Research Paper
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Acta Biochim Biophys Sin
2005,37:680–687
doi:10.1111/j.1745-7270.2005.00100.x
Profiling of Differentially
Expressed Genes in LRRC4 Overexpressed Glioblastoma Cells by cDNA Array
Qiu-Hong ZHANG, Ming-Hua WU,
Li-Li WANG, Li CAO, Ke TANG, Cong PENG, Kai GAN, Xiao-Ling LI, and Gui-Yuan LI*
Cancer
Research Institute, Central South University, Changsha 410078, China
Received: May 8,
2005
Accepted: July 22,
2005
This work was
supported by the grants from the National Natural Science Foundation of China
(No. 30100191 and No. 30270429) and the Natural Science Foundation of Hunan
Province (No. 03JJY3062)
*Corresponding
author: Tel, 86-731-4805090; Fax, 86-731-4805383; E-mail, [email protected]
Abstract Our previous study has shown
that LRRC4 is a novel member of the leucine-rich repeat (LRR) superfamily and
has the potential to suppress brain tumor growth. In order to further analyze
the functions of LRRC4 on the maintenance of normal function and suppression
of tumorigenesis in the central nervous system, we investigated alterations in
gene expression related to neurobiology by the Atlas array in two inducible
dual-stable LRRC4-overexpressing cell lines. Seventeen of 588 genes
spotted on the Atlas membrane showed altered expression levels in LRRC4
transfected U251MG Tet-on cells, which are involved in cell proliferation and
cell cycle progression, tumor invasion and metastasis, and neurotransmitter
synthesis and release. In addition, cell invasion assay results showed that
LRRC4 can inhibit the U251MG cell migration. These studies represent the first
cDNA array analysis of the effects of LRRC4 on the involvement of different
neurobiological genes in U251MG glioblastoma cells and provide new insights
into the function of LRRC4 in glioma.
Key words differential expression;
leucine-rich repeat; LRRC4; glioma; cDNA microarray
The leucine-rich repeat (LRR) superfamily is composed of a very
heterogeneous group of proteins containing leucine-rich motifs, thought to be
involved in highly specific protein-protein interactions or cell adhesion.
Many LRR proteins are involved in the differentiation and development of
normal nervous tissues [1,2]. LRRC4 (GenBank accession number AF196976)
was recently identified and characterized as a novel member of this family,
which displayed significant downregulation in primary brain tumor biopsies
[3], and could inhibit tumorigenesis and cell proliferation of U251MG
glioblastoma cells [4,5]. Its predicted protein shares high homology with
nervous system-expressed LRR proteins such as NGL-1 [6,7] and LRRN6A [7],
which suggests that LRRC4 is a novel gene of relevance in the molecular
and cellular neurobiology of vertebrates, and may play an important role in the
maintenance of normal function and the inhibition of tumorigenesis in the
nervous system. However, the molecular mechanism by which LRRC4 suppresses
glioma tumorigenesis and cell proliferation has not been fully explained.
Gliomas are the most common primary brain tumors, which occur at any age, but
especially in young to middle-aged people, and are comparatively more common
in men [8]. Thus, it is critical to systemically examine the molecular changes
related to neurobiology and to illuminate the LRRC4 mechanism involved
in glioma tumorigenesis.The microarray technique first reported in 1995 by Schena et al.
[9] allows simultaneous parallel expression analysis of thousands of genes.
Information provided by cDNA microarray analysis might be useful for tumor
classification, elucidation of the key factor in tumors, and identification of
genes that might be applied to diagnostic purposes or as therapeutic targets
[10–12].
The Atlas human cDNA expression system provides a convenient and quick method
for profiling the expression of many hundreds of genes at the same time.In order to gain insights into the mechanism by which LRRC4 acts on
glioma and to further unveil the function of LRRC4, this study represents the
first cDNA array analysis of the effects of LRRC4 on the neurobiological genes
differentially expresssed in U251MG glioblastoma cells.
Materials and Methods
Cell culture
U251MG Tet-on-LRRC4 cell lines (P27, P28) were constructed by
our own laboratory [13]. U251MG Tet-on-LRRC4 cells were cultured in RPMI
1640 (Gibco BRL, Grand Island, USA) containing 10% doxycycline-free fetal
bovine serum (BD Biosciences Clontech, Palo Alto, USA) at 37 ?C in an incubator
(Thermo Forma Scientific, Philadelphia, USA) with 5% CO2.
Atlas human neurobiology array
Atlas human neurobiology array 7736-1 was purchased from BD
Biosciences Clontech. The membrane contained 10 ng of each gene-specific cDNA
from 588 known genes and 9 housekeeping genes. Several plasmid and
bacteriophage DNAs and blank spots were also included as negative and blank
controls to confirm hybridization specificity. The 588 known genes spotted on
the Atlas membrane consisted of cDNAs for cell-cycle control proteins,
neurotrophic factor receptors, neurotransmitter-associated proteins, DNA
transcription factors, extracellular cell signaling and communication
proteins, and stress response proteins. A complete list of the genes with
their array positions and GenBank accession numbers is available at http://www.clontech.com.
RNA extraction
Total RNA was extracted from the cell by the standard Trizol method
(Invitrogen, Carlsbad, USA). The RNA sample was digested with DNase I (10 U/mg) to remove DNA
contamination which might lead to false positives during hybridization. After
digestion, DNase I was removed from the sample by phenol-chloroform
extraction, followed by ethanol precipitation. The RNA sample was stored at –70 ?C till use.
The quantity and quality of the purified total RNA was estimated in a UV
spectrophotometer.
cDNA probe synthesis
cDNA was synthesized using a coding DNA sequence (CDS) primer mix
(Atlas human neurobiology CDS primer mix 7736-CDS; BD Biosciences Clontech). [a–32P]2‘-deoxyadenosine
5‘-triphosphate was included in the cDNA synthesis reaction to
facilitate probe labeling. The labeled cDNA was column-purified using the Atlas
nucleospin extraction kit. The purified labeled probes were stored at –20 ?C till use.
Hybridization
The labeled cDNA probes were hybridized to the microarray nylon
membrane (Atlas human neurobiology array 7736-1) according to the
manufacturer’s protocol. ExpressHyb solution was used for hybridization, with
sheared salmon testes DNA as the blocking agent. Along with the probe, Cot-1
DNA was added to block hybridization to repetitive DNA, which might be present
in the array. Hybridization was carried out at 68 ?C for 20 h. Following
hybridization, the membrane was washed three times in washing solution I [2?standard saline citrate (SSC), 1% sodium dodecyl sulfate (SDS)], and
once in washing solution II (0.1?SSC, 0.5% SDS). All the washings were carried out at 68 ?C for 30
min. The membrane was finally rinsed in 2?SSC, wrapped in a Saran wrap and exposed to a phosphor imager
screen. The membrane was exposed overnight at –70 ?C. Two independent
experiments were performed.
Image analysis
The resultant microarray spots were normalized by a two step
normalization process in order to control the background and have uniform
signal intensity. Background normalization was done by checking the signal
intensities of negative controls; normalization for uniform signal intensity
was evaluated against known “housekeeping genes” in the expression
array that have a known and stable binding efficiency. Expression uniformity
among the housekeeping genes was observed in all hybridization experiments. The
qualitative scores of differential expression assigned to each transcript
measurement were according to the following system: the fold increase (+) or
decrease (–) in the range of (+/–) 0–0.5 were considered as No Change (NC); (+/–) 0.6–2.0 as
Marginally Increased (MI) or Marginally Decreased (MD); and (+/–) 2.0 and above
as Increased (I) or Decreased (D).
Western blot analysis
Cells were collected by centrifugation at 12,000 g for 10
min, then the pellet was resuspended in lysis buffer (1% Nonidet P-40, 40 mM
Tris hydrochloride, pH 8.0, 150 mM NaCl) at 4 ?C for 30 min. Protein
concentrations were determined using the bicinchoninic acid (BCA) protein assay
(Pierce, Rockford, USA) examined with a microplate reader (Elx800; Bio-Tek
Instruments Inc., Winooski, USA) at 570 nm. Cell lysates were added to sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer with
complete protease inhibitors (Roche Applied Science, Indianapolis, USA),
separated by SDS-PAGE and transferred to polyvinylidene fluoride (Amersham
Biosciences, Piscataway, USA). The blots were incubated with goat anti-RAP1GAP
(V-19, sc-10331) and anti-CD44 (N-18, sc-7051) (Santa Cruz Biotechnology, Santa
Cruz, USA), rabbit anti-ephrin-B3 (H-170, sc-20724) and anti-Rab5A (S-19,
sc-309) (Santa Cruz Biotechnology), and mouse anti-b-actin (Sigma-Aldrich, St.
Louis, USA) antibody, followed by a horseradish peroxidase conjugated
anti-goat, anti-rabbit or anti-mouse antibody (Santa Cruz Biotechnology),
developed using Supersignal chemiluminescence reagents (Pierce), and exposed
to X-ray film.
In vitro cell invasion assay
The invasion assay of tumor cells was performed using a Transwell
cell culture chamber (Corning Costar No. 3422; Cambridge, USA).
Polyvinylpyrrolidone-free polycarbonate filters with 8 mm pore size were
precoated with 1 mg/40 ml of Matrigel (BD Biosciences Clontech, Palo Alto, USA) containing
fibronectin (FN) on the lower surface, then 2 mg/10 ml of Matrigel
containing FN was applied to the upper surface of the filters. After the
filters were dried at room temperature, they were washed gently with
phosphate-buffered saline. The U251MG Tet-on-LRRC4 cells induced with or
without doxycycline were removed from the culture flask with 0.1% EDTA and suspended
in RPMI 1640 with 0.1% bovine serum albumin at a concentration of 2?106 cells/ml. Cell suspension (100 ml) was added to the upper
compartment of the chamber and incubated for 20 h at 37 ?C in air atmosphere containing
5% CO2. After the cells on the upper side of the filters were
gently wiped off, the filters were fixed in methanol, stained with hematoxylin and
eosin, and mounted on glass slides. The cells that had migrated
to the lower side of the filters were counted under a
light microscope. The numbers of cells in five defined high power
fields (magnification, 200?) were
counted, and the average was determined.
Results
Identification of
differentially expressed genes by LRRC4 regulation
To identify changes in gene expression related to neurobiology, the Atlas
human cDNA array membranes were hybridized with cDNA derived from U251MG
Tet-on-LRRC4 cell lines (P27, P28) in the absence (Dox–) or presence
(Dox+) of doxycycline (2 mg/ml) (Fig. 1). No signals were visible in the blank spots
and negative control spots, indicating that hybridization was highly specific.
Following normalization of the hybridization levels with the housekeeping gene GAPDH
and the b-actin gene, pairwise comparison was conducted using AtlasImage
software (BD Biosciences Clontech). There were 17 genes altered in terms of
their expression levels, of which 6 were upregulated (Table 1) and 11
were downregulated (Table 2) following overexpression of LRRC4 in
U251MG cells (Fig. 2). Interestingly, genes involved in cell
proliferation inhibition and cell cycle arrest, such as the Rap1 GTPase
activating protein 1 (RAP1GAP) gene, the ephrin-B3 gene, the
somatostatin receptor genes, the protein tyrosine phosphatase N (PTPN) gene
and the neurotrophin-3 (NT-3) gene, were upregulated. Conversely, the genes
involved in tumor invasion and metastasis, and neurotransmitter synthesis and
release, including CD44, MMP16, the thymosin b-10 (TB-10)
gene, the annexin A2 and Rab protein genes, the glutamate receptor
metabotropic 5 (mGlu5) gene, were downregulated.
Confirmation of differential
expression
To confirm and validate the results obtained by cDNA array, we
analyzed the expression of selected differentially expressed genes by
conventional molecular methods. Four genes were measured by reverse transcription-polymerase
chain reaction (RT-PCR) and Western blot analysis to verify the accuracy and
the universality of the hybridization data. The RT-PCR (data not shown) and
Western blot results were consistent with the hybridization data in each of the
genes measured (Fig. 3). With the induction of doxycycline (2 mg/ml), it
presented an increase in the expression levels of RAP1GAP and ephrin-B3,
and a reduction in those of Rab5A and CD44 following LRRC4
overexpression.
LRRC4-mediated tumor cell invasion
suppression
Because the LRRC4 overexpression resulted in the
downregulation of genes involved in tumor invasion and metastasis, such as
CD44, MMP16, thymosin b-10 and annexin A2 genes, we next examined whether LRRC4 might affect
the migration of U251MG cells. We used a Transwell chamber in which the upper
and lower wells were separated by a filter coated with Matrigel containing FN.
As shown in Fig. 4, U251MG cells that had not been treated with
doxycycline migrated efficiently (P<0.05, t-test);
this migration was almost completely blocked following LRRC4
overexpression after the addition of doxycycline, indicating that LRRC4
overexpression may suppress U251MG cell invasion.
Discussion
Cell proliferation, differentiation, apoptosis, migration and
interaction are controlled by tightly regulated programs of differential gene
expression. Disturbances in the gene expression profiles occur in both tumor
initiation and progression [14].In this study, we concentrated on the differentially expressed
genes that might be involved in LRRC4 suppressing glioma occurrence and
progression in two inducible U251MG Tet-on-LRRC4 cell lines using an
Atlas human cDNA array.We presented evidence that overexpression of LRRC4 can
elevate the expression levels of certain cell cycle progression regulators,
such as RAP1GAP, ephrin-B3, somatostatin receptors, PTPN and NT-3,
by cDNA array analysis. RAP1GAP is a specific inactivator regulator of Rap1
which is a small GTPase involved in the regulation of cell proliferation,
differentiation and morphology [15]. Alterations in the Rap1 signaling pathway
are important in the development of human gliomas [16,17]. It was demonstrated
that the majority of sporadically occurring astrocytomas display either loss of
tuberin (RAP1GAP) or overexpression of Rap1B [18]. Also, ephrin-B3, a membrane-bound
ligand for the EphB receptor family, plays a critical role in cell cycle arrest
by upregulating the expression of p27 and downregulating the expression
of p19, PCNA and Stant2 [19]. Similar to RAP1GAP and
ephrin-B3, somatostatin receptor expression is a favorable prognostic factor
in human neuroblastoma [20,21]. The Sst2 somatostatin receptor can inhibit cell
proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation [22].
It was identified that there is a correlation between the expression of
dep-1/PTPeta and the somatostatin antiproliferative effects: the expression and
activation of dep-1/PTPeta is required for somatostatin inhibition of glioma
proliferation [23]. In addition, the elevated expression of the NT-3 receptor
TrkC by childhood medulloblastomas is associated with a favorable clinical
outcome of inhibiting tumor growth through the promotion of apoptosis [24].
Our previous study verified that LRRC4 mediates a delay of the cell
cycle, possibly through upregulating the expressions of p21waf1/cip1 and
p27kip1, and downregulating the expressions of CDK2, pRb, EGFR, PCNA and the
ERK1/2 phosphorylation state [5,7]. This evidence suggested that LRRC4 may have
an effect on suppressing cell cycle progression by impacting on Raf/Rap/Ras
pathways.Among the genes differentially expressed, we also focused our
attention on the diminution of the expression of cell adhesion molecules
including CD44, MMP16, TB-10 and annexin A2 involved in tumor invasion and
metastasis. CD44 and MMP are key factors in the migration and invasion of
deadly tumors. Glioma invasion in vitro is also mediated by
CD44-hyaluronan interaction [25–27] and MT1-MMP/CD44/caveolin interaction
[28], which could represent a potential target for anticancer therapies. Thymosin
b-10
has an identified presence in a number of human tumor cell lines derived from
the nervous system [29] and plays a critical role in the regulation of the anchorage-independent
growth and assembly of actin filaments [30,31]. Annexin A2, a calcium and
phospholipid binding protein and a substrate for protein-tyrosine kinases, is
highly expressed in glioblastoma multiforme [32], and is a likely second
messenger in the mitogenic pathways known to be important for the growth of
these tumors [33]. Increased levels of annexin II have been observed in various
cancer cells and tissues, and have been proposed as a marker of malignancy in
vivo [34]. In addition, the annexin II tetramer can serve as a binding
protein for procathepsin B and can cause tumor cell invasion and metastasis
[35]. These findings indicated that LRRC4 might act as a receptor for a certain
trophic factor or for an adhesion molecule participating in the maintenance of normal
brain function and the inhibition of tumorigenesis, like the other LRR
superfamily members. Furthermore, cell invasion assay verified LRRC4
overexpression could markedly suppress the migration and invasion capabilities
of U251MG cells. These findings imply that LRRC4 may inhibit the glioma tumor
cell invasion and metastasis through regulating the expression of the
above-mentioned invasion-related molecules.The cDNA array analysis also revealed a panel of neurobiological
molecules associated with neurotransmitter synthesis and release, which can be
downregulated by LRRC4 overexpression. These kinds of molecules included
a set of Rab proteins and mGlu5. Rab proteins are members of the superfamily of
monomeric GTPase, which belongs to the Ras superfamily of small GTPase. Rab proteins
have emerged as central regulators of vesicle budding, motility and fusion
[36,37]. Most are expressed ubiquitously, such as Rab1A, Rab1B [38–40] and Rab5A
[41,42], but Rab3 showed restricted tissue distributions and appeared to play
specialized roles in regulated secretion or protein sorting in nerve terminals
or endocrine cells [43–46]. Glutamate is an important nutritional amino acid involved in a
number of biochemical pathways and is the main excitatory amino acid
transmitter in the mammalian central nervous system. Glutamate excitotoxicity
has been proposed to be the final common pathway in a number of nervous system
diseases [47–49]. Glioma cells were shown to be impaired in their ability to
remove glutamate from the extracellular space. Moreover, the tumor may actively
induce neuronal death and allow tumor cells to grow by releasing glutamate at
concentrations that can induce widespread neurotoxicity [50]. The alterations
of these molecules indicated LRRC4 may protect nervous system normal function
and suppress glioma tumorigenesis by preventing the synthesis and release of
toxic neurotransmitters.In conclusion, we have identified functionally-related groups of
genes differentially expressed in two dual-stable cell lines overexpressing LRRC4
derived from glioblastoma using a 588-gene cDNA microarray. The observations
made in the present study reveal that LRRC4 possesses at least three
characteristics that impact on the maintenance of normal function and the
inhibition of glioma tumorigenesis in the nervous system. These studies
represent the first cDNA array analysis of the effects of LRRC4 on the
involvement of different neurobiological genes in U251MG glioblastoma cells and
provide new insights into the function of LRRC4 in glioma. Such investigations
should be performed in further studies to elucidate the possible relationship
between LRRC4-regulated genes and LRRC4’s precise role in glioma.
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