Primary glioma tissues and adjacent non-tumor tissues were obtained from 36 adult patients who underwent glioma resection at the Department of Neurosurgery, the First Hospital of Jilin University (Changchun, China). None of the patients had received chemotherapy, immunotherapy and radiotherapy prior to surgery. All samples were immediately frozen in liquid nitrogen and stored at −80°C until use. All patients gave written informed consent before surgery. The study protocol and consent procedures were approved by the Ethics Committee of Jilin University (Changchun, China).

Primary normal human astrocytes (NHA) and 4 human glioma cell lines (U251, U87, U118 and LN18) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and were cultured in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco-BRL, Gaithersburg, MD, USA), 100 U/ml penicillin or 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂.

Total RNA was isolated from the cultured cells and frozen tissues using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. For miR-506 expression, total RNA was reversely transcribed into cDNA using One Step PrimeScript miRNA cDNA Synthesis kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Then the expression levels of miR-506 were quantified using taqman miRna assay kits under the ABI 7900 Fast system (both from Applied Biosystems, Foster City, CA, USA). To quantify STAT3, total RNA was reversely transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Dalian, China). The expression levels of STAT3 were quantified by Real-Time PCR Mixture reagent (Takara) under the ABI 7900 Fast system. The primers for STAT3 mRNA were: forward, 5′-GAAGAATCCAACAACGGC-3′ and reverse, 5′-TCACAATCAGGGAAGCAT-3′. U6 and GAPDH were used as internal controls for miRNAs and mRNAs, respectively. Relative expression was calculated using the 2^−∆∆Ct method.

The miR-506 mimic (miR-506) and corresponding miRNA negative control (miR-NC) were purchased form GenePharma Co., Ltd. (Shanghai, China). The STAT3 overexpression plasmid was designed and synthesized by Ribobio Co. (Guangzhou, China). These molecular products were transfected into U87 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Transfection efficiencies were determined in every experiment at 48 h after transfection.

Cell proliferation was measured by mtt assay. In briefly, 2×10³ transfected cells were seeded into 96-well plates and cultured for 24–72 h. After incubation at 37°C for 4 h, followed by removal of the MTT solution, 150 µl of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to each well. Optical density (OD) was detected at a wavelength of 570 nm. All experiments were performed in triplicate.

For the colony formation assay, 1,000 transfected cells were seeded in 6-well plates and cultured for 14 days at 37°C under 5% CO₂. Then the colonies were fixed with 75% ethanol for 10 min, dried and stained with 0.1% crystal violet solution for 10 min. Then images were captured of the colonies, and the number of colonies was counted under a light microscope (Olympus, Tokyo, Japan).

Cells cycle analysis was performed on U87 cells 48 h after transfection. The transfected cells were harvested, washed, fixed in ice-cold 75% ethanol and stored at −20°C for 12 h. The cells were resuspended in PBS containing 25 mg/ml propidium iodide (PI), 0.1% Triton X-100, and 10 mg/ml Rnase and incubated at 4°C for 30 min in the dark. The cells were then analyzed by fluorescence-activated cell sorting (FACS; BD Biosciences, Mansfield, MA, USA).

The transfected cells (2×10⁴) were seeded into 24-well culture plates and cultured for 24 h at 37°C under 5% Co₂. Then an artificial homogeneous wound was created onto the monolayer with a 20-µl sterile plastic micropipette tip. After wounding, the debris was removed by washing the cells with PBS. To visualize the migrating cells and wound healing, images were captured at 0 and 24 h after wounding.

The transfected cells (2×10⁴) were placed into Transwell chambers (8.0-µm pore size; Corning Inc., Corning, NY, USA) coated with Matrigel (BD Biosciences, Bedford, MA, USA) in serum-free medium. DMEM containing 20% FBS in the lower chamber served as the chemoattractant. After the cells were incubated for 48 h at 37°C with 5% CO₂, the cells that had invaded through the membrane were fixed in 90% alcohol and stained with 0.1% crystal violet for 5 min and then photographed. The number of invaded cells was counted in five randomly selected fields under a light microscope (×200; Olympus).

The complimentary sequence of STAT3 3′UTR for miR-506 (STAT3-Wt) and mutated 3′UTR sequence (STAT3-Mut) were synthesized and inserted into the pGL3-control vector (Ambion, Austin, TX, USA) at the NheI and XhoI restriction sites. For the luciferase assays, 1×10⁵ cells were plated in 24-well plates and cultured for 24 h. Then the cells were co-transfected with 100 ng of STAT3-Wt or STAT3-Mut reporter plasmid, and 100 nm of miR-506 mimic or miR-NC using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. At 48 h after transfection, both firefly and Renilla luciferase activities in the cell lysates were determined using the Dual-Luciferase reporter assay system (Promega, Madison, WI, USA). Renilla luciferase was used for normalization.

Cells were harvested and lysed in ice-cold RIPA buffer (Beyotime, Jiangsu, China) according to the manufacturer's instructions. Concentrations of total cellular protein were quantified using the BCA protein assay kit (Vigorous Biotechnology Beijing Co., Ltd., Beijing, China) according to the manufacturer's instructions. Equal amounts of protein lysates (20 µg each lane) were separated by 8–12% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membrane was incubated at 4°C overnight with the following primary antibodies: anti-STAT3 (1:1,000), anti-cyclin D1 (1:2,000) and anti-Bcl-2 (1:1,000) (all from Santa Cruz Biotechnology), anti-MMP-2 (1:1,000; Cell Signaling Technology) and anti-GAPDH (1:5,000; Santa Cruz Biotechnology), followed by the corresponding secondary antibody labeled with HRP and detected by enhanced chemiluminescence (ECL; Cell Signaling Technology). Protein quantity was detected by GAPDH as a loading control.

Twenty female BALB/c mice (4–5 weeks of age) were obtained from the Experiments Animal Center of Changchun Biological Institute (Changchun, China), and maintained under specific pathogen-free (SPF) conditions. All procedures were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. All animal protocols were approved by the Institutional Animal Care and Use Committee of Jilin University (Changchun, China).

U87 cells (2×10⁶) stably expressing miR-506 or miR-NC were directly injected subcutaneously into the flanks of nude mice (n=10), respectively. Tumor volume (TV) was determined by caliper every week according to the formula: TV (mm³) = 1/2 × width² × length. After 5 weeks of inoculation, all mice were sacrificed and the tissues were removed and weighed. Part of the tumor tissues were harvested for analysis of the expression of miR-506 and STAT3.

Data from at least three independent experiments are expressed as the mean ± SD (standard deviation). The differences between two groups were analyzed using the two-sided Student's t-test, and analysis of more than two groups was performed using one-way ANOVA followed by a Tukey's post hoc test. All data were analyzed using the GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA) and the SPSS 19.0 software (SPSS, Chicago, IL, USA). P<0.05 was used to indicate a statistically significant difference.

The expression of miR-506 was detected in 36 pairs of human glioma and adjacent normal tissues by real-time quantitative RT-PCR (qRT-PCR). As shown in Fig. 1A, we found that the relative expression levels of miR-506 were significantly lower in the glioma tissues than levels in the adjacent normal tissues (P<0.01). In addition to glioma tissues, endogenous expression of miR-506 was detected in four human glioma cell lines (U251, U87, U118 and LN18) and normal human astrocytes (NHAs). It was found that the miR-506 expression in the four glioma cell lines was significantly reduced relative to that in the NHAs (Fig. 1B). The U87 cell line, which possessed the lowest level of miR-506 expression among the four cell lines, was therefore selected for the subsequent studies.

The decreased expression of miR-506 in glioma tissues and cell lines inspired us to hypothesize that miR-506 is a tumor suppressor in glioma. To test the role of miR-506 in glioma growth, miR-506 or miR-NC was transfected into U87 cells and cultured for 48 h, and then miR-506 expression was determined by qRT-PCR. Our results showed that the intracellular level of miR-506 was higher in the U87 cells transfecting with the miR-506 mimic compared with the levels in cells transfected with miR-NC (Fig. 2A). Meanwhile, cell proliferation and colony formation were determined in the U87 cells after transfection of miR-506 or miR-NC. We found that overexpression of miR-506 significantly inhibited cell proliferation (Fig. 2B) and colony formation (Fig. 2C) in the U87 cells (P<0.05). As proliferation is directly connected to cell cycle distribution, the effect of miR-506 on cell cycle progression was also analyzed in the U87 cells. As expected, the percentage of G0/G1 phase cells was increased, and the percentage of S phase cells was decreased in the U87 cells transfected with the miR-506 mimic compared to the percentage in the cells transfected with miR-NC (P<0.05, Fig. 2D). These results suggest that miR-506 inhibits glioma cell growth in vitro.

To reveal the biological role of miR-506 on migration and invasion, cell migration and invasion abilities were determined in the U87 cells transfected with the miR-506 mimic or miR-NC by wound healing and invasion chamber assays, respectively. It was found that overexpression of miR-506 significantly inhibited the migration (Fig. 3A) and invasion (Fig. 3B) capacities in the U87 cells.

To understand how miR-506 regulates cell growth and metastasis, we used two algorithms (TargetScan and miRanda) to help identify miR-506 target genes. STAT3 was selected as the potential target of miR-506, since STAT3 has been found to be involved in the tumorigenesis and metastasis of glioma (19,20). To further confirm whether STAT3 is a direct target of miR-506, a human STAT3 3′UTR fragment containing the binding sites of miR-506 or the mutant sites (Fig. 4A) were cloned into the pGL3 vector, and the miR-506 mimic or miR-NC were co-transfected into U87 cells for luciferase activity. It was found that overexpression of miR-506 markedly suppressed the luciferase activity of the STAT3-Wt 3′UTR, without having an effect on STAT3-Mut 3′UTR in the U87 cells (Fig. 4b). We further found that the mRNA and protein levels of STAT3 were decreased in the U87 cells transfected with miR-506 compared with the miR-NC group (Fig. 4C and D). In addition, we found that overexpression of miR-506 inhibited STAT3 downstream protein expression, such as cyclin D1, Bcl-2 and MMP-2 (Fig. 4D).

We also examined the expression of STAT3 in glioma specimens and the corresponding non-cancerous tissues from 36 glioma patients by qRT-PCR. It was found that the STAT3 mRNA expression level was increased in the glioma tissues compared to that in the paired non-cancerous tissues (Fig. 5A), and was negatively correlated with miR-506 (Fig. 5B; r=−0.632, P<0.001).

We further aimed to ascertain whether overexpression of STAT3 could reverse the suppressive effect of miR-506. U87 cells were transfected with the miR-506 mimic or miR-NC, followed by transfection with the STAT3 overexpression plasmids. The overexpression of STAT3 at the mRNA level (Fig. 6A) and protein level (Fig. 6B) was validated by qRT-PCR and western blotting assay, respectively. In addition, our results revealed that overexpression of STAT3 in the U87 cells attenuated the effect of miR-506 on cell proliferation, colony formation, migration and invasion (Fig. 6C–F). Taken together, these results indicate that the tumor-suppressor role of miR-506 is mediated by targeting STAT3.

The in vitro study indicated that miR-506 inhibits glioma cell growth, we therefore investigated whether miR-506 suppresses tumor growth in vivo. The human U87 cells stably expressing miR-506 or miR-NC were implanted subcutaneously into nude mice to allow tumor formation. At 5 weeks post-injection, the mice were sacrificed, and tumor tissues were extracted. Our results showed that miR-506-expressing U87 tumors were significantly smaller than that of miR-NC-expression U87 tumors (Fig. 7A). The average volume and weight of the miR-506-expressing U87 tumors were significantly decreased compared with the volume and weight of the miR-NC-expressing U87 tumors (both P<0.01, Fig. 7B and C). Furthermore, the expression of miR-506 and STAT3 in xenograft tumor tissues was determined. It was found that miR-506 expression was upregulated (Fig. 7D), while STAT3 expression at the mRNA and protein level was decreased in the miR-506-expressing U87 tumors (Fig. 7E and F). These results indicate that miR-506 suppresses glioma growth in vivo by targeting STAT3.