Glioblastoma tissues and adjacent normal tissues were obtained from the Department of Neurosurgery, The Affiliated Hospital of Taishan Medical University, Shandong, China. All tissues were examined histologically, and pathologists confirmed the diagnosis. Medical ethics committee approved the experiments. The use of human's tissue samples follows internationally recognized guidelines as well as local and national regulations. Informed consent was obtained from each individual.

Human glioblastoma cell line U-87MG was obtained from American Type Culture Collection. Briefly, cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO₂. VSIG4 expressing plasmids/empty vectors (pcDNA3.1) were purchased from Tiangen (Beijing, China). Pre-let-7g and control miR were purchased from Ambion, Inc. (Ambion, Austin, TX, USA). For transfection experiments, the cells were cultured in serum-free medium without antibiotics at 60% confluence for 24 h, and then transfected with transfection reagent (Lipofectamine 2000, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After incubation for 6 h, the medium was removed and replaced with normal culture medium for 48 h, unless otherwise specified.

Western blot analysis was performed as described before (29). Briefly, after incubation with primary antibody anti-VSIG4 (1:500; Abcam, Cambridge, MA, USA), anti-CD133 (1:500; Abcam), anti-EZH2 (1:500; Abcam), anti-c-Met (1:500; Abcam), anti-P4HB (1:500; Abcam), anti-VAMP8 (1:500; Abcam), anti-CX43 (1:500; Abcam), anti-E-cadherin (1:500; Abcam), anti-TGFB1 (1:500; Abcam), anti-vimentin (1:500; Abcam), anti-SNAIL (1:500; Abcam), anti-Notch1 (1:500; Abcam), anti-TLR9 (1:500; Abcam), anti-EphA2 (1:500; Abcam), anti-MLK4 (1:500; Abcam) and anti-β-actin (1:500; Abcam) overnight at 4°C, IRDye™-800 conjugated anti-rabbit secondary antibodies (LI-COR, Biosciences, Lincoln, NE, USA) were used for 30 min at room temperature. The specific proteins were visualized by Odyssey™ Infrared Imaging System (Gene Co., Lincoln, NE, USA).

Cells (10³/ml) in serum-free RPMI-1640/1 mM Na-pyruvate were seeded on 0.5% agar precoated 6-well plates. After 1 week, half the medium was changed every third day. Single spheres were picked and counted.

For U-87MG cell immunofluorescence analyses, U-87MG cells were plated on glass coverslips in 6-well plates and transfected as indicated. At 48 h after transfection, coverslips were stained with CD44 (1:500; Abcam) or antibody anti-VSIG4 (1:500; Abcam). Alexa Fluor 488 goat anti-rabbit IgG antibody was used as secondary antibody (Invitrogen). Coverslips were counterstained with DAPI (Invitrogen-Molecular Probes, Eugene, OR, USA) for visualization of the nuclei. Microscopic analysis was performed with a confocal laser-scanning microscope (Leica Microsystems, Bensheim, Germany). Fluorescence intensities were measured in a few viewing areas for 300 cells per coverslip and analyzed using ImageJ 1.37v software (http://rsb.info.nih.gov/ij/index.html).

Wound healing assay was performed as described before (30).

Migration and invasion assay was performed as described before (29).

The analysis of potential microRNA target site using the commonly used prediction algorithms - miRanda (http://www.microrna.org/).

Total RNA from cultured cells, with efficient recovery of small RNAs, was isolated using the mirVana miRNA Isolation kit (Ambion). Detection of the mature form of miRNAs was performed using the mirVana qRT-PCR miRNA Detection kit and qRT-PCR Primer Sets, according to the manufacturer's instructions (Ambion). The U6 small nuclear RNA was used as an internal control.

It was performed as described before (31). Primers for VSIG4: forward, 5′-GTGTCCAGTTTGGCTAGTGCC-3′; reverse, 5′-GACTGGAGAACAGAAGCAGGC-3′. Primers for GAPDH: forward, 5′-CGGAGTCAACGGATTTGGTCG TAT-3′; reverse, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′.

Northern blot analysis for miRNAs were performed as described previously (32). Probes were labeled with [γ-³²P]-ATP complementary to let-7g-5p and U6 snRNA.

Data are presented as mean ± SEM. Student's t-test (two-tailed) was used to compare two groups (P<0.05 was considered significant), unless otherwise indicated (χ² test).

In an attempt to identify VSIG4 protein expression between glioblastoma tissues and adjacent normal tissues, we performed western blotting in tumor tissues versus normal tissues. Protein was isolated from 6 pairs of glioblastoma tissues and normal tissues (patient nos. 1–6). We found that VSIG4 protein was significantly increased in cancer tissues, compared with adjacent normal tissues (Fig. 1A). It implied that VSIG4 could be an oncogene in glioblastoma.

In order to assess the role of VSIG4 in glioblastoma, we transfected U-87MG cells with VSIG4 expressing plasmids and then western blotting was performed. We found that VSIG4 protein was significantly increased in the cells transfected with VSIG4 expressing plasmids (Fig. 1B). To determine whether VSIG4 can affect GSCs, we performed sphere forming assay to assess formation of stem cell-like population. We found that formations of spheres were increased by VSIG4 in U-87MG cells (Fig. 1C). CD133, EZH2, c-Met and CD44 are robust markers and are of functional importance for GSC for tumor initiation (33–36). In order to detect whether CD133, EZH2, c-Met and CD44 protein expression can be affected by VSIG4, we performed western blotting and immunofluorescence. The results showed that CD133, EZH2, c-Met (Fig. 1D) and CD44 protein were upregulated by VSIG4 (Fig. 1F).

P4HB, VAMP8 and Connexin 43 (CX43) can promote temozolomide (TMZ) resistance in human glioma cells (37–39). To identify whether VSIG4 could have potential to affect temozolomide (TMZ) resistance, we performed western blotting to detect P4HB, VAMP8 and Connexin 43 (CX43) protein. The results showed that P4HB, VAMP8 and Connexin 43 (CX43) protein were upregulated by VSIG4 in U-87MG cells (Fig. 1E).

EMT has been shown to result in cancer cells with stem cell-like characteristics that have a propensity to invade surrounding tissue and display resistance to certain therapeutic interventions (40). In order to assess the role of VSIG4 in EMT of U-87MG, we transfected U-87MG cells with VSIG4 expressing plasmids and then we found that its overexpression caused significant changes in the cell morphology (EMT, phenotype from a cobblestone-like to a spindle-like morphology) (Fig. 2A). To further verify that the changes in cell morphology are caused by EMT, we performed western blotting to detect expression of epithelial and mesenchymal markers in U-87MG cells transfected with VSIG4 expressing plasmids and the cells transfected with empty vectors. The results revealed that epithelial marker (E-cadherin) was inhibited and the mesenchymal markers (TGFB1, Vimentin, SNAIL, ZEB1 and Notch1) were induced by VSIG4 in U-87 MG cells (Fig. 1B).

EMT can result in increased cell invasion and migration (41–43). Thus, we reasoned that VSIG4 could also affect invasion and migration in U-87 MG cells. To identify this reason, we performed would healing, invasion, and migration assays. We found that overexpressing VSIG4 resulted in enhanced migration (Fig. 1C and D) and invasion (Fig. 1E) in the cells.

Having demonstrated that overexpressing VSIG4 promoted formation of stem cell-like population and EMT, next we studied the mechanisms regulating VSIG4 expression in the disease. MicroRNAs (miRs) are a class of small non-coding RNAs (~22 nucleotides) and negatively regulate protein-coding gene expression by targeting mRNA degradation or translation inhibition (44–46). To further confirm whether VSIG4 could be regulated by microRNA, we used the commonly used prediction algorithm - miRanda (http://www.microrna.org/microrna/home.do) to analyze 3′UTR of VSIG4. A dozen of microRNAs were found by the algorithm. Nonetheless, we are interested in let-7g-5p, because let-7g-5p has been proposed as a tumor suppressive gene (47,48). However, its role still keeps emerging in glioblastoma.

Target sites on 3′UTR of VSIG4 are shown in Fig. 3A. We reasoned that let-7g-5p could downregulate VSIG4 expression by targeting its 3′UTR in glioblastoma. Downregulation of let-7g-5p can contribute to upregulation of VSIG4 in glioblastoma. In an attempt to identify the role of let-7g-5p in regulating VSIG4 expression in glioblastoma, we transfected U-87MG cells with pre-let-7g-5p and control miR. After transfection, let-7g-5p expression was detected by real-time PCR and the results showed that let-7g-5p was significantly increased by pre-let-7g-5p in the cells (Fig. 3B).

To confirm the reason, we performed immunofluorescence analyses in U-87MG cells transfected with pre-let-7g-5p and control miR. The results showed that VSIG4 protein was evidently inhibited in the cells transfected with pre-let-7g-5p (Fig. 3C). We next performed western blotting and RT-PCR to detect VSIG4 expression in U-87MG cells transfected with pre-let-7g-5p and control miR. The results showed that VSIG4 protein (Fig. 3D) was significantly downregulated in the cells transfected with pre-let-7g-5p. However, we found that let-7g-5p did not degrade VSIG4 mRNA (Fig. 3E).

In an attempt to identify let-7g-5p expression between glioblastoma tissues and adjacent normal tissues, we performed northern blotting in tumor tissues versus normal tissues. Protein was isolated from 6 pairs of glioblastoma tissues and normal tissues (patient nos. 1–6). We found that let-7g-5p was significantly decreased in glioblastoma tissues, compared with adjacent normal tissues (Fig. 4A). It indicated that let-7g-5p could be a tumor suppressive gene in glioblastoma. In order to assess the role of let-7g-5p in glioblastoma, we transfected U-87MG cells with pre-let-7g-5p and then northern blot analyses were performed. We found that let-7g-5p was significantly increased in the cells transfected with pre-let-7g-5p (Fig. 4B).

To determine whether let-7g-5p could affect stem-like cell characteristics, we performed sphere forming assay to assess the capacity of CSC or CSC-like cell self-renewal in this study. We found that formations of spheres were decreased by let-7g-5p in U-87MG cells (Fig. 4C). We also performed western blotting to detect whether GSCs markers, CD133, EZH2, c-MET, TLR9, EphA2 and MLK4 can be affected by let-7g-5p in the cells. The results showed that CD133, EZH2, c-MET, TLR9, EphA2 and MLK4 protein was significantly decreased by let-7g-5p in U-87MG cells (Fig. 5D and E).

To assess the role of let-7g-5p in U-87MG cells, we transfected U-87MG cells with pre-let-7g-5p and control miR. We found that its overexpression caused slight changes in the cell morphology (MET, phenotype from a spindle-like morphology to a cobblestone-like) (Fig. 5A). To further verify that the changes in cell morphology are caused by MET, we performed western blotting to detect expression levels of epithelial and mesenchymal markers in U-87MG cells transfected with pre-let-7g-5p and the cells transfected with control miR. The results revealed that epithelial marker (E-cadherin) was induced and the mesenchymal markers (TGFB1, Vimentin, SNAIL, ZEB1 and Notch1) were inhibited by let-7g-5p in U-87 MG cells (Fig. 5B).

To identify whether let-7g-5p could inhibit migration and invasion, we performed wound-healing, migration and invasion assays. We found that overexpressing let-7g-5p resulted in decreased migration (Fig. 5C and D) and invasion (Fig. 5E) in the cells.