Glioma tissue samples collected between November 2016 and June 2017 used in the present study were obtained from Nanfang Hospital and Zhujiang Hospital (Southern Medical University, Guangzhou, China), and Guangdong 999 Brain Hospital (Guangzhou, China) after pathological diagnosis, which included 22 grade I–II tumors, 58 grade III–IV tumors (according to the 2007 World Health Organization Classification of Tumours of the Central Nervous System) (10) and 16 relapsed tumors that had undergone TMZ chemotherapy for 6 months. All glioma specimens have been examined and diagnosed by pathological examination at The Department of Pathology of Zhujiang Hospital and Guangdong 999 Brain Hospital. Tissue samples were immediately aliquoted into separate test tubes, frozen on dry ice and stored at −80°C until analysis. Follow-up data was obtained from reviews of patients' medical records. Prior to surgery, no patients had received radiation or chemotherapy. The same procedure occurred for the patients who relapsed. This study was approved by the Ethics Committee of Hainan Medical University (Haikou, China; approval no. 201818). The present study was conducted in accordance with the regulations of the Hainan Institutional Review Board (affiliated to Hainan Provincial Organization Office). All enrolled patients provided written informed consent form before craniotomy. Human nervous glioma cell lines U251 and TMZ-resistant GBM cells (U251TR) were collected from The Department of Pathology, Nanfang Hospital, Southern Medical University. In order to verify that miR-296-3p may at least partially serve a role in multi-drug resistance in glioblastoma by targeting ether-à-go-go, the TMZ-resistant GBM U251TR cell line has been previously constructed from a U251 cell line (11) and stored in the laboratory of the Department of Laboratory of Southern Hospital.

The glioma cell lines, U251 and U251TR, were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; HyClone; Cytiva) and 10 µg/ml TMZ (Sigma-Aldrich; Merck KGaA), and were incubated at 37°C in a humidified 5% CO₂-enriched atmosphere. Human embryonic kidney 293T cells (purchased from the American Type Culture Collection) were also cultured in the same way. The glioma cells were transiently transfected using Lipofectamine^® 3000 Transfection reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The concentrations of miR-181a-5p mimics, miR-181a-5p inhibitors and controls were all 100 nmol/l. RNA fragments (miR-181a-5p mimics/inhibitors) used in transfection assay were purchased from Shanghai GenePharma Co., Ltd. Cells were divided into 6 groups, including: Untreated cells as the control group (2 groups, one for each cell line); cells transfected with miR-181-5p negative control (NC) as the NC group (2 groups, one for each cell line); drug-resistant GBM cells (U251TR) transfected with miR-181-5p mimics as the miR-mimics group; and GBM cells (U251) transfected with miR-181-5p inhibitors as the miR-inhibitors group. Additionally, U251 cells were transfected with the mimic to see whether there was an apoptotic effect. A total of 2×10⁵ cells/well were seeded in a 6-well culture plate and cultured until the cell density reached 70% at 37°C. According to the manufacturer's protocol, the transfection reagent was added into serum-free DMEM, which was added to the culture plate to replace the complete medium and cultured for 48 h at 37°C. The transfection efficiency was observed under a fluorescence microscope (data not shown). The sequences of RNA oligo ribonucleotides were as follows: miR-181a-5p mimics forward, 5′-AACAUUCAACGCUGUCGGUGAGU-3′ and reverse, 5′-UCACCGACAGCGUUGAAUGUUUU-3′; miR-181a-5p mimics-NC forward, 5′-UUCUCCGAACCUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGGUCGGAGAATT-3′; miR-181a-5p inhibitors, 5′-ACUCACCGACAGCGUUGAAUGUU-3′; miR-181a-5p inhibitors-NC, 5′-CAGUACUUUUGUGUAGUACAA-3′.

Total RNA was extracted and isolated from tissues and cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed. The RT mixed reaction solution was prepared on ice, including the following reagents: 2 µl 5X PrimeScript^® Buffer (Takara Biotechnology Co., Ltd.), 0.5 µl PrimeScript RT Enzyme Mix I, 0.5 µl oligo dT primer (50 µM), 0.5 µl random 6-nucleotide primers (100 µM), template RNA (4×10⁵ copies), RNase-free double-distilled water up to 10 µl. The reaction conditions were as follows: 37°C for 15 min for the reverse transcription reaction, and 85°C for 5 sec to inactivate the reverse transcriptase. The synthesized cDNA was stored at −20°C until subsequent use. The expression levels of miR-181a-5p were detected using the SYBR Green Hairpin-it™ miRNAs qPCR Quantitation kit (Shanghai GenePharma Co., Ltd.), in a Fast Real-time PCR 7500 System (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The reaction conditions were as follows: Pre-denaturation at 95°C for 30 sec, followed by 30–40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 30 sec and extension at 72°C for 10 sec. The U6 gene was used as an internal reference. Each assay was performed in triplicate and repeated three times. The relative expression levels were calculated and analyzed using the 2^−ΔΔCq method (12). Primers used in qPCR assays were synthesized by Shanghai GenePharma Co., Ltd. (miR-181a-5p, cat. no. 91151N61; U6, cat. no. 8301151105).

Cells (1×10⁴) were seeded into 96-well plates and divided into six groups: Two blank control groups, two NC groups, miR-181a-5p mimic group and miR-181a-5p inhibitor group, after transient transfection using Lipofectamine as aforementioned. After 24 h, the cells were treated with different concentrations of TMZ, ranging from 10 to 60 µg/ml for 48 h. The assay was undertaken in five replicate wells for each sample. The spectrophotometric absorbance of the samples was calculated as an average. Subsequently, the half maximal inhibitory concentration (IC₅₀) values of TMZ were respectively calculated.

A 96-well plate was used, at a density of 5×10⁴ cells/well. At 24, 48 and 72 h after transfection, 10 µl of CCK-8 solution (Pierce; Thermo Fisher Scientific, Inc.) was added according to the manufacturer's protocol. After 1 h, optical density (OD) at 450 nm value was measured using a microplate reader.

Cells were seeded into a 6-well plate at a density of 5×10⁵ cells/well and cultured for 24 h to create a confluent monolayer. The cells were serum-starved during this assay. The confluent monolayer was scraped with a 200-µl pipette tip along a sterile measuring rule, and washed three times in PBS. The corresponding images under a light microscope (magnification, ×4) were captured at 0 and 24 h. Finally, ImageJ software v1.52a (National Institutes of Health) was used for quantification.

Cell invasion assays were performed using Transwell chambers following the manufacturer's instructions. Briefly, Matrigel (precoating at 37° for 15 min) was diluted using serum-free DMEM. A total of 200 µl of glioma cell (1×10⁵ cells) suspension was seeded into the upper chamber. Subsequently, 600 µl of media supplemented with 20% FBS was added into the lower chamber, followed by incubation for 24 h. A cotton-tipped swab was used to wipe off the cells that did not migrate to the bottom chamber. Cells were stained with 0.1% Crystal Violet Staining Solution (Pierce; Thermo Fisher Scientific, Inc.) at room temperature for 15 min. The chambers were then observed, images were captured and the number of migrated cells were counted under a light microscope (magnification, ×10) in six random fields of view. All assays were independently repeated for three times.

Cells (1×10⁵) were trypsinized and collected by centrifugation at 4°C at 150 × g for 8 min, and apoptosis was analyzed using an Apoptosis Detection kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 250 µl binding buffer was added to the suspended cells, 100 µl of suspension was added into a 1.5-ml Eppendorf tube, along with 5 µl Annexin V-PE and 10 µl 7-AAD, the samples were gently mixed in the dark and left to incubate at room temperature for 15 min before the addition of 400 µl of binding buffer, and analyzed using a FASCAria II (BD Biosciences) flow cytometer and FlowJo software (v7.6.1; FlowJo, LLC) within 1 h.

Following cell lysis with RIPA lysis buffer (Beyotime Institute of Biotechnology), a BCA protein concentration assay kit (Biosharp Life Sciences) and Synergy HTX (BioTek Instruments, Inc.) microplate reader were used to determine the protein concentration. Protein loading buffer (5X; Beijing Zoman Biotechnology Co., Ltd.), was used for equalization of protein loading. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% SDS-PAGE) Gel Quick Preparation kit (Beyotime Institute of Biotechnology) was used for gel production. Samples were boiled at 100°C for 10 min to denature the proteins. Protein samples (100 µg/lane) were separated via SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked with 5% skimmed milk powder for 2 h at room temperature and then rinsed with TBS-Tween (1% Tween-20) for 3 times (10 min each time). The membrane was incubated with primary rabbit antibodies against human F-Box protein 11 (FBXO11; 1:500; cat. no. ab181801; Abcam) and GAPDH (1:1,000; cat. no. AF1186; Beyotime Institute of Biotechnology) for 24 h on a shaker at 4°C, followed by incubation with a HRP-labeled goat anti-rabbit secondary antibody (1:4,000; cat. no. A2028; Beyotime Institute of Biotechnology) at room temperature for 1 h. Signal was detected using enhanced chemiluminescence detection reagent (Beyotime Institute of Biotechnology). Finally, the 4600SF chemiluminescent imaging system (Tanon) was used to semi-quantify the bands. Three independent experiments were performed for each analysis.

TargetScan (www.targetscan.org) was utilized to predict biological targets for miR-181a-5p. Of all these hypothetical targets, FBXO11 was selected for further research.

The 3′-untranslated region (UTR) and mutant 3′-UTR of wild-type FBXO11 were amplified by Wuhan GeneCreate Biological Engineering Co., Ltd., and integrated into the psichenk2.0 (Wuhan Servicebio Technology Co., Ltd.) vector containing the luciferase gene. The plasmids were tested and co-transfected into 293T cells with miR-181a-5p mimics or miR-NC. After 48 h of transfection, the cells were lysed using a Dual-Luciferase^® Reporter Assay kit (Promega Corporation). In the case of firefly luciferase as the internal control, the relative light unit (RLU) value measured by Renilla luciferase was divided by the RLU value measured by firefly luciferase. According to the obtained ratio, the inhibitory effect of miRNA on the target gene was compared, and the luciferase activity was analyzed using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc.).

All statistical analyses were conducted using SPSS 19.0 (IBM Corp.) and GraphPad Prism 8.0 software (GraphPad Software, Inc.). Each experiment was performed in triplicate and repeated for three times. Data were presented as mean ± standard deviation. Independent-samples t-tests were used to analyze significant differences between two groups, while one-way analysis of variance was utilized to estimate significant differences among multiple groups. The least significant difference method was used for multiple comparisons when the variance was equal. The Dunnett's corrective test was employed when the variance was not equal (in the chemotherapeutic drug sensitivity experiments of glioma cells). P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was employed to detect miR-181a-5p expression levels in U251 and U251TR cells and 51 glioma tissues containing primary and relapsed tissues. The data revealed that miR-181a-5p expression was significantly decreased in U251TR cells compared with expression in U251 cells (P<0.01; Fig. 1A). It was also revealed that miR-181a-5p expression was significantly reduced in relapsed GBM tissues after chemotherapy compared with primary GBM tissues (0.5080±0.072 vs. 1.06±0.082, respectively; P<0.01; Fig. 1A). Untransfected U251TR and U251 cells were then used as controls in addition to the mimics NC. miR-181a-5p expression levels were markedly increased in U251TR cells transfected with miR-181a-5p mimics compared with those in the NC group (P<0.01). However, the miR-181a-5p expression levels were significantly decreased in U251 cells transfected with the miR-181a-5p inhibitors compared with the untransfected control group (P<0.01) (all Fig. 1B).

The present study sought to investigate the biological functions of miR-181a. The CCK-8 assays indicated that the miR-181a-5p simulation group had a significantly enhanced sensitivity to TMZ, with a decreased IC₅₀ value in transfected U251TR cells compared with untransfected cells (P<0.01). In contrast, the IC₅₀ value of TMZ in U251 cells transfected with miR-181a-5p inhibitors was notably elevated compared with the control group (P<0.01) (Fig. 2A). The CCK-8 assay demonstrated that the proliferation of drug-resistant glioma cells transfected with the miR-181a-5p mimic was significantly lower compared with that in the NC group and drug-resistant glioma cells (P<0.05; Fig. 2B). The proliferation rate of normal glioma cells transfected with miR-181a-5p inhibitor was higher compared with that in the NC group (P<0.05; Fig. 2B).

The wound healing assay revealed that, compared with the NC and untransfected control group, the U251 cells transfected with the miR-181a-5p inhibitor had a significantly faster 24-h wound closure rate (P<0.001). However, compared with the NC group, the 24 h wound closure rate of U251TR cells transfected with the miR-181a-5p mimic group was significantly less (P<0.001; Fig. 2C). Results of Transwell migration assays revealed that the miR-181a-5p mimics had significantly fewer invasive cells compared with the NC group (P<0.001). In contrast, U251 treatment with miR-181a-5p inhibitor resulted in a significantly higher number of invasive cells compared with the NC group (P<0.001; Fig. 2D). Flow cytometry results showed that compared with the NC and untransfected control group, the percentage of apoptotic U251 cells was significantly higher in the miR-181a-5p mimic group (P<0.001; Fig. 2E). For this experiment, U251 cells were also transfected with the mimic to see whether there was an apoptotic effect. Additionally, similar results were observed in U251TR cells (P<0.001; Fig. 2E). The aforementioned results indicated the inhibitory effects of miR-181a on glioma cells, especially drug-resistant glioma cells.

According to the TargetScan prediction website, miR-181a-5p was predicted to bind to FBXO11, the base complementary region is located at 537–543 of the non-coding region of FBXO11 gene. In the presence of miR-181-5p mimic, the luciferase activity of the FBXO11-wt group was significantly lower than that of the NC group, indicating that miR-181a-5p can target the 3′UTR end of FBXO11 (P<0.05; Fig. 3A). In glioma-resistant cells, the expression of FBXO11 was significantly higher compared with in ordinary glioma cells (P<0.05). When the expression levels of miR-181a-5p were upregulated, the expression levels of FBXO11 in glioma-resistant cells were suppressed compared with untransfected cells (P<0.05) (Fig. 3B). Western blotting demonstrated that FBXO11 expression was markedly upregulated in drug-resistant cells compared with non-resistant cells (Fig. 3C). Additionally, FBXO11 expression was downregulated in drug-resistant glioma cells transfected with miR-181a-5p mimic, while it was upregulated in glioma cells transfected with miR-181a-5p inhibitor compared with in the controls (P<0.001; Fig. 3D).