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Differential expression of microRNAs in Marek’s
disease virus-transformed T-lymphoma cell lines
Yongxiu Yao,
1
Yuguang Zhao,
1
Lorraine P. Smith,
1
Charles H. Lawrie,
2
Nigel J. Saunders,
3
Michael Watson
1
and Venugopal Nair
1
Correspondence
Venugopal Nair
venu.gopal@bbsrc.ac.uk
1
Division of Microbiology, Institute for Animal Health, Compton RG20 7NN, UK
2
LRF Molecular Haematology Unit, Nuffield Department of Clinical Laboratory Sciences,
University of Oxford, Oxford OX3 9DU, UK
3
Bacterial Pathogenesis and Functional Genomics Group, Sir William Dunn School of Pathology,
University of Oxford, Oxford OX1 3RE, UK
Received 22 December 2008
Accepted 15 March 2009
MicroRNAs (miRNAs) are increasingly recognized to play crucial roles in regulation of gene
expression in different biological events, including many sporadic forms of cancer. However,
despite the involvement of several viruses in inducing cancer, only a limited number of studies
have been carried out to examine the miRNA expression signatures in virus-induced neoplasia,
particularly in herpesvirus-induced tumours where virus-encoded miRNAs also contribute
significantly to the miRNome of the tumour cell. Marek’s disease (MD) is a naturally occurring,
rapid-onset CD4
+
T-cell lymphoma of poultry, induced by the highly contagious Marek’s disease
virus (MDV). High levels of expression of virus-encoded miRNAs and altered expression of several
host-encoded miRNAs were demonstrated in the MDV-transformed lymphoblastoid cell line
MSB-1. In order to identify the miRNA expression signature specific to MDV-transformed cells,
we examined the global miRNA expression profiles in seven distinct MDV-transformed cell lines by
microarray analysis. This study revealed that, in addition to the high levels of MDV-encoded
miRNAs, these MD tumour-derived lymphoblastoid cell lines showed altered expression of several
host-encoded miRNAs. Comparison of the miRNA expression profiles of these cell lines with the
MDV-negative, retrovirus-transformed AVOL-1 cell line showed that miR-150 and miR-223 are
downregulated irrespective of the viral aetiology, whereas downregulation of miR-155 was
specific for MDV-transformed tumour cells. Thus, increased expression of MDV-encoded miRNAs
with specific downregulation of miR-155 can be considered as unique expression signatures for
MD tumour cells. Analysis of the functional targets of these miRNAs would contribute to the
understanding of the molecular pathways of MD oncogenicity.
INTRODUCTION
MicroRNAs (miRNAs) are small (approx. 22–25 nt long),
non-coding RNAs that regulate gene expression by base
pairing with the RNA transcripts, targeting them for
translational repression or degradation. All metazoan
genomes encode miRNAs and the latest release (12.0) of
miRBase (http://microrna.sanger.ac.uk) contains 8619
hairpin precursor miRNAs in various species (Griffiths-
Jones et al., 2008). In the past few years, there have been
huge increases in the number of studies on miRNA and
cancer. Profiling of global miRNA expression levels
(miRNome) has generated extensive data on miRNA
expression in various forms of cancer. These studies have
reiterated the important role of miRNAs in all aspects of
cancer biology, including proliferation, apoptosis, inva-
sion/metastasis and angiogenesis (Fabbri et al., 2008; Lee &
Dutta, 2009). Such studies have also provided information
on the developmental lineage, differentiation state and
prognosis of malignant cells (Lowery et al., 2008; Schotte
et al., 2008). Nearly all of these studies have been carried
out on non-infectious forms of cancer. Current estimates
suggest that viruses are involved in 15–20 % of human
cancers worldwide (Javier & Butel, 2008) and oncogenic
viruses have been instrumental in delineating several
molecular pathways of neoplastic transformation. Despite
this, comparatively little is known on global miRNA
expression profiles of virus-induced cancers (Martinez
et al., 2008; Yeung et al., 2008). In many tumours,
particularly those associated with oncogenic herpesviruses
(Cosmopoulos et al., 2008; Cullen, 2006; Gottwein &
Cullen, 2008; Pfeffer et al., 2005; Sullivan & Grundhoff,
2007), high levels of expression of virus-encoded miRNAs
Supplementary figures and tables are available with the online version of
this paper.
Journal of General Virology (2009), 90, 1551–1559 DOI 10.1099/vir.0.009902-0
009902 G2009 SGM Printed in Great Britain 1551
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add further complexity to the miRNome of the trans-
formed cell (Ghosh et al., 2008).
Marek’s disease virus (MDV) is a highly contagious,
oncogenic alphaherpesvirus of the genus Mardivirus;itis
associated with Marek’s disease (MD), a naturally occur-
ring, rapid-onset T-cell lymphoma of chicken (Calnek,
1986). The MDV genome encodes several miRNAs that
map to the MDV-encoded oncogene Meq and the LAT
(latency-associated transcript) regions of the virus
(Burnside et al., 2006; Burnside & Morgan, 2007).
Although the target genes regulated by MDV-encoded
miRNAs are yet to be discovered, high levels of their
expression in MDV-transformed cell lines and tumours
suggest that they play important roles in oncogenesis
(Morgan et al., 2008; Xu et al., 2008). From a small RNA
library generated from MDV-transformed lymphoblastoid
cell line MSB-1 (Akiyama & Kato, 1974), we have
previously demonstrated high levels of expression of
MDV-encoded miRNAs (Yao et al., 2007, 2008). Elevated
levels of expression of some of these miRNAs were also
confirmed by real-time quantitative PCR in these cells (Xu
et al., 2008). Several host miRNAs that are associated
directly with oncogenicity, such as miR-17-92, miR-21 and
let-7i, were also present at a high frequency in the MSB-1
library, suggesting a role for these miRNAs in neoplastic
transformation of these cells (Yao et al., 2008). Although
analysis of miRNAs by cloning (Yao et al., 2008) or high-
throughput sequencing (Burnside et al., 2008) is used to
identify upregulated miRNAs in tumours or transformed
cells, such studies do not provide differential expression
profiles of miRNAs in different cell types. Comparisons of
miRNA expression profiles between neoplastically trans-
formed and normal cells using miRNA microarrays have
enabled the identification of specific miRNA expression
signatures in different types of cancer cell, some of which
have been shown to be useful indicators of cell type, stage
of differentiation and even prognosis of the cancer (Calin
& Croce, 2006; Rosenfeld et al., 2008). Only a limited
number of studies comparing the global miRNA expression
profiles of virally transformed tumour cells have been
carried out, particularly in tumour cells transformed by
oncogenic herpesviruses that themselves encode multiple
miRNAs (Sullivan & Grundhoff, 2007). Here we describe
the results of the comparison of the miRNA expression
profile of seven different MDV-transformed T-lympho-
blastoid cell lines with that of normal chicken splenocyte or
CD4
+
T-cell populations.
METHODS
Transformed cell lines. Small RNA prepared from seven independent
CD4
+
T-lymphoma cell lines derived from MDV-1-induced tumours
was used for miRNA expression profiling. The cell lines studied are
MDCC-MSB1 from a spleen lymphoma induced by the BC-1 strain of
MDV-1 (Akiyama & Kato, 1974), MDCC-HP8 from a GA strain-
induced tumour (Nazerian, 1987) and five cell lines (MDCC-226S,
MDCC-265L, MDCC-273S, MDCC-299K and MDCC-299L) estab-
lished from lymphomas of birds infected with RB-1B virus
(Petherbridge et al., 2004). Reticuloendotheliosis virus T (REV-T
strain)-transformed CD4
+
T-cell line AVOL-1 (Yao et al., 2008) and
avian leukosis virus (ALV) HPRS F42 strain-transformed B-cell line
HP45 (Nazerian, 1987) were included in the experiments as MDV-
negative transformed cell lines. Cell lines were grown at 38.5 uCin5%
CO
2
in RPMI 1640 medium containing 10 % fetal calf serum, 10 %
tryptose phosphate broth and 1 % sodium pyruvate.
Chicken splenocytes, CD4
+
T cells and magnetic cell sorting.
Single-cell suspensions of lymphocytes were prepared from spleen
tissues of uninfected birds by using Histopaque-1083 (Sigma-Aldrich)
density-gradient centrifugation. CD4
+
T cells were isolated by
magnetic cell sorting using mouse anti-chicken CD4 antibodies
(Chan et al., 1988) and goat anti-mouse IgG microbeads (Miltenyi
Biotec). After each antibody treatment, cells were washed three times
with PBS containing 0.5 % bovine serum albumin. At each wash, the
cell suspension was centrifuged at 450 gfor 10 min. Positively stained
cells were sorted through an AutoMACS Pro Separator (Miltenyi
Biotec). Purity of the sorted cells was confirmed to be .99 % by flow
cytometry after labelling with monoclonal anti-goat/sheep IgG–
fluorescein isothiocyanate (Sigma) antibody (data not shown).
Microarray analysis of miRNA expression. Preparation of probes
and hybridization to the arrays were carried out by using methods
described previously (Lawrie et al., 2007, 2008). Briefly, 500 ng
purified miRNA from lymphoblastoid cell lines, normal splenocytes
or CD4
+
T-cell populations was labelled with either Cy3 or Cy5 dye
using an Array 900microRNA RT kit from Genisphere and hybridized
to the mRNA microarray described previously (Lawrie et al., 2008).
The array contains miRNA probe sets (designed from miRBase v. 9.2)
spotted in quadruplicate non-adjacently (the sequences of the probes
are available at http://www.microRNAworld.com). Where the homo-
logous sequences of miRNAs in different species are identical, miRNA
sequences from only one species was spotted on the array. In
addition, probes for the mature MDV-1-encoded miRNAs miR-M2-
3p, miR-M2-5p, miR-M3-3p, miR-M3-5p, miR-M4-3p, miR-M4-5p,
miR-M5-3p, miR-M11-5p and miR-M12-3p (Yao et al., 2008) were
also included on the array. A model design with splenocytes and/or
CD4
+
T cells as reference was used and the expression values are
depicted as log ratios of test and reference samples. Image analysis was
done by using BlueFuse software (BlueGnome).
Statistical analysis of microarray data. Data were normalized
within each microarray by subtracting the mean log
2
ratio from each
measurement. Quantile normalization was then used to standardize
the data across arrays, and a linear model was fitted to each miRNA
using Limma (Smyth, 2005). The resultant P-values were adjusted for
multiple testing by using the Benjamini–Hochberg correction of the
false-discovery rate (Benjamini & Hochberg, 1995). The P-values and
mean expression were calculated for each cell type. Those miRNAs
showing consistent differential expression across all cell types were
subjected to a second analysis where all samples were treated as ‘virus-
infected’, regardless of cell type. A similar analysis in Limma was
performed, resulting in P-values calculated for expression across all
cell types. The data were sorted according to the log
2
ratio and heat
maps were produced by using R(http://www.R-project.org).
Inducible expression of mature miRNAs. Constructs for inducible
expression of miRNAs were generated in the pRTS-1-SVP-Tom(2)
vector, constructed from the pRTS-1 plasmid (Bornkamm et al.,2005)
by replacing the hygromycin B-resistance gene with a puromycin-
resistance gene. The inducible bidirectional promoter in this construct
expresses monomeric red fluorescent protein td-tomato (Shaner et al.,
2004) and the miRNA of interest simultaneously in a tightly regulated,
doxycycline (Dox)-inducible system. The inserted sequence of each
miRNA consists of the stem–loop structure and 100–200 bp of
upstream and downstream flanking genome sequences, a feature
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designed to ensure that the expressed miRNAs are processed as naturally
as possible. The approximately 500 bp fragment of each miRNA was
obtained by RT-PCR using RNA extracted from chicken embryo
fibroblasts by using the following primers: gga-miR-155 (59-
AGATCTCTGATGTCTGTACTCTTTATGAC-39and 59-CTCGAGCC-
CAGTGCCCTTAACTTAG-39); gga-miR-223 (59-AGATCTGCAA-
CGTCTGTCCTGTCC-39and 59-CTCGAGCAGGAACTGTACC-
AGCAG-39). As the sequence of the chicken orthologue was not
available, we used hsa-miR-150 for generating the expression constructs
of miR-150, using RNA prepared from HEK293T cells with primers 59-
AGATCTCTTCCTGCCCTCTTTGATG-39and 59-CTCGAGCA-
ATAGAAACAGGTGTACTTTG-39. More recently, the mature gga-
mir-150 sequence was shown to be identical to the hsa-miR-150
sequence except for a single nucleotide substitution (http://
www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL6541). Agarose gel-
purified PCR fragments were cloned into the BglII and XhoI sites of the
pRTS-1-SVP-Tom(2) vector and confirmed by sequence analysis. For
the expression of cloned miRNAs, HEK293T cells were transfected with
the pRTS-1–miRNA constructs by using Lipofectamine 2000
(Invitrogen) and stable cells were selected with puromycin (1 mgml
21
).
Respective miRNAs induced with the addition of Dox (1 mgml
21
) were
detected by Northern blotting analysis.
Luciferase reporter assays. In order to demonstrate that the Dox-
induced miRNAs in stably transfected HEK293T cells are functional,
we carried out reporter assays on one of the stable cell lines expressing
gga-miR-155 by analysing its effect on the previously characterized
target Pu.1. For this, we generated a reporter construct in which a
110 bp fragment of the chicken Pu.1 39untranslated region (UTR)
transcript (GenBank accession no. NM_205023) that contained the
predicted miR-155-response element (MRE) is inserted downstream
of Renilla luciferase in the psiCHECK-2 vector (Promega) to generate
the Pu.1-39UTR-wt construct. A mutant construct (Pu.1-39UTR-mt)
with mutations in the MRE sequences was also constructed. The
details of the oligonucleotides used for generating the chicken Pu.1-
39UTR reporter constructs are shown elsewhere (Zhao et al., 2009).
HEK293T cells stably expressing gga-miR-223 were used as a control.
Transfection into HEK293T cells for luciferase reporter assays was
carried out in 96-well plates with Lipofectamine 2000 (Invitrogen).
Firefly and Renilla luciferase activities were measured consecutively
with the Dual-Luciferase Reporter Assay system (Promega), using a
Lucy-1 luminometer (Anthos Labtec). In all cases, the constitutively
expressed firefly luciferase activity in the psiCHECK-2 vector served
as a normalization control for transfection efficiency.
Preparation of RNA for Northern blotting. Preparation of RNA
for Northern blotting analysis from splenocytes, CD4
+
and CD4
2
populations of T lymphocytes, MDV- and retrovirus-transformed
cell lines, and HEK293T cells stably selected for recombinant pRTS-1
vectors expressing miRNAs was carried out by using standard
methods as described previously (Yao et al., 2007, 2008). Antisense
oligonucleotides to the miRNAs gga-miR-155, gga-miR-223 and
hsa-miR-150 were used as probes.
RESULTS
In order to determine the miRNA expression signature in
MDV-induced tumours, we compared the global gene
expression profiles of seven MDV-transformed cell lines by
microarray analysis using miRNA probe sets designed from
miRBase v. 9.2. The tests validating the sensitivity,
specificity and reproducibility of these arrays have been
described previously (Lawrie et al., 2008). Ratios of miRNA
expression levels in transformed cell lines normalized to the
reference samples of normal chicken splenocytes or CD4
+
cells were used for the analysis. These studies showed that
the grouping of miRNAs in the seven MDV-1-transformed
cell lines was distinct from that in the MDV-1-negative
lymphocyte cell line AVOL-1 when splenocytes were used
as a reference (Fig. 1a). When purified CD4
+
T cells were
used as a reference, the expression profiles of miRNAs in
four of these cell lines (Fig. 1b) were largely in agreement
with those obtained by using reference splenocytes
(detailed data on the log
2
fold changes and the P-values
for each of the cell lines are provided in Supplementary
Tables S1 and S2, available in JGV Online). The miRNA
profiles of the cell lines in both of these analyses were
generally consistent, although individual cell lines did show
differences (Supplementary Figs S1 and S2).
MDV-1-encoded miRNAs are upregulated in
transformed cell lines
The inclusion of mature probe sequences of nine MDV-1-
encoded miRNA sequences (Yao et al., 2008) alongside the
host miRNA probe sets in the miRNA microarray enabled
assessment of the levels of expression of virus- and host-
encoded miRNAs in these cell lines. Compared with the
MDV-negative REV-T-transformed cell line AVOL-1, all of
the MDV-transformed cell lines showed upregulation of
MDV-1-encoded miRNAs (Fig. 1a), although the expres-
sion levels of individual miRNAs were not uniform in these
cells.
Changes in host miRNA profiles in MD tumour cell
lines
Examination of the global miRNA expression profiles of
the seven MDV-1-transformed cell lines revealed changes
in several host miRNAs (Fig. 1). Major differences in the
host miRNA expression profiles could also be observed
between MDV-1-transformed cell lines and the MDV-
negative cell line AVOL-1, demonstrating the differences in
the molecular oncogenic pathways between these cell lines.
Microarray readouts from our studies did demonstrate
differences between cell lines with regard to the expression
of individual miRNAs. Although such differences could be
important for individual cell lines, our main interest was to
look for miRNA profiles that are conserved in all MDV cell
lines, as this could give insights into the fundamental
molecular pathways of miRNA-mediated gene regulation
in MDV transformation. Our results showed that several
host-encoded miRNAs, such as miR-155, miR-223, miR-
150, miR-451, miR-26a and miR-126, were downregulated
in all MDV-transformed cell lines relative to the levels in
normal splenocytes or CD4
+
T cells (Fig. 1). As miR-223
and miR-150 were also downregulated in retrovirus-
transformed AVOL-1 cells, the reduced expression of
these two miRNAs is thought to be a broader feature of
transformed T cells, irrespective of the viral aetiology.
However, this was not the case with miR-155, the levels
of which were consistently reduced in all of the seven
miRNA expression in MDV-transformed T-cell lines
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MDV-1-transformed lymphoblastoid cell lines whilst the
expression levels in the AVOL-1 cell line were very high
(Fig. 1a), demonstrating that the downregulation of miR-
155 is a feature unique to MDV transformation of T cells.
As all of the MDV-1-transformed cell lines used in this
study have a CD4
+
T-cell phenotype, we also wondered
whether it is appropriate to use whole splenocyte popula-
tions as the reference sample in the analysis. In order to
rule out the possibility that the altered expression profiles
of miRNAs in the MDV-transformed cell lines are not due
to the use of splenocytes as the reference, we also repeated
the analysis of miRNA expression in four MDV-1-
transformed cell lines with purified CD4
+
T cells as the
reference. Overall, the results were largely consistent with
the values obtained by using normal splenocytes as the
reference (Fig. 1b). However, the use of purified CD4
+
cells as the reference resulted in the demonstration of
increased expression of several more host miRNAs, such as
miR-146b, miR-454, miR-7b, miR-34a, miR-18a, miR-
133a, miR-29a and gga-1et-7d (Fig. 1b). As the splenocyte
reference data represent the miRNA expression of multiple
Fig. 1. Heat maps showing clustering of
differentially expressed miRNAs (adjusted
P,0.05) in MDV-transformed cell lines. (a)
miRNAs identified in the seven MDV-trans-
formed cell lines (names of cell lines are shown
below each lane) in comparison to those in the
MDV-1-negative REV-transformed AVOL-1
cell line. The data shown are normalized by
using uninfected splenocytes as the reference.
(b) Heat map showing differentially expressed
miRNAs in four of the above MDV-transformed
cell lines (shown below each lane), normalized
against expression in normal CD4
+
T cells as
the reference. A colour key indicating low
(green) to high (red) values and the P-values is
also shown.
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lymphocyte populations, it was not surprising to see
changes in the miRNA expression patterns when purified
CD4
+
T cells were used as the reference. Data on the log
2
fold changes and P-values for each cell line using
splenocytes or purified CD4
+
T cells as the reference can
be seen in Supplementary Tables S1 and S2, respectively.
Analysis of miRNA expression by Northern
blotting
For further validation of the microarray data demonstrat-
ing the reduced expression of miR-155, miR-223 and miR-
150 in MDV-transformed tumour cell lines, we carried out
Northern blotting analysis comparing the levels of these
miRNAs in four MDV-transformed cell lines with those in
normal lymphocyte populations and retrovirus-trans-
formed cell lines AVOL-1 and HP45. Northern blotting
analysis confirmed the observations of reduced expression
of these three miRNAs in the microarray readouts of
MDV-transformed cells. The levels of gga-miR-155 signal
detected were very low in the normal splenocyte and
CD4
+
/CD4
2
T-cell populations (Fig. 2a). These low levels
were reduced further in all MDV-transformed cell lines
included in the assay. In contrast, stronger gga-miR-155
signals were evident in avian retrovirus-transformed cell
lines HP45 and AVOL-1. The levels of gga-miR-223 were
lower in normal CD4
+
T cells than in whole spleen-cell or
CD4
2
T-cell populations. However, no hybridization
signals for gga-miR-223 expression were evident in any
of the cell lines transformed by either MDV-1 or avian
retroviruses, suggesting that the downregulation of gga-
miR-223 is a broader feature of lymphocyte transforma-
tion, irrespective of the viral aetiology. The expression of
miR-150 also appeared to be restricted to the untrans-
formed cells, as there was no evidence of its expression in
transformed cells. Although miR-150 and miR-223 expres-
sion appeared to be similar in this respect, the levels of
miR-150 were much higher in CD4
+
T cells (Fig. 2a). We
also evaluated the Dox-inducible expression of miR-155,
miR-223 and miR-150 from the pRTS-1 vector
(Bornkamm et al., 2005). Northern blotting analysis of
HEK293T cells expressing the pRTS-1–miRNA constructs
showed high levels of expression of each of the three
mature miRNAs, regulated tightly in a Dox-inducible
manner (Fig. 2b).
For functional evaluation of the efficacy of this inducible
expression system for identifying potential miRNA targets,
we examined the ability of the pRTS-1–miR-155 expression
vector to silence the reporter construct containing the wild-
type or the mutant MRE region of the 39UTR of Pu.1, a
validated target of miR-155 (Zhao et al., 2009). This assay
showed that the relative Renilla luciferase levels of reporter
constructs with wild-type MRE sequences were reduced
specifically by nearly 60 % compared with the mutant MRE
construct (Fig. 3a). This reduction in luciferase levels was
dependent on the induction of miR-155 in these cells by
Dox treatment. The specificity of the reporter assay was
demonstrated further by the absence of reduction in
luciferase levels in cells expressing gga-miR-223 (Fig. 3a).
The tightly regulated nature of the Dox-inducible expres-
sion system used here was demonstrated by the non-leaky
expression of the td-tomato marker gene in the untreated
(Dox2) cells (Fig. 3b).
DISCUSSION
Global changes in miRNA expression profiles using
microarray analysis are used increasingly to identify
specific miRNA expression signatures associated with
several human malignancies (Calin & Croce, 2006, 2007;
Lawrie et al., 2008). Most of these studies have been carried
out on tumour tissues or cell lines derived from various
sporadic forms of cancer (Ozen et al., 2008; Ruike et al.,
2008). These studies have highlighted the direct oncogenic
potential of the cluster of miRNAs such as miR-21, miR-
155 and miR-17-92, providing valuable insights into the
Fig. 2. Northern blotting analysis of differentially expressed
miRNAs. (a) Twenty micrograms of total RNA extracted from the
indicated cells was separated on a 15 % denaturing polyacryl-
amide gel, blotted and hybridized with end-labelled antisense
oligonucleotide probes to gga-miR-155, gga-miR-223 and hsa-
miR-150. The cellular U6 small nuclear RNA served as the loading
control. (b) Dox-inducible expression of miRNAs from pRTS-1
constructs in HEK293T cells. Mature miRNAs (indicated by an
arrow) in Dox+samples are shown. Absence of signals in the
untreated (Dox”) lanes demonstrates the specificity of the miRNA
inducible system. Bands representing each of the three pre-
miRNAs can also be seen.
miRNA expression in MDV-transformed T-cell lines
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molecular pathways of oncogenesis (Wiemer, 2007).
Oncogenic viruses account for a large proportion of
neoplasms in man and animals (Javier & Butel, 2008).
Although the induction of many of these tumours has until
recently been attributed mainly to virus-encoded onco-
proteins, an increasing amount of data indicates that
miRNAs, encoded either by the host or by viruses
themselves in the case of oncogenic herpesviruses
(Cullen, 2006, 2009; Pfeffer et al., 2004, 2005; Sullivan &
Grundhoff, 2007), play significant roles in oncogenesis.
We and others have documented recently that the highly
oncogenic MDV-1 encodes several novel miRNAs
(Burnside & Morgan, 2007; Burnside et al., 2008; Morgan
et al., 2008; Yao et al., 2008). High levels of expression of
these miRNAs in lymphomas and transformed cell lines
have been demonstrated by direct cloning, Northern
blotting and quantitative RT-PCR (Burnside et al., 2006;
Xu et al., 2008; Yao et al., 2008). Although these studies
have been valuable in identifying miRNAs that are
expressed at high levels in these cells, they do not always
provide comprehensive miRNA expression profiles, par-
ticularly of those miRNAs that are downregulated in the
transformed cells. With a view to examining the global
expression of miRNAs in MDV-transformed cells, we
carried out miRNA expression profiling of seven inde-
pendent MDV-transformed tumour cell lines by using
miRNA microarray analysis. Each of these cell lines was
derived from an independent MD lymphoma. As demon-
strated previously with MSB-1 cells (Yao et al., 2007, 2008),
MDV-1-encoded miRNAs were indeed the most abundant
miRNAs in all of the cell lines. High-level expression of
virus-encoded miRNAs appears to be a feature common to
virus-transformed cell lines, as cells transformed by other
oncogenic herpesviruses, such as Kaposi’s sarcoma-asso-
ciated herpesvirus (KSHV) and Epstein–Barr virus, also
showed high levels of expression of virus-encoded miRNAs
(Cai et al., 2005; Lawrie et al., 2008; Pratt et al., 2009).
Higher copy numbers of viral genomes and active
transcription of miRNA genes may account for the higher
expression of virus-encoded miRNAs in the virus-trans-
formed cell lines, although one cannot rule out the
possibility of differential processing of virus-encoded
miRNAs in these cells. The functions and putative targets
of most of the MDV-encoded miRNAs remain unknown.
However, we have shown recently that MDV-miR-M4, one
of the most abundantly expressed virus-encoded miRNAs
in all of the cell lines, is a functional orthologue of miR-155
with the potential to target important lymphocyte-specific
transcription factors such as Pu.1 (Zhao et al., 2009). More
efforts in the future to identify the potential targets of other
MDV-encoded miRNAs (Morgan et al., 2008) will unravel
more molecular pathways of oncogenesis.
Microarray data analysis of the changes in the global
expression profiles of host-encoded miRNAs in MDV-
transformed cell lines could be grouped into (i) those that
are restricted only to some of the MDV-transformed cell
lines and (ii) those that appear to be conserved across all of
the cell lines. The changes in miRNA expression in
individual cell lines, such as the increased expression of
miR-221/miR-222 in MSB-1 cells (Lambeth et al., 2009),
Fig. 3. Functional analysis of inducible miR-155 by using a
reporter assay on the Pu.1 39UTR. (a) Reporter assays in
HEK293T cells expressing gga-miR-155 and gga-miR-223 from
the Dox-inducible promoter in the pRTS-1 vector. Relative Renilla
luciferase levels (RL) of Dox+and Dox”HEK293T cells
transfected with luciferase reporter constructs containing the
wild-type (Pu.1-wt) or mutant (Pu.1-mt) MRE-containing region of
the 39UTR of the chicken Pu.1 transcript (Zhao et al., 2009) are
shown. (b) Fluorescence microscopic image of td-tomato expres-
sion in Dox+(top left) and Dox”(top right) HEK293T cells stably
expressing pRTS-1–gga-miR-155. Bright-field images of the two
cell populations are also shown (bottom left and right).
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are likely to be important in the regulation of respective
target proteins in individual cell lines. However, in this
paper, we focus on the global changes in miRNA
expression common to all MDV-transformed cell lines.
The miRNA profile of the seven MD tumour cell lines
showed changes in the expression of several miRNAs.
These included the downregulation of miR-150, miR-223
and miR-155, confirmed by Northern blotting analyses
(Figs 1 and 2a). Of these, miR-150 and miR-223 were also
downregulated in AVOL-1 cells, demonstrating it to be a
broader feature of lymphocyte transformation, regardless
of the viral aetiology. Reduced expression of miR-150 has
also been reported in human lymphoid malignancies such
as diffuse large B cell lymphoma (Garzon & Croce, 2008;
Landgraf et al., 2007; Lawrie et al., 2008), indicating its
conserved function across different species. Increasing
evidence suggest that miR-150 functions through the
transcription factor c-myb (Garcia & Frampton, 2008; Lin
et al., 2008; Lu et al., 2008; Xiao et al., 2007; Zhou et al.,
2007), and the dysregulation of c-myb and its targets could
be important in T-cell transformation. In the case of miR-
223, although all of the regulatory mechanisms are not yet
understood fully, recent studies have indicated a clear role
for miR-223 in haematopoiesis, as well as in malignancies
(Baek et al., 2008; Garzon & Croce, 2008; Johnnidis et al.,
2008; Merkerova et al., 2008). Whilst identification of the
potential targets is important to understand fully the
molecular pathways, involvement of miR-223 appears to be
logical in MDV-induced lymphocyte transformation.
The downregulation of miR-155 observed by microarray
analysis was unique to MDV-transformed cell lines, as it
was upregulated in the MDV-negative AVOL-1 cell line
(Fig. 1). Although the levels of miR-155 in the normal
lymphocyte populations were low by Northern blotting
analysis, it was clear that MDV-transformed cell lines
showed a distinct reduction in hybridization signals,
especially when compared with the retrovirus-transformed
lymphocyte cell lines HP45 and AVOL-1 (Fig. 2a). Several
recent studies have highlighted the potential multiple roles
of miR-155 in functions ranging from innate immune
responses to oncogenicity (Garzon & Croce, 2008). The
molecular mechanisms that drive down the expression of
miR-155 in MDV-transformed cell lines are not known.
However, some of its functions on targets such as Pu.1
could be rescued by MDV-miR-M4, a highly expressed
MDV-1-encoded functional orthologue of miR-155 (Zhao
et al., 2009). Although the regulatory expression dynamics
of miR-155 and MDV-miR-M4 are not understood fully,
the existence of autoregulatory mechanisms of miR-155
expression mediated through a common set of targets
cannot be ruled out. It is interesting that, in KSHV-infected
primary effusion lymphoma cell lines, miR-155 was also
found to be downregulated in favour of the KSHV-
encoded miR-K12-11 homologue (Skalsky et al., 2007).
The data from this study have enabled us to characterize
the miRNome of MDV-transformed tumours. Although
this has provided valuable insights into the expression
profiles of miRNAs in these cell lines, the major challenge
will be in the identification of the putative targets of the
differentially expressed miRNAs in these cells. Although
bioinformatic predictions of miRNA targets are valuable,
the development of systems for functional characterization
of miRNA targets is important to understand the pathways
of oncogenesis. The tightly regulated, Dox-inducible
miRNA expression system of the differentially expressed
miRNAs that we developed in HEK293T cells will be
valuable in identifying the putative functional targets of
these miRNAs. Demonstration of the expression of mature
miRNAs in a Dox-dependent manner clearly showed the
proper processing of these miRNAs in this system. For
functional validation of the system, we analysed the
putative targeting of miR-155 on one of the validated
target proteins, Pu.1 (Zhao et al., 2009). The tightly
regulated expression of miR-155 and the specific silencing
of the relative luciferase levels with reporter assays with
wild-type 39UTR reporter constructs (Fig. 3) provide a
platform for functional analysis of the putative targets of
differentially expressed miRNAs.
In summary, the data presented here demonstrate that
miRNA expression profiling using microarrays is a
powerful approach for analysing the relative levels of
several miRNAs simultaneously. This study, the first of its
kind in MDV-transformed cell lines, demonstrates that, in
addition to the overexpression of several MDV-encoded
miRNAs, downregulation of some of the host-encoded
miRNAs is also a hallmark of MDV transformation.
Determination of the miRNA profile is a first step towards
identification of the regulatory networks of gene expression
in these cell types.
ACKNOWLEDGEMENTS
The authors thank Dr Georg Bornkamm for providing the pRTS-1
plasmid used in the inducible expression systems. This work was
carried out as part of a BBSRC grant awarded to V. N. We thank all
members of the Avian Oncogenic Virus group for assistance and for
inspiring discussions connected with this work, and Mick Gill for help
with the digital imaging.
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