The study was approved by the Ethics Committee of the First Hospital of Changsha (Changsha, China). A total of 63 glioma tissues and 12 normal brain tissues were obtained from the hospital between July 2010 and March 2012. The tissues were resected during surgery. Written informed consents were obtained from all patients. The clinical information of glioma patients included in the current study is summarized in Table I. The glioma patients were diagnosed according to the World Health Organization (WHO) stage and grading system (14). The Karnofsky performance scale (KPS) runs from 100 to 0, where 100 is ‘perfect health’ and 0 is death. Its purpose is to allow physicians to evaluate a patient's ability to survive chemotherapy for cancer (15). All patients involved in this study received no treatment prior to surgical resection. All tissue samples were immediately snap-frozen and stored in liquid nitrogen until further use.

Normal human astrocytes were purchased from the IBS Cell Bank of Fudan University (Shanghai, China), and cultured in astrocyte media (ScienCell Research Laboratories, Inc., Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified incubator containing 5% CO₂. In addition, human glioma cell lines, including A172, U87, U251 and U373, were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS in a 37°C humidified atmosphere with 5% CO₂.

Cell transfection was conducted in the U87 glioma cell lines using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Briefly, U87 cells were cultured to 70% confluence, and resuspended in serum-free DMEM. Scramble miR (miR-NC), miR-219 mimic for miR-219 upregulation, negative control (NC) inhibitor, miR-219 inhibitor for miR-219 knockdown, and pc-DNA3.1-Sal-like protein 4 (SALL4) open reading frame plasmid for SALL4 upregulation were diluted in OPTI-MEM (Thermo Fisher Scientific, Inc.), which was then added with diluted Lipofectamine 2000. All mimics, inhibitors and plasmids were purchased from Yearthbio (Changsha, China). Following incubation for 20 min at room temperature, the mixture was added into the U87 cell suspension. Following incubation at 37°C and 5% CO₂ for 6 h, the transfection mixture was replaced with DMEM with 10% FBS. MiR-NC was the corresponding control for miR-219 mimic, and NC inhibitor was the corresponding control for miR-219 inhibitor.

Total RNA was extracted from tissues and cell lines using TRIzol reagent (Thermo Fisher Scientific, Inc.). RNA quality and concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) RNA was converted into cDNA using PrimeScript 1st Strand cDNA Synthesis kit (Takara Bio, Inc., Tokyo, Japan). For miR-219 expression detection, the miRNA Q-PCR Detection kit (GeneCopoeia, Rockville, MD, USA) was used to conduct qPCR on an ABI 7300 Plus thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). U6 gene was used as an internal control. For mRNA expression detection, SYBR Green I Real-Time PCR kit (Biomics Biopharma, Nantong, China) was used to conduct qPCR on an ABI 7300 Plus system. The primers for SALL4 were: Forward, 5′-AGCACATCAACTCGGAGGAG-3′ and reverse, 5′-CATTCCCTGGGTGGTTCACTG-3′. The primers for the internal control gene GAD PH were: Forward, 5′-CTGGGCTACACTGAGCACC-3′ and reverse, 5′-AAGTGGTCGTTGAGGGCAATG-3′. The reaction conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The relative expression levels were analyzed by the 2^−ΔΔCq method (16). According to the mean value of miR-219 levels that served as the cutoff value, glioma tissues were divided into the high and low miR-219 expression groups.

U87 cells were lysed in ice-cold buffer containing 0.5 mol/l Tris-HCl, pH 7.4, 1.5 mol/l NaCl, 2.5% deoxycholic acid, 10% NP-40 and 10 mmol/l EDTA (Sigma-Aldrich; Merck, Darmstadt, Germany) with cocktail protease inhibitors (Thermo Fisher Scientific, Inc.). Cell lysates were then obtained by centrifugation at 16,000 × g for 20 min at 4°C. Protein concentrations were determined by Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and 50 µg protein was subjected to 12% SDS-PAGE, followed by immunoblotting onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked for 1 h in phosphate-buffered saline with Tween 20 (PBST) containing 5% nonfat dry milk (Yili, Beijing, China). Subsequently, the membrane was incubated at room temperature for 3 h with primary antibodies: Rabbit polyclonal to SALL4 antibody (1:50, ab29112, Abcam, Cambridge, MA, USA) and rabbit polyclonal to GAPDH antibody (1:50, ab9485, Abcam). The membrane was then washed three times with PBST, then immersed in PBST containing horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000, ab7090, Abcam) at room temperature for 1 h. After washing in PBST four times (10 min/wash), an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) was used to perform chemiluminescence detection. The relative protein expression was determined using Image J software v.1.48 (National Institutes of Health, Bethesda, MD, USA), represented as the density ratio vs. GAPDH.

Cell proliferation was measured by MTT assay. U87 cells in 200 µl serum-free DMEM were plated into 96-well plates at a density of 5,000 cells per well. After cells were adhered, the medium was replaced with DMEM containing 10% FBS, and incubated at 37°C for 12, 24, 48 and 72 h, respectively. Next, 20 µl MTT solution (5 mg/ml) was added into each well. Following incubation at 37°C for 4 h, the MTT solution was replaced with 150 µl dimethyl sulfoxide to dissolve the tetrazolium crystals for 10 min. Absorbance at 570 nm was detected with a microplate reader (Thermo Fisher Scientific, Inc.).

U87 cells were plated at 1×10⁵ cells/well in 24-well plates with 1 ml DMEM and incubated for 24 h. Scratch wounds were generated using a 200 µl pipette tip. The medium was then replaced with fresh DMEM with 10% FBS. The wound size was recorded by camera at 0 and 24 h, respectively. Quantification was implemented by Image J v1.48 software.

In this assay, 24-well-plate-matched Bio-coat cell invasion chambers (BD Biosciences, Franklin Lakes, NJ, USA) were used to measure the invasion of glioma cells. Briefly, 1.0×10⁵ U87 cells were seeded in the upper transwell chamber in serum-free medium, with 500 µl DMEM containing 10% FBS in the lower chamber. Following incubation at 37°C for 24 h, the cells that did not migrate through the pores were carefully wiped out with a cotton-tipped swab. The cells that had migrated were then fixed in 90% alcohol, followed by staining with 0.1% crystal violet (Sigma-Aldrich; Merck). Subsequent to washing with PBS for three times, invading cells were observed and photographed using an inverted microscope (Olympus Corp., Tokyo, Japan).

Bioinformatics analysis was performed to predict the potential target genes of miR-219 using TargetScan online software (version 7.1; www.targetscan.org), according to the manufacturer's instructions. SALL4 was shown as the putative target gene of miR-219, and this association was then confirmed using a luciferase reporter gene assay.

Luciferase reporter gene assay was conducted to confirm the association between miR-219 and its potential target, SALL4. Initially, the mutant type (MT) of SALL4 3′-untranslated region (UTR) lacking complementarity with the miR-219 binding sequence was constructed using QuickChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA), according to the manufacturer's instructions. The wild type (WT) or mutant type (WT) 3′UTRs of SALL4 were also constructed and inserted into the multiple cloning sites in the psiCHECK2 luciferase reporter vector (Promega Corp., Madison, WI, USA). Lipofectamine 2000 was used to co-transfected U87 cells with WT-SALL4-3′UTR or MT-SALL4-3′UTR reporter plasmids, and miR-219 mimics or miR-NC. At 48 h after co-transfection, the luciferase activities were examined using a dual-luciferase reporter assay system (Promega Corp.), according to the manufacturer's protocol. The Renilla luciferase activity was normalized to the firefly luciferase activity.

Data in the present study are expressed as the mean ± standard deviation of three independent experiments. The difference between two groups was analyzed using Student's t-test, while differences among more than two groups were analyzed using analysis of variance. Associations between SALL4 expression and the clinicopathological characteristics in glioma patients were assessed using the χ² test. Statistical analysis was performed using SPSS version 19.0 (IBM Corp., Armonk, NY, USA). A P-value that was <0.05 was considered to indicate a statistically significant difference.

In the current study, the expression of miR-219 was examined in a total of 63 glioma tissues and 12 normal brain tissues. The RT-qPCR data revealed that the miR-219 levels were significantly decreased in glioma tissues, when compared with the normal brain tissues (Fig. 1A; P<0.001). In addition, high-grade gliomas (WHO grade III–IV) presented significantly lower levels of miR-219 as compared with those in low-grade gliomas (WHO grade I–II) (Fig. 1B; P<0.001). Furthermore, miR-219 was significantly downregulated in several common human glioma cell lines (U87, A172, U373 and U251) compared with the normal astrocytes (P<0.001; Fig. 1C). Therefore, miR-219 is concluded to be downregulated in glioma, and may contribute to glioma progression.

Subsequently, the clinical significance of miR-219 expression in glioma was investigated. Glioma tissues were divided into the high and low miR-219 expression groups, according to the mean value of miR-219 levels that served as the cutoff value. It was observed that low miR-219 expression was significantly associated with an advanced WHO grade and KPS index, but not with the age or sex of patients (Table I). Accordingly, these results suggested that decreased expression of miR-219 is significantly associated with glioma progression in glioma patients.

In order to upregulate the expression of miR-219, U87 cells were transfected with miR-219 mimic. Transfection with miR-NC was used as the control group. Following transfection, the miR-219 levels were significantly higher in the miR-219 group compared with those in the miR-NC group (Fig. 2A). MTT, wound healing and transwell assays were then conducted to determine the cell proliferation, migration and invasion, respectively. As indicated in Fig. 2B-D, ectopic expression of miR-219 significantly decreased the proliferation, migration and invasion of U87 cells, when compared with the miR-NC group. These findings suggest that miR-219 may function as a tumor suppressor in glioma.

Next, bioinformatics analysis was performed to predict the potential target genes of miR-219. As shown in Fig. 3A, SALL4 was observed to be a putative target of miR-219. To verify the targeting association between miR-219 and SALL4, luciferase reporter plasmids containing WT or MT SALL4 3′UTRs were constructed (Fig. 3B and C). The results of luciferase reporter gene assay demonstrated that the luciferase activity was significantly inhibited in U87 cells co-transfected with the WT-SALL4-3′UTR luciferase reporter plasmid and miR-219 mimic, when compared with the control group, but the luciferase activity was unchanged compare with the controlwhen transfected with MT-SALL4-3′UTR luciferase reporter plasmid and miR-219 mimic (Fig. 3D). These findings indicate that SALL4 is a target gene of miR-219, and that miR-219 directly binds to the 3′UTR of SALL4 mRNA in U87 cells.

As miRs generally inhibit the expression of their target genes at the post-transcriptional level, the present study then investigated the effects of miR-219 on the protein expression of SALL4 in U87 cells. As shown in Fig. 4A, upregulation of miR-219 significantly decreased the protein expression of SALL4. Next, U87 cells were transfected with miR-219 inhibitor to knockdown its expression, with NC inhibitor transfection used as the control group. Following transfection, the miR-219 levels were significantly reduced in the miR-219 inhibitor group, when compared with the NC inhibitor group (Fig. 4B). Western blot analysis data revealed that downregulation of miR-219 significantly increased the protein expression of SALL4 in U87 cells as compared with that in the NC inhibitor group (Fig. 4C). Therefore, these findings suggest that miR-219 negatively regulated the protein expression of its target gene SALL4 in U87 cells.

As the earlier experiments observed that miR-219 was downregulated in glioma tissues and that it negatively regulated SALL4 in glioma cells, the expression of SALL4 in glioma tissues and cell lines was subsequently investigated. The results of RT-qPCR indicated that SALL4 was significantly upregulated in glioma tissues as compared with the normal brain tissues (Fig. 5A). Furthermore, the high-grade gliomas (WHO grade III–IV) exhibited higher SALL4 levels in comparison with those in low-grade gliomas (WHO grade I–II; Fig. 5B). Similarly, SALL4 expression levels were also higher in glioma cell lines compared with normal astrocytes (Fig. 5C and D). These observations reveal that the increased expression of SALL4 in glioma may be caused by the downregulation of miR-219.

The current study further examined whether SALL4 was involved in the miR-219-mediated proliferation, migration and invasion of U87 cells. miR-219-overexpressing U87 cells were transfected with SALL4 expression plasmid, in order to upregulate its expression. Following transfection, the mRNA and protein levels of SALL4 were significantly higher in the miR-219 + SALL4 group compared with those in the miR-219 group (Fig. 6A and B). MTT, wound healing and transwell assays were subsequently conducted to determine cell proliferation, migration and invasion, respectively. As indicated in Fig. 6C-E, the proliferation, migration and invasion of U87 cells were significantly upregulated in the miR-219 + SALL4 group when compared with those in the miR-219 group. Accordingly, overexpression of SALL4 attenuated the suppressive effects of miR-219 upregulation on the malignant phenotypes of U87 cells, suggesting that miR-219 serves a suppressive role in glioma cells via directly targeting SALL4.