MiR-181b-5p Downregulates NOVA1 to Suppress Proliferation, Migration and Invasion and Promote Apoptosis in Astrocytoma
Migration and Invasion and
Promote Apoptosis in Astrocytoma. PLoS ONE 9(10): e109124. doi:10.1371/journal.pone.0109124
MiR-181b-5p Downregulates NOVA1 to Suppress Proliferation, Migration and Invasion and Promote Apoptosis in Astrocytoma
Feng Zhi 0
Qiang Wang 0
Danni Deng 0
Naiyuan Shao 0
Rong Wang 0
Lian Xue 0
Suinuan Wang 0
Xiwei Xia 0
Yilin Yang 0
Chad Creighton, Baylor College of Medicine, United States of America
0 1 Modern Medical Research Center, Third Affiliated Hospital of Soochow University , Changzhou, Jiangsu , China , 2 Department of Neurosurgery, Third Affiliated Hospital of Soochow University , Changzhou, Jiangsu , China
MicroRNAs (miRNAs) are small, short noncoding RNAs that modulate the expression of numerous genes by targeting their mRNA. Numerous abnormal miRNA expression patterns are observed in various human malignancies, and certain miRNAs can act as oncogenes or tumor suppressors. Astrocytoma, the most common neuroepithelial cancer, represents the majority of malignant brain tumors in humans. In our previous studies, we found that the downregulation of miR-181b-5p in astrocytomas is associated with a poor prognosis. The aim of the present study was to investigate the functional role of miR181b-5p and its possible target genes. miR-181b-5p was significantly downregulated in astrocytoma specimens, and the reduced expression of miR-181b-5p was inversely correlated with the clinical stage. The ectopic expression of miR-181b-5p inhibited proliferation, migration and invasion and induced apoptosis in astrocytoma cancer cells in vitro. The NOVA1 (neuro-oncological ventral antigen 1) gene was further identified as a novel direct target of miR-181b-5p. Specifically, miR181b-5p bound directly to the 3'-untranslated region (UTR) of NOVA1 and suppressed its expression. In clinical specimens, NOVA1 was overexpressed, and its protein levels were inversely correlated with miR-181b-5p expression. Furthermore, the changing level of NOVA1 was significantly associated with a poor survival outcome. Similar to restoring miR-181b-5p expression, downregulating NOVA1 inhibited cell growth, migration and invasion. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p. Our results indicate that miR-181b-5p is a tumor suppressor in astrocytoma that inhibits tumor progression by targeting NOVA1. These findings suggest that miR-181b-5p may serve as a novel therapeutic target for astrocytoma.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This work was supported by National Natural Science Foundation of China (31071046, 81302197), Changzhou Social Development Project
(CS20092015, CS20102010), Changzhou Health Bureau Project (ZD200903, ZD201007), Changzhou Science Technology Bureau Guiding Project (CY20119004). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Astrocytomas are the most common primary brain tumors in
the central nervous system . The prognosis of patients with
astrocytoma is closely related to the WHO tumor grade, and
patients with glioblastoma, the most common histological type,
have a poor prognosis, with a median survival of approximately 15
months . Thus, efforts to better understand the biological basis
of astrocytoma progression may provide important clinically
relevant insights into disease management.
MicroRNAs (miRNAs) are a class of highly conserved,
singlestranded, small, noncoding RNA molecules known as endogenous
regulators of post-transcriptional gene expression that regulate
expression through translational repression and messenger RNA
cleavage . It has been widely accepted that miRNAs play
pivotal roles in various biological processes, including
development, cell proliferation, differentiation, apoptosis and metabolism
. Accumulating evidence also suggests that miRNAs participate
in the tumor angiogenesis, invasion and metastasis of human
malignancies, acting as oncogenes or tumor suppressors depending
on their targets, information that may provide insight into the
diagnosis and prognosis of human cancers . Our previous
studies indicated that miR-181b-5p is downregulated in
astrocytoma and that this reduced expression of miR-181b-5p is
associated with a poor survival outcome, suggesting that
miR181b-5p may act as a tumor suppressor during astrocytoma
development and/or progression . miR-181b-5p belongs to the
miR-181 family, which includes miR-181a-5p, miR-181b-5p and
miR-181c-5p. However, whether miR-181b-5p is a tumor
suppressive or oncogenic miRNA remains controversial, and the
regulatory mechanism underlying miR-181b-5p-mediated
function remains to be elucidated in different cancers. Recently,
miR181b-5p was found to inhibit glioma cell proliferation, migration,
invasion and tumorigenesis by targeting IGF-1R  and to reduce
chemoresistance to temozolomide in glioma cells by targeting
MEK1 . As it is well known that miRNAs regulate the
expression of multiple target genes and affect a variety of cellular
pathways, further research is thus required to fully understand
their contributions to this malignancy.
In this study, we clearly demonstrate that miR-181b-5p
functions as a tumor suppressor miRNA in astrocytoma. The
tumor-suppressive effect was mediated via the repression of
NOVA1 (neuro-oncological ventral antigen 1). Our findings
provide valuable clues toward understanding the mechanisms of
astrocytoma pathogenesis and present an opportunity for the
development of new effective clinical therapies.
Materials and Methods
Human tissue samples
Surgically excised tumor specimens from 90 patients with
astrocytomas and normal adjacent tissues (NATs) from 25
astrocytoma patients were collected in the Department of
Neurosurgery of the Third Affiliated Hospital of Soochow
University, China. The cases included 8 patients with grade I,
26 with grade II, 33 with grade III and 23 with grade IV
astrocytomas. The histological grading was performed on the basis
of World Health Organization (WHO) criteria. The collected
tissues were immediately snap-frozen in liquid nitrogen and stored
at -80uC. Written informed consent was obtained from all of the
patients or their representatives before the study, which was
approved by the Research Ethics Board of the Third Affiliated
Hospital of Soochow University.
The human astrocytoma cell lines U251 and U87 were
purchased from Cell Resource Centre of the Shanghai Institutes
for Biological Sciences of the Chinese Academy of Sciences. All of
the cells were cultured in Dulbeccos Modified Eagles Medium
(Invitrogen, USA) supplemented with 10% fetal bovine serum
(Hyclone, USA) in a humidified 37uC incubator that was
maintained at 5% CO2.
Oligonucleotides and cell transfection
The oligonucleotide miR-181b-5p mimic (pre-miR-181b-5p),
mimic negative control (pre-ncRNA), miR-181b-5p inhibitor
(antimiR-181b-5p) and inhibitor negative control (anti-ncRNA) were
purchased from GenePharma (Shanghai, China). A small
interfering RNA (siRNA) targeting NOVA1 (si-NOVA1) and a
NOVA1 vector encoding the entire coding sequence without the
39-UTR were designed and synthesized by Invitrogen (Invitrogen,
USA). A scrambled siRNA (si-NC) and a control vector were
included as negative controls. miRNAs, siRNAs and vectors were
transfected into cells at the indicated concentrations using
Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according
to the manufacturers instructions. Total RNA was isolated at 24 h
post-transfection. Total cellular protein was isolated at 48 h after
RNA isolation and quantitative real-time PCR
Total RNA was extracted from cultured cells or clinical samples
using TRIzol Reagent (Invitrogen) according to the
manufacturers protocols. The RNA molecules were then treated with
RNasefree DNase (TaKaRa, Dalian, China) using a standardized
protocol. The levels of mature miR-181b-5p were quantified
using Taqman miRNA probes (Applied Biosystems), as previously
reported . The U6 small nuclear RNA was selected as a
control. For the analysis of NOVA1 and b-actin, quantitative
realtime PCR (qRT-PCR) was performed using an Applied
Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster
City, CA, USA) with SYBR green dye (Invitrogen). The sequences
of the sense and antisense primers used for the amplification of
NOVA1 and b-actin were as follows: NOVA1 (sense),
5TACTGAGCGAGTGTGCTTGAT-3, NOVA1 (antisense),
5GTCTGGGGTTGTAGAATGCTG-3; b-actin (sense),
5-AGGGAAATCGTGCGTGAC-3, and b-actin (antisense),
5CGCTCATTGCCGATAGTG-3. All of the reactions were
performed in triplicate.
Total cellular protein was isolated using RIPA lysis buffer
(Sigma-Aldrich Inc., St Louis, MO) at 48 h after transfection.
Western blotting analyses were performed using conventional
protocols, as previously described . Briefly, a rabbit
monoclonal b-actin antibody (Cell Signaling Technology, USA), rabbit
polyclonal NOVA1 antibody (Abcam, UK) and secondary rabbit
IgG-HRP (Sigma, USA) were applied at 1:1,000, 1:500 and
1:5,000 dilutions, respectively. The Quantity One analysis
program (Bio-Rad, USA) was used to obtain the quantitative data.
The entire human NOVA1 39-untranslated region (39-UTR)
was amplified by PCR using human genomic DNA as the
template. The PCR products were inserted into the p-MIR-report
plasmid (Ambion). Efficient insertion was confirmed by
sequencing. For luciferase reporter assays, cells were cultured in 6-well
plates, and each well was transfected with 2 mg of firefly luciferase
reporter plasmid, 2 mg of b-galactosidase expression vector
(Ambion) and equal amounts of pre-ncRNA, pre-miR-181b-5p,
anti-ncRNA or anti-miR-181b-5p using Lipofectamine 2000
(Invitrogen). The b-galactosidase vector was used as a transfection
control. At 24 h post-transfection, the cells were assayed using
luciferase assay kits (Promega, Madison, WI, USA). The data
depicted represent three independent experiments performed on
Cell viability assay
Cells in the logarithmic phase of growth were seeded at 2000
per well in 96-well plates and cultured for 12, 24, 48 and 72 h.
Next, 20 ml MTT (5 mg/mL) was added to each test well and
incubated for 4 h at 37uC. The supernatant was discarded, and
150 ml of dimethyl sulfoxide (DMSO) was added to each well to
solubilize the crystals for 10 min at room temperature. The optical
density (OD) was measured at a wavelength of 570 nm.
Cells were cultured to 95% confluence in six-well plates. Cell
layers were scratched using a 20-mL tip to form wound gaps,
washed twice with PBS and cultured. The wound healing was
photographed at different time points, and each wound was
analyzed by measuring the distance migrated by the cells in three
Figure 1. miR-181b-5p is downregulated in astrocytoma tissue samples. The miR-181b-5p expression was normalized to U6. (A) Relative
expression of miR-181b-5p in NAT and astrocytoma tissues. (B) Relative expression of miR-181b-5p in different WHO grades of cancer tissues.
Cell invasion assay
A cell invasion assay was performed using BD Matrigel invasion
chambers (BD Biosciences) following the manufacturers protocol.
Cells were suspended in serum-free DMEM culture medium at a
concentration of 46105 cells/mL and then added to the upper
chamber (46104 cells/well) with a Matrigel-coated filter. Simul
taneously, the lower chambers were filled with 10% FBS as a
chemoattractant, and the cells were incubated in the chambers for
48 h. At the end of the experiments, the cells on the upper surface
of the membranes were removed using a cotton swab, and the cells
on the lower surface were fixed and stained with 0.1% crystal
violet. Five visual fields of each insert were randomly chosen and
counted under a microscope (IX71, Olympus, Japan). Image-Pro
Insight (Version 8.0.21) software was used to count the migrated
and non-migrated cells. The mean number of migrating or
invading cells was expressed as a percentage relative to the control.
Cells were harvested and washed in cold PBS. Combined
Annexin V and propidium iodide staining was performed using
the Annexin V-FITC apoptosis detection kit (BD Biosciences, San
Jose, CA) according to the manufacturers protocol. After the flow
cytometric analysis of the cells, apoptosis profiles were obtained
with the Cell Quest Pro software.
The data shown are presented as the mean 6 SD of at least
three independent experiments. The differences were considered
statistically significant when p ,0.05. A survival curve was
estimated using the Kaplan-Meier method in SPSS 13.0, and the
resulting curves were compared using the log-rank test.
Aberrant expression of miR-181b-5p in human
To assess the expression of miR-181b-5p in astrocytomas, a
qRT-PCR analysis was performed on 25 NAT samples and 90
astrocytoma tissue samples. The results showed that the expression
of miR-181b-5p was consistently lower in the astrocytoma tissues
compared with the NAT samples (Figure 1A). We then divided the
astrocytoma samples into 4 groups according to their tumor grade.
As shown in Figure 1B, the expression of miR-181b-5p in the
high-grade astrocytomas was significantly lower than in the
lowgrade astrocytomas. The expression of miR-181b-5p was
progressively decreased from the WHO grade I to the WHO grade IV
astrocytomas. These data support the notion that low
miR-181b5p expression is closely related to astrocytoma progression and
that this miRNA may act as a tumor suppressor in astrocytoma.
miR-181b-5p overexpression inhibits cell proliferation,
migration and invasion and induces apoptosis in vitro
To investigate the biological functions of miR-181b-5p in
astrocytoma cells, U251 cells were transfected with equal
concentrations of pre-ncRNA, pre-miR-181b-5p, anti-ncRNA or
anti-miR-181b-5p and analyzed for cell growth. The expression of
miR-181b-5p was significantly increased by the introduction of
pre-miR-181b-5p, whereas anti-miR-181b-5p abolished the
miR181b-5p levels in U251 cells (Figure 2A). The proliferation assay
revealed that the cells that were transiently transfected with
premiR-181b-5p proliferated at a significantly reduced rate; in
contrast, the cells transfected with anti-miR-181b-5p proliferated
at a significantly enhanced rate (Figure 2B). The relative cell
survival rate of the pre-miR-181b-5p-transfected cells at 72 h was
66.5%, and the relative cell survival rate of the
anti-miR-181b-5ptransfected cells at 72 h was 138.7%. To further detect whether
miR-181b-5p is associated with the migration ability of
astrocytoma, we analyzed the effect of miR-181b-5p expression on the
migratory and invasive behavior of U251 cells. The overexpression
of miR-181b-5p decreased the migration capacity of astrocytoma
cells, whereas the inhibition of miR-181b-5p promoted migration
(Figure 2C). Furthermore, the overexpression of miR-181b-5p
induced by pre-miR-181b-5p transfection reduced the invasive
ability of U251 cells, whereas the knockdown of miR-181b-5p
significantly enhanced this ability (Figure 2D). Next, we used
Annexin V and PI double-staining FACS analysis to investigate
the effects of miR-181b-5p overexpression on astrocytoma cell
apoptosis. As shown in Figure 2E, the overexpression of
miR181b-5p induced by transfection with pre-miR-181b-5p resulted in
a significant increase in apoptotic cells, whereas the inhibition of
miR-181b-5p slightly reduced the number of apoptotic cells. The
biological functions of miR-181b-5p on cell proliferation,
migration, invasion and apoptosis were also investigated in U87 cells. As
shown in Figure S1 in File S1, overexpression of miR-181b-5p
significantly inhibited the proliferation (Figure S1A in File S1),
migration (Figure S1B in File S1), invasion (Figure S1C in File S1)
and promoted cellular apoptosis (Figure S1D in File S1) in U87
cells, while the inhibition of miR-181b-5p had opposite effects.
These results showed that miR-181b-5p expression contributes to
the regulation of astrocytoma cell proliferation and motility in
NOVA1 is a direct target of miR-181b-5p
To fully understand the mechanisms of miR-181b-5p action in
astrocytoma, we used three computational algorithms, miRanda,
TargetScan and Pictar, to search for potential targets of
miR181b-5p and selected NOVA1 for further analysis. The predicted
interaction between miR-181b-5p and its target binding sites
within the NOVA1 39-UTR is illustrated in Figure 3A. To
determine whether the negative regulatory effects of miR-181b-5p
on NOVA1 expression were mediated through binding to the
presumed complementary sites at the 39-UTR of NOVA1, we
fused the entire NOVA1 39-UTR into a downstream position of a
firefly luciferase reporter plasmid. The resulting plasmid was
introduced into U251 cells combined with a transfection control
plasmid (b-gal) as well as pre-ncRNA, pre-miR-181b-5p,
antincRNA or anti-miR-181b-5p. As shown in Figure 3B,
miR-181b5p overexpression significantly decreased the luciferase reporter
activity (normalized against b-gal activity) compared with the
prencRNA treatment, whereas the inhibition of miR-181b-5p
significantly increased the reporter activity. However, transfection
with the parental luciferase plasmid (without the NOVA1
39UTR) or with the mutant luciferase plasmid (the NOVA1 39-UTR
with mutations in the seed complementary site) did not affect the
luciferase reporter activity. We next investigated whether
miR181b-5p could regulate NOVA1 at both the mRNA and protein
levels. miR-181b-5p inhibitors or mimics were transfected into
cancer cells, and the levels of NOVA1 mRNA and protein were
monitored. A qRT-PCR analysis revealed that the inhibition of
miR-181b-5p in U251 cells led to increased expression of
endogenous NOVA1 mRNA compared with the control;
conversely, the enhanced expression of miR-181b-5p decreased
the expression of endogenous NOVA1 mRNA compared with the
control (Figure 3C). The expression levels of NOVA1 protein were
assessed by a western blot analysis at 48 h after transfection. All of
the cells transfected with pre-miR-181b-5p exhibited reduced
NOVA1 expression relative to the cells transfected with
pre-ncRNA, whereas the cells transfected with anti-miR-181b-5p
exhibited enhanced NOVA1 expression relative to the cells
transfected with anti-ncRNA (Figure 3D). These results strongly
demonstrate that NOVA1 is a target gene of miR-181b-5p, which
directly recognizes the 39-UTR of the NOVA1 transcript to
downregulate its expression. As NOVA1 is the direct target gene
of miR-181b-5p, we asked whether NOVA1 was upregulated in
human samples. Figure 3E displays representative western blots of
NOVA1 in astrocytomas. The expression of NOVA1 in the
astrocytomas was significantly higher than that in the NAT
samples, and the expression levels increased as the tumor grade
increased. The relative expression changes of NOVA1 in
astrocytomas are shown in Figure 3F. To determine whether
reduced miR-181b-5p expression correlates with levels of NOVA1
expression in tumor tissues, Spearmans correlation analysis was
carried out. The result showed that there was an inverse
correlation between the expression levels of NOVA1 and
miR181b-5p (r = 20.60) in human astrocytomas (Figure 3G). In our
previous studies, we found that the reduced expression of
miR181b-5p was significantly associated with poor survival outcome,
so we asked whether NOVA1 upregulation was correlated with
patient survival. As shown in Figure 3H, patients with high
NOVA1 expression levels exhibited poorer survival outcomes than
patients with low NOVA1 expression levels (p = 0.035). These
results further confirm the negative regulation of NOVA1 by
Knockdown of NOVA1 inhibits cellular proliferation,
migration and invasion and induces apoptosis in vitro
To determine the effects of NOVA1 on cancer cell proliferation,
migration, invasion, and apoptosis, we performed the targeted
knockdown of NOVA1 expression using RNAi in U251 cells. A
cell proliferation assay showed that NOVA1 knockdown
significantly inhibited cell growth (Figure 4A). Consequently,
woundhealing and Transwell assays examining migration and invasion
were performed. Decreased migrating and invading U251 cells
were observed when transfected with si-NOVA1 compared to the
controls (Figure 4B-4C). Lastly, an apoptosis assay showed that
NOVA1 knockdown promoted apoptosis (Figure 4D). These
results were similar in U87 cells, as shown in Figure S2 in File S1.
Overexpression of NOVA1 reverses the inhibitory effects
As shown above, overexpression of miR-181b-5p inhibited
proliferation, migration, and invasion of astrocytoma cells. We also
validated NOVA1 as a direct target of miR-181b-5p. Similar to
miR-181b-5p overexpression, we found that reduced expression of
NOVA1 significantly inhibited cell proliferation, migration and
invasion, similar to those induced by miR-181b-5p. To further
elucidate whether the tumor suppressive effect of miR-181b-5p
was mediated by repression of NOVA1 in astrocytoma cells, a
NOVA1 expression vector which encoded the entire coding
sequence without the 39-UTR was transfected into U251 cancer
cells. As shown in Figure 5, ectopic expression of NOVA1
significantly prevented the suppression of proliferation (Figure 5A)
and apoptosis (Figure 5B) induced by miR-181b-5p, indicating
that NOVA1 was directly responsible for the biological effects
induced by miR-181b-5p overexpression.
Accumulating evidence has indicated that the aberrant
expression of miRNAs may be a common mechanism involved in the
development of various cancers . Regardless, further
investigation of cancer-specific miRNAs and their targets is necessary to
fully understand their role in the pathogenesis of tumors and may
be important for the design of novel therapeutic targets .
In our previous study, we established a unique molecular
diagnostic signature for astrocytomas that included miR-21-5p,
miR-24-3p, miR-30c-5p, miR-106a-5p, miR-124-3p, miR-137
and miR-181b-5p . miR-106a-5p and miR-181b-5p are two of
the most significantly downregulated miRNAs in astrocytomas,
and their low expression levels are significantly associated with a
poor survival outcome; this observation triggered our interest in
investigating their function and their target genes during
astrocytoma development. In our recent work, we proved that
miR-106a-5p inhibits the proliferation and migration of
astrocytoma cells and promotes apoptosis by targeting FASTK .
However, the potential role of miR-181b-5p as an oncogene or a
tumor suppressor in cancer development remains controversial.
Figure 4. NOVA1 downregulation inhibited cell proliferation, migration and invasion and promoted apoptosis in vitro. (A)
Downregulation of NOVA1 decreased cell growth (* p,0.05, ** p,0.01). The experiment was repeated three times. (B) Downregulation of NOVA1
decreased cell migration ability. (C) Downregulation of NOVA1 decreased cell invasion ability. The images shown are representative images from
three independent experiments, and a statistical analysis was performed (mean 6 SD, *** p,0.001). (D) Downregulation of NOVA1 promoted
Figure 5. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p. (A) Cell proliferation assay. An MTT cell viability assay
was performed at 0, 24, 48 and 72 h after the transfection of U251 cells with control vector + pre-ncRNA, control vector + pre-miR-181b-5p, NOVA1
vector + pre-ncRNA, and NOVA1 vector + pre-miR-181b-5p. The experiment was repeated three times. (B) Cell apoptosis assay. U251 cells were
transfected with control vector + pre-ncRNA, control vector + pre-miR-181b-5p, NOVA1 vector + pre-ncRNA, and NOVA1 vector + pre-miR-181b-5p.
The experiment was repeated three times, and representative data were shown.
miR-181b-5p promotes cell proliferation, increases cell migration
and invasion and inhibits apoptosis in prostate cancer  and
gastric cancer by targeting TIMP3 . miR-181b-5p also
enhances drug resistance in breast cancer . In contrast,
this miRNA inhibits cell proliferation and colony formation and
induces apoptosis in gastric cancer by targeting CREB1  and
reduces drug resistance in pancreatic cancer by inhibiting CYLD
and BCL-2 . Previous studies have reported that
miR181b-5p functions in gliomas to suppress growth by targeting the
IGF-1R oncogene  and modulates glioma cell sensitivity to
temozolomide by targeting MEK1 . However, it is now well
known that miRNAs regulate the expression of multiple target
genes and affect a variety of cellular pathways. Here, consistent
with our previous studies, we found that miR-181b-5p was
downregulated in human astrocytoma tissues. The ectopic
expression of miR-181b-5p suppressed cell proliferation, migration
and invasion and induced apoptosis in vitro. On the basis of a
bioinformatic analysis, we further confirmed NOVA1 as a direct
target of miR-181b-5p. Therefore, this study may provide new
therapeutic strategies for astrocytoma prevention and treatment.
NOVA1 belongs to the Nova family of neuron-specific
RNAbinding proteins, which were originally identified as targets in an
autoimmune neurologic disease characterized by the failure of
motor inhibition. NOVA1 is essential for the proper development
of the mammalian motor system and for the survival of
motoneurons by controlling the alternative processing of a wide
array of mRNAs that are important for synaptic activity .
NOVA1 is known as a splicing factor that regulates the inclusion
or exclusion of exons depending on the position of NOVA binding
relative to splice sites: when NOVA1 binds to the 39-region of the
cassette exon, it promotes the inclusion of this exon . NOVA1
also regulates the alternative splicing of pre-mRNAs encoding the
inhibitory neurotransmitter receptor subunits GABA(A)Rgamma2
and GlyRalpha2 by directly binding to intronic elements, resulting
in the enhancement of exon inclusion . The antagonistic role
of NOVA1 is exemplified in the alternative splicing of D2R
(dopamine D2 receptor) pre-mRNA as a pre-mRNA-binding
protein . NOVA1 is also an essential regulator of Z+_agrin,
which induces AChR clusters through interaction with the agrin
receptor (Lrp4), leading to phosphorylation of the muscle-specific
receptor tyrosine kinase MuSK . Furthermore, NOVA1 might
mediate neuronal responsiveness, and its expression might
positively correlate with neural repair after ischemia/reperfusion
insults in the rat brain . In our study, miR-181b-5p directly
targeted NOVA1 expression in astrocytoma cells, playing
important roles in disease progression. The overexpression of
miR-181b5p and corresponding reduced expression of NOVA1 decreased
the oncogenic potential of cells, as evidenced by decreases in the
proliferation rate and cell migration and increased apoptosis
damage. The mechanism by which miR-181b-5p decreases the
oncogenic potential of cells is most likely through the inhibition of
NOVA1. NOVA1 is expressed only in the central nervous system
(CNS) in mammals and has been shown to regulate ,700
alternatively spliced exons by binding to YCAY clusters in target
pre-mRNAs [25,30]. Importantly, most of these targets are genes
involved in synaptic function and axon guidance; thus, NOVA1
regulation plays an important role in shaping neuronal wiring and
function . The aberrant expression of NOVA1 caused by
miR-181b-5p may lead to the inappropriate alternative splicing of
oncogenes, which may facilitate astrocytoma pathogenesis. To the
best of our knowledge, this study is the first report showing the
direct regulation of NOVA1 by miR-181b-5p.
In summary, our data indicate that miR-181b-5p is a tumor
suppressor gene in astrocytomas. The overexpression of
miR181b-5p inhibits astrocytoma cell proliferation, migration and
invasion and promotes apoptosis. Moreover, we show that
NOVA1 is a direct target of miR-181b-5p. Although much
remains to be elucidated in terms of the role of miR-181b-5p in
the pathogenesis of astrocytomas, miR-181b-5p represents a new
potential therapeutic target for the treatment of this cancer.
File S1 Supporting figures. Figure S1, The role of
miR-181b5p in U87 cell proliferation, migration, invasion and apoptosis in
vitro. (A) The role of miR-181b-5p in cell proliferation. An MTT
cell viability assay was performed at 0, 24, 48 and 72 h after the
transfection of U87 cells with equal concentrations of pre-ncRNA,
pre-miR-181b-5p, anti-ncRNA and anti-miR-181b-5p. For
comparison, the expression levels of miR-181b-5p in
pre-miR-181b-5por anti-miR-181b-5p transfected cells were compared with their
respective negative controls (* p,0.05, *** p,0.001). The
experiment was repeated three times. (B) Wound-healing assays of
U87 cells treated with equal concentrations of pre-ncRNA,
premiR-181b-5p, anti-ncRNA and anti-miR-181b-5p. The wound
gaps were photographed and measured. The images shown are
representative images from three independent experiments. (C)
Transwell assays of U87 cells treated with equal concentrations of
pre-ncRNA, pre-miR-181b-5p, anti-ncRNA and
anti-miR-181b5p. The images shown are representative images from three
independent experiments, and a statistical analysis was performed
(mean 6 SD; *** p,0.001). (D) The role of miR-181b-5p in
apoptosis in U87 cells. U87 cells were transfected with equal
concentrations of pre-ncRNA, pre-miR-181b-5p, anti-ncRNA and
anti-miR-181b-5p. The experiment was repeated three times, and
representative data are shown. Figure S2, NOVA1 downregulation
inhibited cell proliferation, migration and invasion and promoted
apoptosis in U87 cells. (A) Downregulation of NOVA1 decreased
U87 cell growth (** p,0.01, *** p,0.001). The experiment was
repeated three times. (B) Downregulation of NOVA1 decreased
U87 cell migration ability. (C) Downregulation of NOVA1
decreased U87 cell invasion ability. The images shown are
representative images from three independent experiments, and a
statistical analysis was performed (mean 6 SD; *** p,0.001). (D)
Downregulation of NOVA1 promoted apoptosis.
Conceived and designed the experiments: FZ XWX YLY. Performed the
experiments: FZ QW DND RW LX. Analyzed the data: FZ DND QW
LX SNW. Contributed reagents/materials/analysis tools: QW DND NYS
SNW XWX. Wrote the paper: FZ QW YLY XWX.
1. Wen PY , Kesari S ( 2008 ) Malignant gliomas in adults . N Engl J Med 359 : 492 - 507 .
2. Gabayan AJ , Green SB , Sanan A , Jenrette J , Schultz C , et al. ( 2006 ) GliaSite brachytherapy for treatment of recurrent malignant gliomas: a retrospective multi-institutional analysis . Neurosurgery 58 : 701 - 709 ; discussion 701 - 709 .
3. Esteller M ( 2011 ) Non-coding RNAs in human disease . Nat Rev Genet 12 : 861 - 874 .
4. Bartel DP ( 2004 ) MicroRNAs: genomics , biogenesis, mechanism, and function. Cell 116 : 281 - 297 .
5. Ryan BM , Robles AI , Harris CC ( 2010 ) Genetic variation in microRNA networks: the implications for cancer research . Nat Rev Cancer 10 : 389 - 402 .
6. Lu J , Getz G , Miska EA , Alvarez-Saavedra E , Lamb J , et al. ( 2005 ) MicroRNA expression profiles classify human cancers . Nature 435 : 834 - 838 .
7. Zhi F , Chen X , Wang S , Xia X , Shi Y , et al. ( 2010 ) The use of hsa-miR-21, hsamiR-181b and hsa-miR-106a as prognostic indicators of astrocytoma . Eur J Cancer 46 : 1640 - 1649 .
8. Shi ZM , Wang XF , Qian X , Tao T , Wang L , et al. ( 2013 ) MiRNA-181b suppresses IGF-1R and functions as a tumor suppressor gene in gliomas . RNA 19 : 552 - 560 .
9. Wang J , Sai K , Chen FR , Chen ZP ( 2013 ) miR-181b modulates glioma cell sensitivity to temozolomide by targeting MEK1 . Cancer Chemother Pharmacol 72 : 147 - 158 .
10. Zhi F , Zhou G , Shao N , Xia X , Shi Y , et al. ( 2013 ) miR-106a-5p Inhibits the Proliferation and Migration of Astrocytoma Cells and Promotes Apoptosis by Targeting FASTK . PLoS One 8 : e72390 .
11. Lewis BP , Shih IH , Jones-Rhoades MW , Bartel DP , Burge CB ( 2003 ) Prediction of mammalian microRNA targets . Cell 115 : 787 - 798 .
12. Krek A , Grun D , Poy MN , Wolf R , Rosenberg L , et al. ( 2005 ) Combinatorial microRNA target predictions . Nat Genet 37 : 495 - 500 .
13. John B , Enright AJ , Aravin A , Tuschl T , Sander C , et al. ( 2004 ) Human MicroRNA targets . PLoS Biol 2 : e363 .
14. Zhi F , Gong G , Xu Y , Zhu Y , Hu D , et al. ( 2012 ) Activated beta-catenin Forces N2A Cell-derived Neurons Back to Tumor-like Neuroblasts and Positively Correlates with a Risk for Human Neuroblastoma . Int J Biol Sci 8 : 289 - 297 .
15. Calin GA , Croce CM ( 2006 ) MicroRNA signatures in human cancers . Nat Rev Cancer 6 : 857 - 866 .
16. Chitwood DH , Timmermans MC ( 2010 ) Small RNAs are on the move . Nature 467 : 415 - 419 .
17. He L , Yao H , Fan LH , Liu L , Qiu S , et al. ( 2013 ) MicroRNA-181b expression in prostate cancer tissues and its influence on the biological behavior of the prostate cancer cell line PC-3 . Genet Mol Res 12 : 1012 - 1021 .
18. Guo JX , Tao QS , Lou PR , Chen XC , Chen J , et al. ( 2012 ) miR-181b as a potential molecular target for anticancer therapy of gastric neoplasms . Asian Pac J Cancer Prev 13 : 2263 - 2267 .
19. Bisso A , Faleschini M , Zampa F , Capaci V , De Santa J , et al. ( 2013 ) Oncogenic miR-181a/b affect the DNA damage response in aggressive breast cancer . Cell Cycle 12 : 1679 - 1687 .
20. Lu Y , Roy S , Nuovo G , Ramaswamy B , Miller T , et al. ( 2011 ) Anti-microRNA222 (anti-miR-222) and -181B suppress growth of tamoxifen-resistant xenografts in mouse by targeting TIMP3 protein and modulating mitogenic signal . J Biol Chem 286 : 42292 - 42302 .
21. Chen L , Yang Q , Kong WQ , Liu T , Liu M , et al. ( 2012 ) MicroRNA-181b targets cAMP responsive element binding protein 1 in gastric adenocarcinomas . IUBMB Life 64 : 628 - 635 .
22. Takiuchi D , Eguchi H , Nagano H , Iwagami Y , Tomimaru Y , et al. ( 2013 ) Involvement of microRNA-181b in the gemcitabine resistance of pancreatic cancer cells . Pancreatology 13 : 517 - 523 .
23. Cai B , An Y , Lv N , Chen J , Tu M , et al. ( 2013 ) miRNA-181b increases the sensitivity of pancreatic ductal adenocarcinoma cells to gemcitabine in vitro and in nude mice by targeting BCL-2 . Oncol Rep 29 : 1769 - 1776 .
24. Ratti A , Fallini C , Colombrita C , Pascale A , Laforenza U , et al. ( 2008 ) Posttranscriptional Regulation of Neuro-oncological Ventral Antigen 1 by the Neuronal RNA-binding Proteins ELAV . Journal of Biological Chemistry 283 : 7531 - 7541 .
25. Ule J , Stefani G , Mele A , Ruggiu M , Wang X , et al. ( 2006 ) An RNA map predicting Nova-dependent splicing regulation . Nature 444 : 580 - 586 .
26. Dredge BK , Stefani G , Engelhard CC , Darnell RB ( 2005 ) Nova autoregulation reveals dual functions in neuronal splicing . EMBO J 24 : 1608 - 1620 .
27. Park E , Iaccarino C , Lee J , Kwon I , Baik SM , et al. ( 2011 ) Regulatory roles of heterogeneous nuclear ribonucleoprotein M and Nova-1 protein in alternative splicing of dopamine D2 receptor pre-mRNA . J Biol Chem 286 : 25301 - 25308 .
28. Ruggiu M , Herbst R , Kim N , Jevsek M , Fak JJ , et al. ( 2009 ) Rescuing Z + agrin splicing in Nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing . Proceedings of the National Academy of Sciences 106 : 3513 - 3518 .
29. Li H , Sun C , Wang Y , Gao Y , Liu Y , et al. ( 2013 ) Dynamic expression pattern of neuro-oncological ventral antigen 1 (Nova1) in the rat brain after focal cerebral ischemia/reperfusion insults . J Histochem Cytochem 61 : 45 - 54 .
30. Zhang C , Frias MA , Mele A , Ruggiu M , Eom T , et al. ( 2010 ) Integrative modeling defines the Nova splicing-regulatory network and its combinatorial controls . Science 329 : 439 - 443 .
31. Ule J , Ule A , Spencer J , Williams A , Hu JS , et al. ( 2005 ) Nova regulates brainspecific splicing to shape the synapse . Nat Genet 37 : 844 - 852 .