miR-517a is up-regulated in glioma and promotes glioma tumorigenesis in vitro and in vivo

miR-517a has been reported to act as an oncogenic miRNA in human hepatocellular carcinoma and lung cancer. However, the roles and underlying molecular mechanism of miR-517a in glioma remain unclear. In the present study, the expression of miR-517a in clinical glioma tissues and glioma cell lines was examined by quantitative real-time PCR (qRT-PCR). Transfected with knockdown or forced expression of miR-517a, the effects of miR-517a on cell proliferation, migration, and invasion were detected through in vitro and in vivo tumorigenesis assays. Here, we report that miR-517a expression was up-regulated in glioma tissues when compared with normal brain tissues, and up-regulation of miR-517a level is tightly correlated with the status of pathology classification of glioma. A functional assay found that overexpression of miR-517a in glioma cells markedly promoted or suppressed cell proliferation, colony formation, migration and invasion, respectively. Moreover, we revealed that the knockdown of miR-517a dramatically suppressed glioma cell growth, migration, and invasion in vitro and in vivo. Furthermore, we found that knockdown of miR-517a significantly induced apoptosis. Therefore, miR–517a acts an oncogenic miRNA that promotes tumor progression in glioma, and thus may become a promising therapeutic candidate for glioma.


Introduction
Malignant gliomas are the most common primary tumors of the central nervous system. They are characterized by high invasion, migration, and proliferation abilities [1][2][3]. Despite recent advances in our understanding of the molecular mechanisms of the disease -and improvement in therapeutic strategiesthe median survival of GBM patients still remains dismal, with less than 1 year after diagnosis [4]. Thus, it is quite urgent to understand the molecular mechanisms by which glioma initiates, progresses, invades, and recurs in order to develop effective prognostic biomarkers and novel therapies. miRNAs are endogenous, small, non-coding RNAs that regulate gene expression by antisense complementarity to specific mRNA. MiRNAs are short non-coding RNAs that interfere with target gene expression through either inhibition of mRNA translation or promotion of mRNA degradation [5]. Recent works have suggested that deregulation of a group of small non-coding RNAs called miRNAs is involved in initiation and progression of gliomas, including miR-335 [6], miR-218 [7], miR-15b, miR-152 [8], miR-92b [9], miR-145 [10], and miR-124 [11]. miR-517a is considered to be a novel oncomiR and was observed to be elevated in human hepatocellular carcinoma (HCC) [12]; expression of miR-517a increased proliferation, migration, and invasion of HCC cells in vitro [12]. A recent study reported that miR-517a accelerated lung cancer cell proliferation, migration, and invasion through inhibiting forkhead box J3 (FOXJ3) expression [13]. However, the regulatory mechanism of miR-517a in glioma remains largely unclear.
In the present study, we investigated the putative role of miR-517a in the progression of glioma. We found that the expression of miR-517a was frequently increased in clinical glioma tissue samples compared with normal brain tissues, and up-regulation of miR-517a level is tightly correlated with the status of pathology classification. Furthermore, through in vitro and in mouse xenograft model, we investigated the function of miR-517a in glioma, including the proliferation, migration, invasion, and apoptosis.

Clinical samples
We obtained 60 snap-frozen glioma and 20 normal brain samples from Nanxishan Hospital of Guangxi Zhuang Autonomous Region, including 20 grade II tumors, 20 grade III tumors, and 20 grade IV tumors according to the WHO classification. All specimens had confirmed pathological diagnosis and were classified according to the WHO criteria (Table 1). These glioma cases were from 38 males and 22 females with age ranging from 19 to 72 years. The purpose and content of the present study were explained fully to all subjects, whose signed informed consent was obtained prior to enrollment. Written informed consent was obtained from each patient. Our study was conducted in accordance with the World Medical Association's Helsinki guidelines and was approved by the Research Ethics Committee of Nanxishan Hospital of Guangxi Zhuang Autonomous Region, China.

Cell lines
The LN18, U87, and U118 glioma cell lines and primary normal human astrocytes (NHAs) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (GIBCO-BRL) medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, U.S.A.) at 37 • C/5% CO 2 . All cell lines were authenticated by short tandem repeat DNA profiling.

Stable expression or knockdown of miR-517a in glioma cell lines
Lentiviral vectors which overexpressed or knockdown miR-517a were purchased from GenePharma (Shanghai, China). A lentiviral vector expressing scrambled RNA was used as the control and the sequence was 5 -TTAACGTCGATGTCCGT-3 . Glioma cell lines were infected with lentiviral vector. Total RNA from these cell clones was isolated, and levels of miR-517a were quantified using RT-qPCR.

MTT assay
A cell proliferation assay was conducted with MTT kit according to the manufacturer's instruction. Triplicate wells were measured in each treatment group.

Colony formation assay
For the cells to form colonies, a total of 600 LN18 or U118 cells transfected with miR-517a mimic or scramble mi-RNA were placed onto a fresh six-well plate and maintained in media containing 10% FBS, replacing the medium every 4 days. After 2 weeks, the colonies were fixed with methanol and stained with 0.1 % crystal violet (Sigma, St. Louis, MO). The visible colonies were manually counted. Triplicate wells were assessed for each treatment group.

Transwell assay
Matrigel-coated invasion chambers (8 μm, BD Biosciences, San Jose, CA, U.S.A.) were prepared according the manufacturer's instructions. Transfected GBM cells were seeded in the upper compartment and allowed to migrate for 24 h in an incubator. Cells were seeded in 1% FBS DMEM medium in the upper chamber and 10% FBS medium in the well below the insert. The cells that had invaded the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of cells that had invaded through the Matrigel was counted in six random fields for triplicate membranes under the microscope.

Flow-cytometric analysis
After the double staining with FITC-Annexin V and propidium iodide (PI) was done using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's recommendations, the cells were analyzed with a flow cytometry (FACScan ® ; BD Biosciences) equipped with a CellQuest software (BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic cells and apoptotic cells, and then the relative ratio of early apoptotic cells were compared with control transfectant from each experiment. Cells for cell cycle analysis were stained with PI using the CycleTEST™ PLUS DNA Reagent Kit (BD Biosciences) following the protocol and analyzed by FACScan.

Statistical analysis
The Student's t test (two-tailed), one-way ANOVA, and Mann-Whitney U test were conducted to analyze the in vitro and in vivo data by SPSS 20.0 software. P-values less than 0.05 were considered significant.

Up-regulation of miR-517a in human glioma tissues and cell lines
To explore the expression and significance of miR-517a in glioma, we first examined the expression level of miR-517a in 60 snap-frozen glioma tissues and 20 normal brain tissues by quantitative real-time PCR (qRT-PCR). Compared with normal brain tissues, significant increase of miR-517a was observed in glioma tissues ( Figure 1A). Meanwhile,  we also observed that increased levels of miR-517a in glioma patients were positively correlated with the status of pathology classification (WHO II vs WHO IV; P<0.01). A gradually increased miR-517a expression was found from grade II to IV samples ( Figure 1A). In addition, qRT-PCR was performed on a panel of three human glioma cell lines (LN18, U87, and U118) and primary NHA. Up-regulation of miR-517a could also be observed in glioma cell lines than that of NHA ( Figure 1B). These results suggested that miR-517a could be closely related to human glioma and up-regulation of miR-517a might be related to glioma oncogenesis and progression.

Overexpression of miR-517a promotes growth of glioma cells in vitro
To further explore its biological roles in glioma, miR-517a overexpression lentiviral vector (Lv-miR-517a) and lentiviral empty vector (miR-NC) were transfected into human glioma LN18 and U118 cells. After transfection, the expression level of miR-517a was significantly up-regulated compared with the control group (Figure 2A). We then performed colony formation assay to determine the cell proliferation in each group, and showed that up-regulation of miR-517a significantly increased LN18 and U118 cells proliferation ( Figure 2B,C). Additionally, MTT assay indicated that up-regulation of miR-517a also substantially promoted the proliferation ability in LN18 and U251 cells ( Figure  2D). These results demonstrated that miR-517a could promote the proliferation phenotype of glioma cells in vitro.

Overexpression of miR-517a induced the invasion and migration of glioma cells in vitro
Next, we examined the effects of miR-517a on the ability of LN18 and U118 cells to invade and migrate. Cell migration and invasion abilities were determined in the LN18 and U251 cells transfected with the Lv-miR-517a or miR-NC by a transwell chamber or a Boyden chamber, respectively. Transwell assays with Matrigel demonstrated that LN18 and U118 cells-overexpressing miR-517a were found to have significantly increased migration and invasion abilities than control cells (Figure 3). Together, these data suggest that miR-517a is critical for glioma cells invasion and migration in vitro.

Knockdown of miR-517a inhibits glioma cell proliferation, migration, and invasion
To determine whether miR-517a had an effect on the malignant phenotype of glioma cells, we constructed lentiviral-mediated knockdown of miR-517a in the U87 cell line, which exhibited the highest miR-517a expression level among cell lines in our study. The miR-517a expression levels were knocked down more than 70% in U87 cell line relative to the control group ( Figure 4A). Colony formation assay and MTT assay showed that knockdown of miR-517a decreased the proliferation ability in U87 cells ( Figure 4B,C). In addition, the transwell assays demonstrated that LN18 cells knockdown of miR-517a were found to have significantly reduced the migration and invasion abilities than control cells ( Figure 4D).

Knockdown of miR-517a induces apoptosis in vitro
To illustrate the mechanisms of how miR-517a modulates glioma cell growth, flow cytometry was applied to detect the effects of miR-517a on cell apoptosis. Annexin V/PI double staining demonstrated that the proportion of apoptosis LN18 cells transfected with anti-miR-184 was significantly higher than that in the anti-miR-NC group ( Figure 5A).   Concordantly, the expression of caspase-3 cleavage and Bad were activated, the expression of Bax and Bcl-2 were markedly reduced in LN18 cells transfected with anti-miR-517a ( Figure 5B).

Down-regulation of miR-517a inhibits glioma cell tumorigenesis in vivo
To explore whether the level of miR-517a expression could affect tumorigenesis, LN18 cells stably transfected with lentiviral-mediated anti-miR-517a or empty vector were inoculated into female nude mice. Eighteen days after the injection, the tumors formed in the anti-miR-517a group were substantially smaller than those in the control group ( Figure 6A,B). Moreover, the mean tumor weight at the end of the experiment was markedly lower in the anti-miR-517a group compared with the empty vector group ( Figure 6C). Tumors formed from lentiviral anti-miR-517a-transfected LN18 cells exhibited decreased positivity for Ki67 than those from control cells ( Figure  6D). These findings indicate that knockdown of miR-517a inhibits tumor growth in vivo.

Discussion
Gliomas are well-known for not only rapid proliferation but also invading the surrounding brain tissue, leading to extremely poor prognosis. Similar to other tumors, some environmental carcinogenic factors may also be associated with the occurrence of glioma [14]. miRNAs have emerged as key factors involved in several biological processes, including development, differentiation, cell proliferation, and tumorigenesis [15,16]. Thus, it is possible that miRNAs may shed light on the potential mechanism of glioma. In the present study, our data provided the first evidences that expression of miR-517a was increased in glioma tissues compared with normal brain tissues. We further found that miR-517a expression was positively correlated with the status of pathology classification. These results suggest that miR-517a may promote the progression of glioma.
Chromosome 19 miRNA cluster (C19MC) on 19q13.41 have been considered so far to be the largest human miRNA gene cluster, which harbor 43 miRNA genes and extends over 100 kb long region [17]. Recent miRNA-related studies show that miR-517a has been proposed to be promoted in tumor metastasis of HCC and lung cancer [12,13]. Whereas, in bladder cancer miR-517a was down-regulated, and ectopic restoration of miR-517a significantly inhibited cell proliferation by promoting apoptosis [18]. These studies suggest that miR-517a may have have different roles in different cancers. To comprehensively understand the effect of miR-517a on glioma cancer cells, we performed in vitro proliferation and invasion assays to identify the migration and invasion ability of glioma cells. Importantly, our study showed that knockdown of miR-517a significantly inhibited tumorigenesis as well as the tumor growth of glioma cells in vivo. These data further indicate that miR-517a indeed acts as a novel oncogenic miRNA in glioma cells.
Apoptosis, a fundamental process essential for development and maintenance of tissue homeostasis, is a major mechanism to eliminate cancer cells [19]. Thus, an effective strategy for cancer treatment seems to target signaling intermediates in the apoptosis-inducing pathways [20]. Here, we showed that knockdown of miR-517a induces apoptosis in LN18 cells. Consistently, the expression of caspase-3 cleavage and Bad were up-regulated, the expression of Bax and Bcl-2 were markedly down-regulated in LN18 cells transfected with anti-miR-517a, which is consistent with apoptosis induction. These data add further evidence for the oncogenesis role of miR-517a in glioma.
In summary, our results showed that miR-517a expression was frequently up-regulated in glioma specimens and cell lines, and increased levels of of miR-517a level was tightly correlated with the malignant progression. Our study in vitro and in vivo suggested that miR-517a acts as a novel oncogenic miRNA that promotes tumor cell proliferation, migration, and invasion of glioma cells by inducing apoptosis. The present study provides novel insights into the mechanism underlying the development and progression of glioma, and suggests that miR-517a may become a promising therapeutic candidate for glioma.

Funding
The authors declare that there are no sources of funding to be acknowledged.