RT-qPCR (Fig. 4A). Cellular proliferation in the two cell lines was analyzed using the MTT assay, and it was observed that miR-194-5p ASO promoted the cellular proliferation of U373 and U118 cells (Fig. 4B).