Brain glioma cell U87 MG-Luc2 was purchased from ATCC (ATCCHTB-14-LUC2), hereinafter referred to as U87 cells and has been authenticated by STR profiling (Beijing Microread Genetics Co., Ltd.). The cells were cultured in DMEM complete medium containing 10% fetal calf serum (Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.) with a culture condition of 37°C, 5% CO₂ and 95% relative humidity.

This study was approved by the Ethics Committee of Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Art and Science (Xiangyang, China).

When cell attachment growth density of U87 reached 90%, the cells were digested with 0.25% trypsin. U87 cells were transferred into DMEM medium and cultured in incubators at 37°C, 5% CO₂ then synaptic retraction was observed under a microscope. The culture was collected for the third generation use.

U87 cells with an attachment growth density of 90% were collected and digested with 0.25% trypsin. The U87 brain glioma stem cells were sorted by adding tumor stem cell culture medium (Jiangsu Promocell Biotechnology Co., Ltd.). The medium was replaced every two days, centrifugation was performed at 800 × g for 5 min at 4°C before replacement, and then half of the medium was replaced. After 3 weeks, the suspension cells were collected and passaged in accordance with the above method.

Construction and transfection of miR-124 expression vector. The miR-124 mimic, miR-124 inhibitor and miR-control expression vectors were designed and produced by Shanghai Gene Pharmaceutical Technology Co., Ltd. The cells were digested by trypsin for 24 h before transfection, and U87 stem cells were transfected when the cells were fused to approximately 80%, the specific steps referred to the specification of the kit. The cells were cultured in 37°C, 5% CO₂ incubator for 48 h, and the medium was changed every 6 h. The transfection results were detected by RT-qPCR. Lipofectamine™2000 transfection kit was purchased from Shanghai Yanjin Biotechnology Co., Ltd.

The concentration of cell suspension was adjusted to 1×10⁷, and the total RNA was extracted by adding suspension and TRIzol lysate at 3:1. After extraction, 1.5% agarose gel electrophoresis was used to analyze the integrity of RNA, micro-amount of nucleic acid analyzer was used to detect the purity of extracted RNA. A260/A280 value between 1.8 and 2.0 was considered to meet the experimental requirements. RT-qPCR reaction was performed after the completion of RNA extraction. Reverse transcription system: oligo dt primer 1.0 µl, 5X PrimeScript Buffer 4.0 µl, 2.5 U/µl polyA polymerase 1 µl, dNTP mixture 1.0 µl, total RNA 2 µg, RNase-free distilled water was added to 10 µl. Reaction condition was: 40°C for 15 min, 85°C for 5 sec; PCR amplification was carried out after the completion of reverse transcription reaction. PCR amplification system was: cDNA template 2 µl, SYBR-Green Mix 25 µl, upstream primer 0.5 µl, downstream primer 0.5 µl, double-steamed water was added to 50 µl. PCR reaction program was: 30 cycles of predenaturation at 95°C for 3 min, denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, elongation at 72°C for 60 sec, elongation at 72°C for 5 min after the cycle was complete. GAPDH was used as the internal parameter of the reaction. Samples were set in triplicate for each test, and 2^−ΔCq was used to analyze the results (9). The primer sequence was designed and produced by Hepeng (Shanghai) Biotechnology Co., Ltd. (Table I).

The expression of Nogo-A and NgR protein was detected by western blot analysis. The total protein was extracted from the cell by RIPA total protein lysate (Wuhan Aspen Biological Company) and the concentration of extracted protein was determined by BCA, and the BCA protein quantitative detection kit was purchased from Beyotime Institute of Biotechnology. Then, 40 µg extracted protein solution was used for SDS-PAGE electrophoresis, and buffer was added at a 1:4 dilution. β-actin protein was used as an internal reference, the concentration gel electrophoresis was performed at a constant 80 V for 40 min, and separation gel electrophoresis was performed at a constant 120 V for 90 min. Trans-membrane was conducted at a constant 100 V for 100 min and blocked at 37°C for 60 min after the completion of electrophoresis. After trans-membrane, the immunological reaction was carried out. Incubation was performed with primary rabbit anti-human Nogo-A polyclonal antibody (1:200; cat. no. 10740-1-AP; ProteinTech Group, Inc.) at 4°C for 16 h. It was washed the next day in lukewarm water 3 times, 10 min each time with PBS. Then, incubation was performed with secondary goat anti-rabbit polyclonal IgG (1:500; cat. no. SA00001-2; ProteinTech Group, Inc.) at 37°C for 60 min. ECL luminescent reagent was used for visualization and fixation. QuantityOne software was used to analyze the strip after film scanning. Protein relative expression level was calculated as: band gray value/internal parameter gray value. Western blot detection kit was purchased from Beyotime Institute of Biotechnology.

U87 brain glioma stem cells were prepared into a single cell suspension of 4×10⁶/ml, performed routine vaccination, and cultured in 96-well cell culture plate. Then, 20 µl MTT solution (5 mg/ml) was added after 6 h, and cells were cultured at 37°C for 4 h continuously. Supernatant containing impurities was removed, dimethylsulfoxide was added, and the plate was placed on a horizontal vibration table. The absorbance values at wavelength 570 nm were measured at 12, 24, 48 and 72 h. The MTT test kit was purchased from Shanghai LMAI Bioengineering Co., Ltd.

U87 brain glioma stem cells were prepared into a single cell suspension of 1×10⁶/ml. CD133⁺ cells were separated by immunomagnetic beads, washed, re-suspended, counted in serum-free medium, and the cell morphology was observed. The immunomagnetic beads kit was purchased from Shanghai Chem Biotechnology Co., Ltd.

SPSS19.0 [AsiaAnalytics (formerly SPSS China)] was used for statistical analysis. Measurement data were expressed as mean ± standard deviation. ANOVA and the LSD post hoc test were used for multigroup comparison. The t-test was used for comparison between two groups and χ² test was used for rate comparison. The correlation between miR-124 and Nogo-A, and NgR protein expression was analyzed by Spearman's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

The relative expression levels of miR-124 in the miR-124 mimic, miR-124 inhibitor and miR-control groups were 1.83±0.14, 0.89±0.09 and 1.34±0.12, respectively. There was statistical difference between the three groups (P<0.05). The relative expression of miR-124 in cells of miR-124 mimic group was significantly higher than that of miR-124 inhibitor and miR-control groups (P<0.05), while the relative expression of miR-124 in cells of miR-124 inhibitor group was lower than that of miR-control group (P<0.05; Fig. 1).

Expression of Nogo-A and NgR protein in U87 brain glioma stem cells after transfection. The relative expression levels of Nogo-A protein in the miR-124 mimic, miR-124 inhibitor and miR-control groups were 0.392±0.011, 0.569±0.013 and 0.483±0.012, respectively, the relative expression levels of NgR protein was 0.533±0.012, 0.711±0.014 and 0.601±0.014, respectively. There was significant difference in Nogo-A and NgR protein between the three groups (P<0.05), the relative expression of Nogo-A and NgR protein in cells of miR-124 mimic group were significantly lower than those of miR-124 inhibitor and miR-control groups (P<0.05), while the relative expression of Nogo-A and NgR protein in cells of miR-124 inhibitor group were higher than those of miR-control group (P<0.05; Table II and Fig. 2).

The results of MTT proliferation in vitro showed that the absorbance values in the three groups were significantly different at each time point (P<0.05). The absorbance values of the cells in the miR-124 mimic and miR-control groups were significantly lower than those in the miR-124 inhibitor group at each time point (P<0.05), while the values of the cells in the miR-124 mimic group was significantly lower than that in miR-control group (P<0.05; Fig. 3).

The levels of CD133⁺ cells in the miR-124 mimic, miR-124 inhibitor and miR-control groups were 3.98±0.45, 36.12±1.34 and 51.25±1.48%, respectively. There was statistical difference between the three groups (P<0.05), the level of CD133⁺ cells in miR-124 mimic group was significantly lower than that in miR-124 inhibitor and miR-control groups (P<0.05), while the level of CD133⁺ cells in miR-124 inhibitor group was higher than that in miR-control group (P<0.05; Fig. 4).

The results of correlation analysis showed that there was a negative correlation between miR-124 and the expression of Nogo-A (r=−0.855, 95% CI: −0.9908 to −0.7954, P<0.001) and NgR (r=0825, 95% CI: −0.9845 to −0.6776, P<0.001; Fig. 5).