Human GBM cell lines (U251, T98G, LN229, SHG44 and A172) were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Normal human astrocyte cells (NHA) cells were obtained from the American Type Culture Collection. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and incubated under humidified conditions at 37°C and 5% CO_2.

Human GBM samples and adjacent normal brain samples were collected from 50 patients undergoing surgical resection at Tianjin Medical University General Hospital (Tianjin, China) between June 2013 and June 2017. These glioma samples were from 33 males and 17 females with age ranging from 23–75 years (median, 49 years). All GBM samples were examined by two senior pathologists. Written informed consent was obtained from all patients prior to enrollment in the study; the study was approved by The Institutional Review Board of Tianjin Medical University General Hospital.

The edgeR software package (Bioconductor) in R Studio 3.5.1 (https://www.rstudio.com/) was used to analyze the aberrantly expressed lncRNAs in normalized gene expression profile data from The Cancer Genome Atlas (TCGA) GBM database (24,25). RNA sequencing data of GBM tissues and normal brain tissues were collected from the TCGA database (http://cancergenome.nih.gov), and 162 GBM cases were detected in all. For the normalized gene expression profile data, the edge R package of R software was used to analyze significantly aberrantly expressed lncRNAs at the level: moderately to GBM samples vs. normal samples. A log fold change >2 and false-discovery rate P<0.05 was selected as significantly cutoff values. Significantly enriched gene sets were investigated. The clinical data were obtain from Gene Expression Profiling Interactive Analysis (GEPIA) dataset (http://gepia.cancer-pku.cn/). GO term enrichment analysis was identified using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.8 (https://david.ncifcrf.gov/).

Total RNA was extracted from clinical tissues and GBM cell lines using TRIzol^® reagent according to the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.). A NanoDrop spectrophotometer was used to determine the concentration of extracted RNA. RT-qPCR was performed in triplicate on an ABI 7500 HT fast real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using the SYBR^® Premix Ex Taq™ II kit (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. Primers were: Unigene56159 forward, 5′-GTGAAAAGAAACATTCGAGTGT-3′, and reverse, 5′-TGAAGTAAGCAGGAAAGGGGGA-3′; miR-194-5p forward, 5′-AGTGTGACGTGACATCCGT-3′, and reverse, 5′-GCAGCTCAGTAACAGTCCGC-3′; PCNA forward, 5′-TTTGGTGCAGCTCACCCTG-3′, and reverse. 5′-CGCGTTATCTTCGGCCCTTA-3′; MMP-2 forward, 5′-CAGGACATTGTCTTTGATGGCATCGC-3′, and reverse, 5′-TGAAGAAGTAGCTATGACCACCGCC-3′; MMP-9 forward, 5′-ATCCCCCACCTTTACCA-3′, and reverse 5′-TCAGAACCGACCCTACAA-3′; U6 forward, 5′-TGTGGGCATCAATGATTTGG-3′ and reverse, 5′-ACACCATGTATCCGGGTCAAT-3′; GAPDH forward 5′-CCATGTTCGTCATGGTGTG-3′ and reverse, 5′-GGTGCTAAGCAGTTGGTGGTG-3′. The cycling conditions were: 95°C for 10 min, then 40 cycles at 95°C for 15 sec, and 60°C for 60 sec. U6 was used as a control to normalize the miR-194-5p expression. Relative expression levels were calculated using the 2^−ΔΔCq method and normalized to the internal reference gene (26).

Unigene56159 small interfering RNA (siRNA) and the negative control (si-NC), miR-194-5p mimic or inhibitor, and their respective negative control (miR-NC; 20 µM)were obtained from Shanghai GenePharma Co., Ltd. Sequences were: Unigenge56159 siRNA forward, 5′-GGAGUGAGAUGUCAAAUAACA-3′, and reverse, 5′-UUAUUAGACAUCACACUCCAU-3′; si-NC forward, 5′-UUCUACGAAUGUGUCACCUTT-3′, and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′; miR-194-5p mimics forward, 5′-UGUAACAGCAACUCCAUGUGGA-3′, and reverse, 5′-CACAUGGAGUUGCUGUUACAUU-3′; miR-194-5p inhibitor forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACUUCGGAGAATT-3′; miR-NC forward, 5′-CAGUACUUUUGUGUAGUACAA-3′ and reverse, 5′-UUAACUAAUAUUUCAUCCAUA-3′. The Lipofectamine^® 2000 kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transfection according the manufacturer's protocol at 37°C for 4 h. Then the supernatant was removed and fresh medium was added. The sample was collected for experiment at 24 h after transfection.

The TargetScan database (www.targetscan.org) and the starBase database (http://starbase.sysu.edu.cn/) were used to investigate target miRNAs interacting with Unigene56159 through complementary sequences. From the statistically relevant microRNAs, the top 5 in terms of their prediction score were selected, including miR-194-5p, miR-124-3p, miR-130a-3p, miR-148a-3p and miR-543: miR-194-5p achieved the highest score in two databases. The human Unigene56159 Luc-reporter (Genepharm, Inc.) was transfected into the ligation site of the Unigene56159 3′-untranslated region (UTR) PCR product. U251 cells were cultured in 6-well plates at 3×10⁵ cells/wells and co-transfected with pmirGLO-Unigene56159-3′UTR-wild-type (WT) or pmirGLO-Unigene56159-3′UTR-mutant (MUT), and miR-194-5p or miR-NC mimics. Then cells was incubated in a 37°C, 5% CO₂ humidified atmosphere for 4 h and then supernatant was removed. At 48 h post-transfection, luciferase activity was detected using the Luciferase Assay system (Promega Corporation), and normalized to Renilla luciferase activity.

Transfected cells were collected 24 h post-transfection and cultured at a density of 2×10³ cells/well in 96-well plates. Proliferation assays were performed using a Cell Counting Kit-8 (Beyotime Institute of Biotechnology) according to manufacturer's protocols, and measured at an absorbance of 450 nm at 0, 24, 48 and 72 h (Infinite F50, Tecan Group, Ltd.).

U251 and T98G Cells (~200) were seeded into 6-well plates and cultured in 10% FBS at 37°C for 12 days to allow for colony formation. Subsequently, cells were fixed with 4% polyoxymethylene for 10 min at room temperature before being stained with 10% Giemsa (30 min) (Sigma-Aldrich; Merck KGaA). The number of colonies (>50 cells) was calculated under light microscope (Nikon Corp., Tokyo, Japan).

U251 and T98G cells (5×10⁴) were seeded in the upper chambers of Transwell plates precoated with Matrigel^® (Corning Life Sciences) in serum-free DMEM (Gibco; Thermo Fisher Scientific, Inc.). DMEM supplemented with 20% FBS was added to the lower chambers and the cells were incubated at 37°C in a 5% CO₂ humidified atmosphere for 24 h. Following incubation, non-invasive cells in the upper chamber were removed using a cotton swab. The invasive cells in the lower chamber were fixed using 4% paraformaldehyde for 10 min and stained with hematoxylin and eosin for 5 min at room temperature. Stained cells were manually counted under a light microscope (Nikon Corporation; magnification ×100).

Total protein was extracted from patient tissue samples (approximately 250 mg/case) and cell lines (U251 and T98G) using RIPA buffer (Pierce; Thermo Fisher Scientific, Inc.). Protein concentrations were determined using the BCA protein assay kit (Bio-Rad Laboratories, Inc.), and 40 µg protein was separated by 10% SDS-PAGE. Separated proteins were transferred onto a PVDF membrane (EMD Millipore; Merck KGaA) and blocked for 1 h at room temperature with TBS containing 5% non-fat milk (w/v). The membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-proliferating cell nuclear antigen PCNA (Rabbit polyclonal antibody, cat. no. 10205-2-AP, 1:500; Wuhan Sanying Biotechnology), rabbit MMP-2 (Rabbit polyclonal antibody, cat. no 10373-2-AP, 1:500; Wuhan Sanying Biotechnology), rabbit anti-MMP-9 (Rabbit polyclonal antibody, cat. no 10375-2-AP, 1:500; Wuhan Sanying Biotechnology) and mouse anti-GAPDH (Mouse Monoclonal Antibody, cat. no. sc-47724, 1:1,000; Santa Cruz Biotechnology, Inc.), as the loading control. Images of the western blots were captured using a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Inc.).

U251 and T98G cell (1×10⁵) were culture on cell slides (glass slides stained with 0.1% poly-L-Lysine overnight)and then fixed with 4% paraformaldehyde for 20 min and incubated with 0.1% Triton X-100 for 10 min at room temperature. Slides were subsequently washed with PBS twice for 5 min and incubated with 5% BSA for 1 h at room temperature (CAS Number:9048-46-8, Sigma Aldrich; Merck KGaA), then incubated with primary antibodies against MMP-2 (Rabbit polyclonal antibody, cat. no. 10373-2-AP, 1:100; Wuhan Sanying Biotechnology) at 4°C overnight. Following primary antibody incubation, slides were incubated with fluorescence-labeled rabbit secondary antibody (Rhodamine TRITC-conjugated Goat Anti-Rabbit IgG, Catalog No.: SA00007-2, 1:100, Wuhan Sanying Biotechnology) at room temperature for 1 h. The nuclei were stained with DAPI for 10 min. Slides were visualized using a fluorescent microscope (magnification ×400).

One-way ANOVA with post hoc Tukey's test or Student's t-test was used to compare between groups. Survival curves were drawn using the log-rank test with GraphPad Prism 5.0 (GraphPad Software, Inc.). The correlation between Unigene56159 expression and the clinicopathological characteristics of patients with glioma was analyzed using the χ² test or Fisher's exact test. Statistical analysis was performed using SPSS 19.0 (IBM Corp.) and data was displayed as mean ± SD. Experiments were independently conducted in triplicate. P<0.05 was considered to indicate a statistically significant difference.

A log2-fold change (FC) of >2 and a false discovery rate (P<0.05) were selected as the cut-off values based on the Benjamini-Hochberg method (27). The expression of Unigene56159 was obtained and 151 cases of valid data collected. The top 20 differentially expressed lncRNAs meeting this criteria were collected, and these lncRNAs were identified according to the level of log2FC (Fig. 1A). Among the differentially expressed lncRNAs, Unigene56159 expression levels were the highest in GBM, demonstrating markedly upregulated expression levels compared with normal brain tissue (Fig. 1A and B). To determine putative functions of Unigene56159, the associated gene expression profiles collected from the GO database were analyzed. The most prominent biological processes included cell migration, cell adhesion and zinc-finger activity (Fig. 1C). Moreover, the clinical data collected from the TCGA database (GEPIA) revealed that high levels of Unigene56159 in GBM were associated with a poorer overall survival compared with low Unigene56159 expression levels according to the median survival time of patients (http://gepia.cancer-pku.cn/) (Fig. 1D). The expression level of Unigene56159 was also significantly associated with patient's Karnofsky performance scale scores from the data of our clinical sample (n=50; Table I; P=0.018). Subsequently, RT-qPCR analysis was used to assess Unigene56159 expression levels in GBM and adjacent normal brain tissues for 50 patients from the present study. Unigene56159 expression was significantly increased in the GBM tissue compared with normal brain tissue (Fig. 1E). These findings suggested that Unigene56159 may function as an oncogene for GBM progression.

The expression of Unigene56159 was evaluated in GBM cell lines (U251, LN229, T98G, A172 and SHG44) compared with the normal astrocyte cell line (NHA) by RT-qPCR assay (P-values=0.0101, 0.0435, 0.0094, 0.02436 and 0.03435; Fig. 2A). It was noted that the Unigene56159 level were higher in U251 and T98G than in other cell lines. To identify the effect of Unigene56159 on the proliferative and invasive ability of GBM, U251 and T98G cells were selected and transfected with either si-Unigene56159 to knockdown Unigene56159 gene expression or with the si-NC. The transfection efficiency of si-Unigene56159 was high, exhibiting significantly decreased expression levels in the si-Unigene56159-transfected cells compared with the si-NC in both U251 and T98G cell lines (Fig. 2B). In both GBM cell lines, the knockdown of Unigene56159 resulted in a significant decrease compared with si-NC group at the similar time points in their proliferative capacity following 24–72 h (Fig. 2C). Furthermore, Unigene56159 silencing significantly reduced the number of colonies (>50 cells) (Fig. 2D), in addition to the invasive capacity in both U251 and T98G cell lines compared with respective si-NC transfected cells (Fig. 2E). These data demonstrated a suppressive function of both proliferative and invasive processes following Unigene56159 silencing in GBM cells in vitro.

Invasion and proliferation serve a vital role in tumor progression (28,29). Using data from the TCGA database, the expression of Unigene56159 was obtained and 151 cases of valid data collected and a significant positive correlation was identified between Unigene56159 expression and expression levels of the tumor proliferation marker; PCNA (r=0.3034; P=0.0003) and invasion markers MMP-2 (r=0.3072; P=0.0001) and MMP-9 (r=0.2936; P=0.0012; Fig. 3A).

To further determine whether reduced Unigene56159 expression may affect the proliferative and invasive capacity of GBM, mRNA and protein expression levels of PCNA, MMP-2 and MMP-9 were assessed using RT-qPCR and western blot analysis, respectively, and MMP-2 levels were also detected by immunofluorescence. As shown in Fig. 3B, Unigene56159 knockdown decreased the protein level of proliferation and invasion markers and inhibited the mRNA expression of PCNA, MMP-2 and MMP-9 (Fig 3C). Then PCNA, MMP-2 and MMP-9 were costained in U251 and T98G cells with immunofluorescence staining assays, the result further showed that unigene56159 silencing suppressed the MMP-2 expression in glioma cells (Fig. 3D). These results demonstrated that Unigene56159 silencing significantly decreased the expression levels of both proliferation- and invasion-related biomarkers. Taken together, these results suggested that Unigene56159 may be associated with both proliferation and invasion in GBM.

The complementary sequence between Unigene56159 and miR-194-5p was identified using both the TargetScan database and starBase database. A dual-luciferase reporter assay was subsequently used to identify the putative miR-194-5p target site (Fig. 4A). Transfection efficiency was evaluated by increasing or decreasing miR-194-5p expression levels in U251 and T98G cell lines (Fig. 4B). Notably, miR-194-5p significantly decreased the luciferase activity of the Unigene56159-3′UTR-WT U251 cells, but not the Unigene56159-3′UTR-MUT U251 cells (Fig. 4C). To determine the role of Unigene56159 on the expression of miR-194-5p, U251 and T98G cells were transfected with siRNA-Unigene56159. The expression of miR-194-5p significantly increased in both U251 and T98G cell lines upon Unigene56159 knockdown compared with si-NC transfected cells (Fig. 4D). To further explore this, miR-194-5p expression levels in the 50 GBM samples were compared with normal brain samples and it was found that the expression was significantly decreased in GBM compared with normal tissue (P<0.001; Fig. 4E). In addition, the level of Unigene56159 expression was significantly negatively correlated with miR-194-5p in GBM patient samples (r=−0.4046; P=0.0036; Fig. 4F). These data confirmed that miR194-5p represses Unigene56159 in glioma cells.

There were 162 cases of miR-194-5p expression with survival time in TCGA database. The Kaplan–Meier curve demonstrated that a high level of miR-194-5p was positively correlated with the overall survival of patients with glioma from GEPIA database (Fig. 5A). In GBM cell lines, miR-194-5p was found to be significantly decreased compared to NHA cells (Fig. 5B); based on this, U251 cells were used to conduct rescue experiments. The level of miR-194-5p increased when transfected with si-Unigene56159 compared with si-NC. However, after adding the miR-194-5p inhibitor in the si-Unigene56159 group, the miR-194-5p level decreased compared with miR-inhibitor NC (Fig. 5C). Furthermore, a colony formation assay indicated that a miR-194-5p inhibitor impeded the suppression of proliferative ability following Unigene56159 knockdown (Fig. 5D). Tumor invasive ability was also decreased following Unigene56159 knockdown, whereas co-transfection with miR-194-5p inhibitors impeded these effects in U251 cells (Fig. 5E). Altogether, these data demonstrated that miR-194-5p may abrogate the malignant behavior of Unigene56159 in GBM cells.