Background
As the most common and aggressive primary brain tumor, glioma is characterized with extremely poor prognosis outcomes [
1]. The overall survival of low-grade glioma patients is approximately 60 months, and that for high-grade glioma is only about 13 months [
2]. Invasive growth happens more frequently in glioma patients, which makes complete tumor resection very difficult, leading to a high rate of lethality [
3]. Despite of the considerable progresses achieved in neuro-oncology, the mortality of glioma still remains high [
4]. A previous study indicated that abnormal expression of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), as well as their interaction in gliomas, could be new molecular targets for glioma therapy [
3]. Thus, a better understanding of molecular mechanisms underlying glioma genesis is beneficial to the treatment of glioma.
LncRNAs are a prominent class of RNAs sized 200+ nucleotides, which could control gene expression at transcriptional and posttranscriptional levels, inactivating or stabilizing proteins [
5]. Lots of studies have shown that the dysregulation of lncRNAs may influence cell proliferation and apoptosis, and engage in tumorigenesis and drug resistance. LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) was reported as an oncogenic lncRNA in several types of human cancers. For instance, its upregulation has been documented in kidney cancer, ovarian cancer, lung cancer, breast cancer and glioma [
6]. NEAT1 could facilitate the initiation of hepatocellular carcinoma and predict poor prognosis outcome [
7]. NEAT1 knockdown in glioma stem cells could restrain cell invasiveness and promote cell apoptosis resulted from the activation of let-7e [
8]. In addition, Chen et al. suggested that through affecting the WNT/β-Catenin pathway, NEAT1 contributed to the worsening of glioblastoma [
9]. In brief, lncRNA NEAT1 was suggested as a tumor promotor in many malignancies, including glioma.
On the other hand, miRNAs are highly conserved small single-stranded non-coding RNAs, which could directly regulate gene expression through inhibiting the translation or promoting the degradation of mRNA [
10]. Mounting evidence suggested that the dysregulation of miRNAs contributes to the development of cancers, such as miR-132. Chen et al. reported the inhibitory effects of miR-132 on the proliferation, metastasis and radio-resistance of glioma cells [
11]. Li et al. also demonstrated that miR-132 inhibited the tumorigenicity of glioma cells and promoted their apoptosis [
12]. The suppressive influence that miR-132 imposed on glioma deserves further studies.
Sex determining region Y-box protein 2 (
SOX2), a transcription factor involves in pluripotency induction and maintenance of cell stemness, has been proved to be aberrantly expressed in various solid tumors including breast cancer, lung cancer, prostate cancer, glioblastomas and melanomas [
13].
SOX2 is important for the survival of glioma stem cells and closely associated with the relapse of glioma after chemotherapy or radio-therapy in adults [
14]. Dong et al. suggested that
SOX2 was an oncogene in glioblastoma multiforme and
SOX2 down-regulation could suppress the proliferation, invasion and migration of cancer cells [
15]. Although NEAT1, miR-132 and
SOX2 have been proved to play crucial roles in glioma, their regulatory mechanism and interaction still need to be further studied.
Therefore, we investigated the interaction between lncRNA NEAT1, miR-132 and SOX2 in glioma and identified their roles in glioma. NEAT1 could indirectly regulate SOX2 expression through targeting miR-132. NEAT1 and SOX2 knockdown successfully reduced the invasiveness of glioma cells. These findings can provide new insights into glioma treatment.
Methods
Tissue samples
Resected brain tumors were collected from Jingjiang People’s Hospital from Jan 2012 to Jan 2016. Tissue samples included 14 glioma samples and 5 adjacent non-tumor samples. Tumor samples were pathologically graded as 7 low grade tumors (stage I and stage II) and 7 high grade tumors (stage III and stage IV) according to the WHO criteria. All tissues were directly preserved in liquid nitrogen and stored at − 80 °C. In this study, all investigation and experiments have obtained patients’ consent and been approved by the Ethic Committee for Clinical Research of Jingjiang People’s Hospital. The baseline characteristics of the included patients are shown in Additional file
1: Table S1.
Microarray profiling
Total RNA was extracted from 14 fresh human glioma tissues and five normal tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA) and purified with a RNeasy Mini Kit (Qiagen, Valencia, CA). Besides, sample preparation and microarray hybridization were performed based on the manufacturer’s standard protocols with minor modifications. LncRNAs with differential expressions in glioma tissues were picked out by the whole genome microarray expression profiling with the criteria of log2 (fold change) > 2 and adjusted P < 0.01. Sample labeling, microarray hybridization and washing were all performed according to the manufacturer’s standard protocols. Briefly, Cyanine-3-CTPlabeled cRNA was hybridized onto the lncRNA microarray chips (Affymetrix Human Genome U133 Plus 2.0 Array with platform GPL570). The lncRNA microarray analysis was performed by OE Biotech, Shanghai, China. R program was used to cluster the differentially expressed genes. Euclidean distance function was used for the two-dimensional unsupervised hierarchical clustering. The analysis of the expression difference between glioma and normal tissues was performed by limma package with Benjamini and Hochberg False Discovery Rate method.
Cell line culture
Cell lines were acquired from BeNa Culture Collection (Beijing, China), including glioma cell lines (U87, U251, SHG-44 and U-118MG), human astroglia cell line (HA), and human embryonic kidney cell line (HEK-293). Cells were maintained in high-glucose Dulbecco’s Modified Eagle medium (DMEM, Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) in 5% CO2 at 37 °C.
QRT-PCR
Total RNA extraction was performed using TRIzol reagent (Invitrogen). 200 ng of RNA quantified by NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA) was reverse transcribed using ReverTra Ace qPCR RT Kit (Toyobo, Japan) and then amplified with target gene-specific primers by the THUNDERBIRD SYBR qPCR Mix (Toyobo). Sequences of the primers (all primers were designed and synthesized by Shanghai GenePharma company) are listed in Table
1. The reaction process for PCR was initial denaturation (94 °C, 2 min), denaturation (94 °C, 30 s), primer annealing (54 °C, 30 s), primer extension (72 °C, 1 min), 30 cycles and further extension (72 °C, 10 min). The expression levels of NEAT1,
SOX2 and miR-132 were calculated by 2
− △ △ CT method. NEAT1 and
SOX2 expression levels were normalized to GADPH while miR-132 level was normalized to U6. Each experiment was performed in triplicate and each measure was done in triplicate too.
Table 1
Primers for qRT-PCR
NEAT1 forward | 5’-TGGCTAGCTCAGGGCTTCAG-3’ |
NEAT1 reverse | 5’-TCTCCTTGCCAAGCTTCCTTC-3’ |
SOX2 forward | 5’-ATTTATTCAGTTCCCAGTCCAAGC-3’ |
SOX2 reverse | 5’-CCCTCTCCCCCCACGC-3’ |
miR-132 forward | 5’-TGGATCCCCCCCAGTCCCCGTCCCTCAG-3’ |
miR-132 reverse | 5’-TGAATTCGGATACCTTGGCCGGGAGGAC-3’ |
GAPDH forward | 5’-TGAACGGGAAGCTCACTGG-3’ |
GAPDH reverse | 5’-TCCACCACCCTGTTGCTGTA-3’ |
U6 forward | 5’-CTCGCTTCGGCAGCACA-3’ |
U6 reverse | 5’-AACGCTTCACGAATTTGCGT-3’ |
Western blot analysis
Whole cell lysate of cells was employed to extract total proteins using RIPA buffer (Beyotime, Shanghai, China). Briefly, RIPA buffer was thawed and mixed well. Before using, PMSF was added to RIPA buffer and reached a final concentration of 1 mM. Cells were washed using PBS. -150 μl RIPA buffer was added to every well of the plate. After centrifugation (10,000 g, 5 min), the supernatant was collected for western blot assay. 100 μg of total protein was quantified by Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA) and electrophoretically transferred to a PVDF membrane (Life Technologies, Gaithersburg, MD, USA). The membrane was incubated in turn with TBST supplemented 5% skimmed milk (room temperature, 1 h), primary antibody (4 °C, overnight), and secondary antibody (room temperature, 1.5 h). Anti-SOX2 (BA3292) and anti-β-actin (BM3873) from Bosterbio (Wuhan, China) were diluted to 1:200 and used as primary antibodies. HRP-linked anti-human IgG (BM1921, 1:200, Bosterbio) was the secondary antibody. The visualization was enhanced by ECL Plus (Life Technologies, Gaithersburg, MD, USA). β-actin was the internal control. Each experiment was performed in triplicate and each measure was done in triplicate too.
Cell transfection
Negative control (NC), miR-132 mimics, miR-132 inhibitor, si-NC, si-NEAT1, si-
SOX2, pcDNA3.1 plasmids, pcDNA3.1-NEAT1 and pcDNA3.1-
SOX2 were all obtained from Genepharma. The cell suspension was prepared by pancreatin digested U87 and U251 cells and complete medium. Then the cells were incubated in six-well plates (1 × 10
6 cells/well) for 18–24 h. Three hours before transfection, glioma cells at 80–90% confluence were incubated with fresh medium without serum and antibiotics. Transfection was performed using Lipofectamine 2000 (~ 0.6 μg Lipofectamine reagent/1 μg DNA, Life Technologies). Sequences of purchased siRNA, mimics and inhibitor are listed in Table
2.
Table 2
Purchased siRNA and mimics/inhibitor
si-NC | 5’-CCCACCAGUUUGAGAC UCCACAAAU-3’ |
si-NEAT1 | 5’-GGTCTGTGTGGAAGGAGGAAGGCAG-3’ |
si-SOX2 | 5’-CUGCAGUACAACUCCAUGAUU-3’ |
miR-132 mimics | 5’- UGUCAGUUUGUCAAAUACCCCA-3’ |
miR-132 inhibitor | 5’- TGGGGTATTTGACAAACTGACA − 3’ |
Dual luciferase reported assay
HEK-293 cells were cultured in 12-well plate and co-transfected with pcDNA3.1 plasmid vectors carrying either wild or mutated NEAT1 or SOX2 3’UTR sequences together with miR-132 mimics or mimics NC. The NEAT1 and SOX2 3’ UTR constructs were purchased from GenePharma (Shanghai, China). Firefly and Renilla luciferase activities were measured 48 h after transfection using the dual-luciferase reporter assay (Promega, Madison, WI, USA). The luciferase activity was calculated as the ratio of firefly luciferase intensity/renilla luciferase intensity.
MTT assay
Transfected glioma cells were seeded in a 96-well plate (1 × 104 cells/well) to incubate for 24, 48, 72 and 96 h. Then 10 μl of 5 mg/mL MTT prepared in PBS (pH = 7.4, Sigma-Aldrich, St. Louis, MO, USA) was added to each well for incubation (4 h). After the supernatants were removed, dimethyl sulfoxide (DMSO, Thermo Fisher Scientific, Waltham, MA, USA) was added (100 μl/well) and the absorbance value (at 490 nm) was measured by a microplate reader. Each experiment was performed in triplicate and each measure was done in triplicate too.
Transwell assay
To investigate cell migration ability, Transwell assay was conducted using a 24-well insert with 8 μm pores (Corning Incorporated, Corning, NY, USA). Cells were firstly dissociated by pancreatin, then resuspended in 100 μl serum-free medium (with 1% FBS), and finally placed in the upper chamber. In the lower chamber, 500 μl 10% FBS medium was added. The cell density was 5 × 105 cells/ml. After incubation at 37 °C for 6 h, the cells on the upper side of membrane were scraped off, while those on the lower side were observed using a microscope. Before observation, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min. As for assessing cell invasion, 50 μl diluted Matrigel™ (1:8, Sigma-Aldrich, St Louis, MO, USA) was added to the upper chamber of the transwell. 500 μl 10% FBS medium was added in the lower chamber. The cell density was 5 × 105 cells/ml was added in upper chamber resuspended in 100 μl 10% FBS medium. After incubation at 37 °C for 36 h, the cells on the upper side of membrane were scraped off, while those on the lower side were observed using a microscope. Each experiment was performed in triplicate and each measure was done in triplicate too.
Statistical analyses
The form of mean ± standard errors was used to express the data. To compare the differences among two or multiple groups, paired Student’s t test or One-Way ANOVA were applied. P < 0.05 signified statistical significance. The data analysis software used was GraphPad Prism 6.0.
Discussion
As an oncogene, lncRNA NEAT1 played a role in glioma by targeting miR-132 and then indirectly promoting SOX2 expression. NEAT1 and SOX2 knockdown could restrain the viability and invasiveness of glioma cells. To sum up, NEAT1 knockdown inhibited the aggression of glioma through down-regulating SOX2 by targeting miR-132.
The effects of NEAT1 in human cancers have been thoroughly studied. For instance, NEAT1 was considered to be a competing endogenous RNA to modulate the expression of
ATAD2 by suppressing miR-106b-5p in papillary thyroid cancer [
16]. NEAT1 could also be activated by
Oct4’s binding to its promoter, which therefore led to lung cancer progression and poor prognosis outcome [
17]. In addition, NEAT1 could enhance the radio-resistance of cervical cancer by suppressing miR-193b-3p-CCND1 interaction [
17]. So, generally, NEAT1 could regulate cancer progression by interrupt the crosstalk between miRNA and miRNA’s target mRNA. In glioma, NEAT1 expression was elevated and positively associated with the grade of glioma. The over-expression of NEAT1 in glioma was also identified by other researchers. He et al. found that higher NEAT1 expression was closely related to higher WHO grade of glioma, which was in line with our results [
18]. The up-regulation of NEAT1 in glioma stem cells was also reported by Gong et al. [
8]. The aberrant expression of NEAT1 might be a biomarker of the occurrence and exacerbating of glioma.
With a high expression in tumor tissues and cells, NEAT1 exerted tumorigenesis-promotive function in glioma. The knockdown of NEAT1 could inhibit glioma cell growth and metastasis. Accumulated evidences supported our findings. For instance, it was revealed that NEAT1 down-regulation could suppress the development of glioma in vitro through restraining the mobility and viability of glioma cells as well as inducing apoptosis, which was similar to our findings [
9,
19]. According to Yang et al.’s study, silencing NEAT1 reduced the proliferation but promoted the apoptosis of glioma cells CD133 and U87 [
20]. Our results also confirmed the inhibitory effect of NEAT1 knockdown on the viability and invasion of glioma cells. In consequence, NEAT1 knockdown was effective in reducing aggression and proliferation of glioma cells.
In addition to NEAT1,
SOX2 was also over-expressed in glioma as well as other human tumors.
SOX2 has been identified as an oncogene that promotes squamous cancer development by promoting cancer cell viability, proliferation, migration and invasion
etc [
21‐
28]. Silencing
SOX2 in glioma cells had similar inhibitory effects as NEAT1 knockdown. The tumor promotive effect of
SOX2 in glioma was reported in many previous studies. Berezovsky et al. demonstrated the up-regulation of
SOX2 in glioblastoma and found that it had a higher expression in glioma with higher grade, in agreement with our study that
SOX2 expression elevated with the increasing of glioma grade [
29]. In addition, Dong et al. found that the restoration of
SOX2 attenuated the anti-tumor effect of miR-429 in glioblastoma [
15]. Gangemi et al. also demonstrated that
SOX2 knockdown in glioblastoma tumor-initiating cells suppressed cell growth and tumorigenicity [
30]. Considering the strong association between the high
SOX2 level and glioblastoma development, some researchers believed that
SOX2 can be a target for glioma therapy in future [
31]. Therefore,
SOX2 positively affected cancer development in glioma.
In our study, we identified that NEAT1 could indirectly regulate the expression of
SOX2 through targeting miR-132. NEAT1 knockdown could down-regulate
SOX2 by up-regulating miR-132, thus suppressing glioma cell growth and invasiveness. MiR-132 was considered as a suppressor in various malignancies, such as lung cancer [
32], colorectal cancer [
33], breast cancer [
34] and ovarian cancer [
35]. MiR-132 was reported to inhibit hepatocellular carcinoma cell proliferation and promote cell apoptosis by suppressing Shh/Hedgehog signaling [
36]. In addition, epidermal growth factor and
TALIN2 could be suppressed by miR-132, therefore inhibit prostate cancer metastasis [
37]. In pancreatic cancer, miR-132 inhibited Akt signaling and suppressed pancreatic cancer cell proliferation [
38]. However, studies on its effect in glioma were not too much. Some studies proved that miR-132 was an anti-oncogene that inhibited migration and invasion of glioma [
39,
40]. Nevertheless, its target relationship with NEAT1 has never been revealed except for this study. The interactions between NEAT1, miR-132 and
SOX2 in glioma were also novel findings. NEAT1 was found to function as a competing endogenous RNA in other studies, which shared a similar regulatory mechanism with our study as it regulated gene expressions through binding to miRNAs. For instance, Zhang et al. found that NEAT1 could sponge miR-485 and enhance the expression of
STAT3 in hepatocellular carcinoma [
7]. In glioma, NEAT1 could specifically bind to miR-449b-5p and miR-181d-5p to inhibit their transcriptions, leading to the over-expression of oncogenes
c-Met and
SOX5, respectively [
19,
41]. Here, we uncovered that NEAT1 could modulate
SOX2 expression in glioma by sponging miR-132.
Although we identified the promotive function of NEAT1 in glioma and investigated its relationship with miR-132 and SOX2, the limitations should be pointed out and made up in the future. For example, the effect of miR-132 on the viability and invasion of glioma cells needed to be further investigated. The in vivo experiments were essential to verify the in vitro results. Clinical analysis could be conducted to evaluate the association between the expression of NEAT1 or SOX2 and the prognosis outcome of glioma patients.