The human glioma cell line A172 was purchased from the Chinese Academy of Sciences Cell Bank. All cells were cultured in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C with 5% CO₂ in a humidified chamber. Hypoxic conditions were induced by incubating the cells in a modular incubator chamber with a gas mixture containing 1% O₂, 5% CO₂ and 94% N₂ at 37°C. Forty-five human glioma tissues including 4 World Health Organization (WHO) grade I tumors, 8 grade II tumors, 13 grade III tumors and 20 grade IV tumors, and 2 normal brain tissues of decompression operation were obtained from the Department of Neurosurgery of Qilu Hospital of Shandong University. The glioma specimens were verified and classified according to the WHO classification standard of tumors by two experienced clinical pathologists. The present study was approved by the Institutional Review Board of Shandong University. Written informed consent was obtained from all patients, and the hospital ethics Committee approved the experiments.

Mature miR-Let-7f mimics, the scrambled mimic control, the miR-Let-7f inhibitor, the scrambled inhibitor control, the miR-584-5p mimic and POSTN siRNA were designed and synthesized by RiboBio. Cell transfections and co-transfections were performed using Lipofectamine 2000 when the cells reached 70% confluency according to the manufacturer's instructions. Untransfected cells were used as the blank control, while cells transfected with the scrambled oligos were used as the negative controls. The transfection efficiency was verified by quantitative real-time PCR. Forty-eight hours after transfection, the glioma cells were harvested for subsequent experiments.

A172 cells were seeded into 96-well culture plates at a density of 3,000 cells/well. Cell proliferation was analyzed at 24, 48, 72 and 96 h after transfection using a Cell Counting Kit-8 (CCK-8). A volume of 10 µl of CCK-8 solution was added to each well, and the cells were incubated for another 1 h in a humidified incubator at 37°C. Optical density was measured at 450 nm using a microplate reader. Each assay was performed in triplicate.

The transfected cells (1×10⁵) were seeded in 6-well plates, incubated overnight, and at 90% confluency, the cell monolayer was scratched with a sterile pipette tip. The scratched plates were cultured in DMEM containing 1% FBS. Images were captured at 0 and 12 h along the scrape line under a microscope. Cell migration assays were performed using Transwell chambers (8-µm; Corning). In total, 5×10⁴ transfected cells in FBS-free medium were seeded in the upper chamber. Medium containing 10% FBS was added to the lower chamber. After 12 h, the cells that did not migrate or invade were removed using cotton buds. The cells migrating on the lower surface were fixed and stained with crystal violet.

Total RNA was extracted from the tissue samples using TRIzol. Then, total RNA (50 ng) was reverse-transcribed with miR-Let-7f stem-loop RT primers or with the U6 RT primers using a ReverTra Ace qPCR RT kit to generate cDNA. Real-time PCR was performed using a SYBR Premix Ex Taq™ kit with miR-Let-7f or U6 PCR primers. The reactions were performed using a LightCycler 2.0 instrument. U6 expression was used as the endogenous control. The absolute expression levels were calculated as concentration ratios using a Roche LightCycler^® 2.0 system.

Paraffin-embedded human glioma tissue samples were sectioned and dewaxed. Endogenous peroxidase activity was quenched by incubating the slides in methanol containing 3% hydrogen peroxide for 30 min, after which the sections were incubated for 2 h at room temperature with normal goat serum and subsequently incubated at 4°C overnight with primary antibody (1:300 CD34; Abcam). Next, the sections were incubated with horseradish peroxidase-linked second antibody, followed by reaction with diaminobenzidine and counterstaining with PAS staining kit and then Mayer's hematoxylin.

VM formation was evaluated using a commercial Matrigel matrix (BD Biosciences, France). A172 glioma cells were digested and resuspended at 5×0⁴ cells/ml in DMEM containing 1% FBS. Wells of 96-well tissue culture plates were coated with Matrigel (50 µl/well; BD Biosciences) which was allowed to polymerase at 37°C for 30 min. The glioma cell suspension was then plated at 100 µl/well onto the surface of Matrigel and incubated at 37°C. Cells were photographed using an Olympus inverted microscope.

All experiments were performed three times. The statistical analysis and experimental graphs were generated using SPSS 17.0 and GraphPad Prism software. Descriptive statistics including the means ± SD, Mann-Whitney test, Kaplan-Meier plots, log-rank tests were used to analyze the significant differences. p<0.05 and p<0.01 were considered to indicate a statistically significant result.

To examine whether the VM correlates with the WHO grades of human glioma, we assessed the VM in 45 human glioma specimens with different grades and 2 normal brain tissues by immunochemical staining (Table I). As shown in Fig. 1A, the VM was not found in the normal brain tissues and WHO grade I astrocytoma tissues, except pilocytic astrocytoma as its vascular hyperplasia. We also found that the VM rarely existed in WHO grade II glioma tissues (Figs. 1B and 2B). However, VM was very common in high-grade (WHO III and IV) gliomas, particularly in GBM (Fig. 1C and D). Accordingly, the VM-positive rate of human glioma was observably associated with its WHO grades (Fig. 2B).

To analyze whether VM in clinical samples associated with the clinical survival information of the 45 patients, Kaplan-Meier estimates were used in the present study. As expected, the subgroup of VM negative glioma patients presented a significantly prolonged postoperative survival time (Fig. 2A). Since our previous research found miR-Let-7f was identified as a potent anti-glioma miRNA (23), we hypothesized that miR-Let-7f was probably involved in the regulation process of VM. To assess the relationship between miR-Let-7f and VM, we verified the levels of miR-Let-7f in these 45 human glioma specimens by quantitative real-time PCR. The results showed that miR-Let-7f expression levels were negatively correlated with VM (Fig. 2C). These data were particularly strong for the VM suppressive effect of miR-Let-7f in glioma. However, its underlying mechanism of anti-VM effects remains unknown.

Migration is known to play an important role in tumor VM, and in an earlier study we showed that miR-Let-7f regulated the glioma cell migration (23). To determine whether miR-Let-7f could affect VM, we used the miR-Let-7f inhibitor and mimics. miRNA inhibitors are synthetic, 2′-O-methyl-modified, single-stranded molecules that interfere with miRNA function by sequestering them via irreversible binding (24,25). Mimics are synthetic, double-stranded, modified RNA molecules that imitate the functions of endogenous miRNAs (26). As a proof of principle, the transient transfection of 80 nM inhibitor or mimics did not affect glioma cell viability (Fig. 2D). In addition, we observed a significant pro-migratory effects of the miR-Let-7f inhibitor and anti-migratory effects of its mimics in wound healing and Transwell assays of A172 cells (Fig. 3A and B) in agreement with our findings previously reported. Furthermore, VM tube formation assay results confirmed that miR-Let-7f knockdown significantly promoted the VM formation and overexpression of miR-Let-7f completely blocked it (Fig. 3C). It suggests that miR-Let-7f may hinder the VM forming capacity of glioma cells by anti-migratory effects.

Several studies have demonstrated the hypoxia-induced angiogenesis and VM, and the pro-migratory effects of hypoxia. In this regard, we supposed that miR-Let-7f was involved in the regulation of hypoxia-induced VM formation. After 6 h hypoxia treatment, A172 glioma cells revealed an excessively enhanced VM formation (Fig. 4B, upper).

To understand the mechanisms implicated in the VM suppressive role under hypoxia condition of miR-Let-7f, we investigated the time-dependent effects of miR-Let-7f knockdown and overexpression in A172 glioma cells. First, we observed a significant VM suppression effect of miR-Let-7f mimics, particularly even under hypoxic conditions in A172 cells. While miR-Let-7f inhibitor significantly promoted A172 glioma cell VM under normoxic and hypoxia conditions (Fig. 4). Notably, the VM had a dynamic process with increased observation time. The undisturbed VM formation initiated at 4 h, peaked at 6 h and vanished after 12 h in normoxia control A172 cells. In addition, miR-Let-7f inhibitor further enhanced the VM structures, particularly revealing the effect of hypoxia. In contrast, miR-Let-7f mimics completely blocked the VM formation throughout the experiment whether with hypoxia or not.

Taken together, our results clearly demonstrated that miR-Let-7f knockdown markedly promoted the VM forming capacity of human glioma cells and aggravate the hypoxia-induced VM promoting effects. While miR-Let-7f overexpression antagonized the VM formation, particularly the hypoxia-induced VM on human glioma cells.

As we previously reported, miR-Let-7f inhibited glioma migratory ability by directly targeting POSTN (23). It suggested that miR-Let-7f may block the VM forming capacity of glioma cells by regulating the POSTN-dependent migration. To investigate whether miR-Let-7f and POSTN are linked, we utilized a POSTN siRNA. siRNA transfected A172 glioma cells almost lost the migratory and VM forming ability as miR-Let-7f overexpressed cells (Fig. 5, middle), and the VM promoting effect of miR-Let-7f inhibitor was totally blocked (Fig. 5, lower). In addition, the hypoxia effect was also blocked by POSTN knockdown (Fig. 5, right). Hence we could confirm that miR-Let-7f antagonizes the hypoxia-induced VM promoting effects by targeting POSTN.