A total of 20 glioma tissues resected from patients with glioma (11 male and 9 female; mean age ± SD, 55.3±12.5 years; age range, 23–85 years) and 14 normal tissues from brains of traffic accident victims (8 male and 6 female; mean age ± SD, 51.2±15.4 years; age range, 18–82 years) were acquired. The tissues were obtained from the Department of Neurosurgery of Chenzhou First People's Hospital (Chenzhou, China) between January 2016 and December 2018. Samples were frozen in liquid nitrogen immediately after collection. Based on the World Health Organization (WHO) classification system (16), two clinical pathologists pathologically diagnosed and divided the glioma specimens into two groups: Low-grade and high-grade. The low-grade group contained 9 stage I–II tumors, while the high-grade group comprised 11 stage III–IV tumors. Patients had not received chemotherapy or radiotherapy prior to surgical resection. Experiments were performed according to the principles of the Declaration of Helsinki. Experimental protocols were approved by Chenzhou First People's Hospital and written informed consent was obtained from all patients or their relatives prior to participation in the study.

Normal human astrocytes (NHA), glioma LN229, A172 and U251 cell lines, and a glioblastoma cell line of unknown origin, U87 (cat. no. HTB14), were purchased from the American Type Culture Collection. Cells were cultured in DMEM (HyClone; Cytiva) supplemented with 10% FBS (HyClone; Cytiva), and maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Small interfering RNA (siRNA/si) sequences targeting SNHG7 (si-SNHG7-1, 5′-GCCGCUUGUGUU CUUGAUUTT-3′; and si-SNHG7-2, 5′-CCTCTGGTGCCUCGUUCUGGAAACGAUTT-3′) and scrambled si-negative control (NC; 5′-CCCAGUAAUAGGACACCAATT-3′) were purchased from Guangzhou RiboBio Co., Ltd. The miR-138-5p inhibitor (5′-CACACUCGGGUCGAAUGAAGGA-3′), miR-138-5p mimic (5′-AGCUGGUGUUGUGAAUCAGGCCG-3′), scrambled mimic NC (5′-UUCUCCGAACGUGUCACGU-3′) and scrambled inhibitor NC (5′-CAGUACUUUUGUGUAGUA CAA-3′) were obtained from Shanghai GenePharma Co., Ltd. Cell transfection was performed using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Total RNA was extracted from cells and tissues using TRIzol^® and TRIzol™ LS reagent (both Invitrogen; Thermo Fisher Scientific, Inc.), respectively, according to the manufacturers' protocols. Total RNA was reverse transcribed into cDNA to analyze lncRNA and mRNA expression using the PrimeScript™ RT reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. qPCR was subsequently performed using a TB Green™ Premix Ex Taq™ II kit (Takara Bio, Inc.), with GAPDH as the internal control. The PCR was performed using the following conditions: Initial denaturation at 95 °C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec, with final annealing and extension at 60°C for 60 sec and 95°C for 15 sec. Total RNA was reverse transcribed into cDNA to determine miRNA expression using a miRcute Plus miRNA First-Strand cDNA kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's protocol. qPCR was subsequently performed using a miRcute Plus miRNA qPCR kit (SYBR Green) (Tiangen Biotech Co., Ltd.), with U6 as the internal control. qPCR was performed on an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following primer pairs (Sangon Biotech Co., Ltd.) were used for qPCR: SNHG7 forward, 5′-GTTGGGGTGTTGGCATTCTTGTT-3′ and reverse, 5′-TGGTCAGCCTGGTCACTCTGG-3′; EZH2 forward, 5′-AATCAGAGTACATGCGACTGAGA-3′ and reverse, 5′-GCTGTATCCTTCGCTGTTTCC-3′; GAPDH forward, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′; miR-138-5p forward, 5′-AGCTGGTGTTGTGAATCAGGCCG-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; and U6 forward, 5′-CTC GCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGA ATTTGCGT-3′. The miR-138-5p reverse primer was obtained using the miRcute Plus miRNA qPCR kit (SYBR Green). Expression levels were quantified using the 2^−ΔΔCq method (17).

Nucleic and cytoplasmic fragments were extracted from cells using a PARIS™ kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Briefly, collected cells were lysed in cell fractionation buffer, then the nucleic and cytoplasmic fractions were isolated by centrifugation. The supernatant containing the cytoplasmic fraction was transferred into a clean tube in the absence of RNase. Disruption buffer was used to lyse the pellet containing the nucleic fraction, which was subsequently mixed with the cytoplasmic fraction and lysis/binding solution. Subsequently, 100% ethanol was added the mixture, and the sample was filtered and washed with washing solution. The elution solution was used to obtain cytoplasmic and nucleic mRNAs. U6 and GAPDH were used as the positive controls for the nucleic and cytoplasmic extracts, respectively.

Prediction of putative binding sites complementary to SNHG7 in miR-138-5p was performed using the lncRNASNP2 database (http://bioinfo.life.hust.edu.cn/lncRNASNP). Putative targeting sequences between miR-138-5p and EZH2 were predicted using TargetScan 7.2 (http://www.targetscan.org).

Glioma cells were cultured in 96-well plates and transfected as aforementioned. Following incubation for 24, 48 or 72 h, 20 µl CCK-8 (Dojindo Molecular Technologies, Inc.) was added to each well for 4 h. The absorbance was measured at a wavelength of 490 nm using a Elx800 microplate reader (BioTek Instruments, Inc.; Agilent Technologies, Inc.). According to the absorbance values, cell proliferation was calculated as a percentage of the control.

Following transfection for 48 h, U87 and A172 cells were collected for cell cycle analysis. Briefly, cells were fixed in 75% ethanol overnight at −20°C and then stained with PI (BD Biosciences) for 30 min in the dark at 37°C. The cell cycle distribution was analyzed using a flow cytometer (Attune NxT; Thermo Fisher Scientific, Inc.). All data were analyzed using FlowJo v10 software (FlowJo LLC). All experiments were independently performed in triplicate.

Total protein was extracted from glioma cells using a Protein Extraction kit (Nanjing KeyGen Biotech Co., Ltd.) and quantified using a BCA protein assay kit (Sigma-Aldrich; Merck KGaA). Proteins (40 µg/lane) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked with 5% skimmed milk for 1.5 h at room temperature. Membranes were subsequently incubated with the following primary antibodies at 4°C overnight: Rabbit polyclonal anti-EZH2 (1:2,000; cat. no. 21800-1-AP; ProteinTech Group, Inc.) and rabbit polyclonal anti-GAPDH (1:1,000; cat. no. 25778; Santa Cruz Biotechnology, Inc.). Following the primary antibody incubation, the membranes were incubated with an HRP-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab6721; Abcam) at room temperature for 2 h. Protein bands were visualized using the ECL reagent (EMD Millipore).

Following the prediction of the putative binding sites of miR-138-5p in SNHG7/EZH2, pMIR-REPORT vectors (Guangzhou RiboBio Co., Ltd.) containing wild-type (WT) or mutant (MT) 3′-UTR sequences of SNHG7 or EZH2 were constructed. Cells were co-transfected with miR-138-5p mimic or mimic NC and SNHG7/EZH2-WT or SNHG7/EZH2-MT for 48 h using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Relative luciferase activity was measured using a Luc-Pair™ Duo-Luciferase assay kit (Shanghai Yeasen Biotechnology Co., Ltd.).

The RIP assay was performed using a Magna RNA-Binding Protein Immunoprecipitation kit (EMD Millipore) according to the manufacturer's protocol. Whole cell lysates were incubated with anti-IgG negative control antibody (derived from normal mice; EMD Millipore) or anti-Argonaute 2 (Ago2) antibody (from humans; 1:50; EMD Millipore) and RIP buffer containing magnetic beads. Each sample was subsequently incubated with proteinase K buffer. The immunoprecipitated RNAs were isolated and SNHG7 and miR-138-5p expression was detected using RT-qPCR, as aforementioned.

Transfected cells were cultured in 6-well plates overnight in medium supplemented with 10% FBS at 37°C. Subsequently, cells were incubated for 14 days at 37°C. Following the incubation, cells were fixed with methanol for 10 min at room temperature and stained with 0.1% crystal violet for 20 min at room temperature. Colonies were counted under a light microscope.

Statistical analysis was performed using SPSS 21.0 software (IBM Corp.) and data are presented as the mean ± SD of three independent experiments. Statistical differences between two groups were determined using an unpaired two-tailed Student's t-test, while statistical differences between multiple groups were determined using a one-way ANOVA followed by Tukey's post hoc test. Spearman's rank correlation was used for correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

To determine the role of SNHG7 in glioma, the expression levels of SNHG7 in tissues from normal brains, low-grade glioma (stage I–II) and high-grade glioma (stage III–IV) were analyzed. The results revealed that SNHG7 expression levels were significantly upregulated in glioma tissues compared with in normal tissues; in addition, the expression levels of SNHG7 were significantly increased in high grade glioma (stage III–IV) tissues compared with in low-grade glioma (stage I–II) tissues (Fig. 1A). Thus, SNHG7 expression may be positively associated with tumor grade. The expression levels of SNHG7 were also determined in cell lines, including NHAs, U251, LN229, U87 and A172 cells. Compared with in NHAs, SNHG7 expression was significantly upregulated in glioma cell lines, including in U251 (P<0.05) and LN229 (P<0.05), and especially in U87 (P<0.01) and A172 (P<0.01) (Fig. 1B). Therefore, the latter two cell lines (U87 and A172) were chosen for subsequent experiments. RT-qPCR analysis revealed that SNHG7 expression was successfully knocked down in U87 and A172 cell lines following transfection with both si-SNHG7-1 and si-SNHG7-2 (Fig. 1C). The results of the CCK-8 assay demonstrated that SNHG7-knockdown significantly decreased cell proliferation in U87 and A172 cell lines compared with in cells transfected with si-NC (Fig. 1D). In addition, the results of the colony formation assay further indicated that SNHG7-knockdown significantly decreased the number of colonies formed from glioma cells compared with those formed from glioma cells transfected with si-NC (Fig. 1E). Flow cytometry analysis of the cell cycle distribution revealed that SNHG7-knockdown arrested U87 and A172 cells in the G₁ phase (Fig. 1F).

To determine the underlying mechanism of SNHG7 in glioma, RT-qPCR was used to analyze SNHG7 expression in the nucleus and cytoplasm. The results demonstrated that SNHG7 was mainly localized in the cytoplasm of U87 and A172 cell lines (Fig. 2A). Based on these results, it was hypothesized that SNHG7 may function as a ceRNA. Bioinformatics analysis using the lncRNASNP2 database predicted that miR-138-5p contained potential binding sites for SNHG7 (Fig. 2B). miR-138-5p expression levels were upregulated in glioma cells following the transfection of U87 and A172 cells with si-SNHG7-1 compared with si-NC (Fig. 2C). miR-138-5p expression was significantly increased following transfection with the miR-138-5p mimic compared with the miR-NC (Fig. 2D). The results of the dual luciferase reporter assay demonstrated that the relative luciferase activity was significantly decreased following the co-transfection of cells with the miR-138-5p mimic and SNHG7-WT, but not SNHG7-MT vectors (Fig. 2E). These findings indicated the potential association between SNHG7 and miR-138-5p. It was subsequently investigated whether miR-138-5p and SNHG7 existed in the same RNA-induced silencing complex using a RIP assay. The results indicated significant enrichment of SNHG7 in the anti-Ago2 group compared with the anti-IgG group; similar results were obtained for miR-138-5p (Fig. 2F). Additionally, miR-138-5p expression was analyzed in 14 normal brain tissues, nine low-grade glioma (stage I–II) tissues and 11 high-grade glioma (stage III–IV) tissues. miR-138-5p expression levels were significantly downregulated in glioma tissues compared with in normal tissues, and significantly lower expression levels of miR-138-5p were observed in high-grade tissues compared with in low-grade tissues (Fig. 2G). Furthermore, Pearson's correlation analysis identified a negative correlation between miR-138-5p and SNHG7 expression in glioma tissues (Fig. 2H). These findings suggested that SNHG7 may directly target miR-138-5p.

To further investigate whether SNHG7 exerted biological effects on glioma cells by targeting miR-138-5p, miR-138-5p expression was knocked down in U87 and A172 cells by transfection with a miR-138-5p inhibitor, and the transfection efficiency was analyzed using RT-qPCR (Fig. 3A). Compared with the transfection with si-SNHG7 alone, miR-138-5p expression was downregulated to a further extent in U87 and A172 cells co-transfected with si-SNHG7 and miR-138-5p inhibitor (Fig. 3B). The results of the CCK-8 assay demonstrated that transfection with the miR-138-5p inhibitor reversed the SNHG7-knockdown-induced inhibition of proliferation in U87 and A172 cells (Fig. 3C). Notably, the same trend was also observed in the colony formation assay (Fig. 3D). Compared with the si-SNHG7 group, the number of cells in the G₁ phase of the cell cycle was decreased in the si-SNHG7 and miR-138-5p inhibitor group (Fig. 3E). Overall, these results suggested that transfection with the miR-138-5p inhibitor may reverse the effect of SNHG7-knockdown in glioma cells.

miRNAs are known to act at the post-transcriptional level by directly regulating target gene expression and modulating subsequent protein expression (18). To determine whether SNHG7 regulated EZH2 expression by functioning as a ceRNA and sponging miR-138-5p, bioinformatics analysis was first performed using TargetScan, which predicted the presence of potential miR-138-5p targeting sites in EZH2 (Fig. 4A). According to the results of the dual luciferase reporter assay, the relative luciferase activity was significantly decreased by co-transfection of the miR-138-5p mimic and EZH2-WT vector in U87 and A172 cells, while the relative luciferase activity was unaffected in U87 and A172 cells co-transfected with miR-138-5p mimic and EZH2-MT (Fig. 4B). EZH2 mRNA expression in glioma and normal tissues was subsequently determined using RT-qPCR. The results revealed that EZH2 mRNA expression was significantly upregulated in glioma tissues compared with normal tissues; moreover, compared with in low-grade glioma (stage I–II) tissues, EZH2 mRNA expression was significantly upregulated in high-grade glioma (stage III–IV) tissues (Fig. 4C). In addition, EZH2 mRNA expression was negatively correlated with miR-138-5p expression (Fig. 4D). The role of miR-138-5p in modulating the effects of SNHG7 on EZH2 were further investigated. SNHG7-knockdown markedly downregulated the expression levels of EZH2; however, the expression levels were reversed following the transfection with the miR-138-5p inhibitor (Fig. 4E). These findings suggested that SNHG7 may serve as a ceRNA and may sponge miR-138-5p to modulate EZH2 expression and promote glioma cell proliferation (Fig. 4F).