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ATM is down-regulated by N-Myc–regulatedmicroRNA-421Hailiang
Hua,1, Liutao Dua, Gindy Nagabayashia, Robert C. Seegerb, and
Richard A. Gattia,c,1
aDepartment of Pathology and Laboratory Medicine, and
cDepartment of Human Genetics, David Geffen School of Medicine at
the University of California,Los Angeles, CA 90095; and bDivision
of Hematology-Oncology, and Saban Research Institute, Children's
Hospital Los Angeles, Keck School of Medicine,University of
Southern California, Los Angeles, CA 90027
Edited by George Klein, Karolinska Institute, Stockholm, Sweden,
and approved December 8, 2009 (received for review July 13,
2009)
Ataxia-telangiectasia mutated (ATM) is a high molecular
weightprotein serine/threonine kinase that plays a central role in
themaintenance of genomic integrity by activating cell cycle
check-points and promoting repair of DNA double-strand breaks.
Little isknown about the regulatorymechanisms for ATMexpression
itself.MicroRNAs are naturally existing regulators that modulate
geneexpression in a sequence-specific manner. Here, we show that
ahuman microRNA, miR-421, suppresses ATM expression by target-ing
the 3′-untranslated region (3′UTR) of ATM transcripts.
Ectopicexpression of miR-421 resulted in S-phase cell cycle
checkpointchanges and an increased sensitivity to ionizing
radiation, creatinga cellular phenotype similar to that of cells
derived from ataxia-telangiectasia (A-T) patients. Blocking the
interaction betweenmiR-421 andATM 3′UTRwith an antisensemorpholino
oligonucleo-tide rescued the defective phenotype caused by miR-421
overex-pression, indicating that ATMmediates the effect ofmiR-421
on cellcycle checkpoint and radiosensitivity. Overexpression of the
N-Myctranscription factor, an oncogene frequently amplified in
neuro-blastoma, induced miR-421 expression, which, in turn,
down-regulated ATM expression, establishing a linear signaling
pathwaythat may contribute to N-Myc-induced tumorigenesis in
neuroblas-toma. Taken together, our findings implicate a previously
unde-scribed regulatory mechanism for ATM expression and
ATM-dependent DNA damage response and provide several
potentialtargets for treating neuroblastoma and perhaps A-T.
neuroblastoma | S-phase checkpoint | radiosensitivity | DNA
repair
Ataxia-telangiectasia mutated (ATM) kinase plays a hier-archical
regulatory role in the double-strand break (DSB)-induced DNA damage
response in which ATM transduces a DSBdamage/repair signal to
downstream effector machinery by phos-phorylating critical protein
substrates (1–4). ATM mutations,which usually result in loss of ATM
protein expression (5), lead tothe autosomal recessive progressive
neurodegenerative diseaseataxia-telangiectasia (A-T) (6, 7). Both
homozygotes and hetero-zygotes are at an increased risk for cancer
(8). ATM has beenreported to be regulated by a transcription
factor, E2F-1, (9) andtheATM gene is also reported to be subject to
epigenetic silencingsuch as by methylation of the ATM promoter (10,
11), suggestingthat ATM can also be up-regulated at the
transcriptional levelunder some circumstances. MicroRNAs regulate
gene expressionthrough inhibition of translation or degradation of
the targetedmRNA (12, 13). Physiological functions of microRNAs
havebeen observed in normal and lineage-targeted development (14)as
well as in the context of human cancers (15). In this study,we
demonstrate that miR-421 targets the 3′-untranslated region(3′UTR)
of ATM and down-regulates its expression, whereasmiR-421 expression
is driven by theN-Myc transcription factor, anoncogene that is
frequently amplified in neuroblastoma cells.
ResultsMiR-421 Suppresses ATM Expression by Targeting 3′UTR of
ATM. Toexplore the possibility that microRNAs might regulate
ATMexpression, we searched the 3′UTR of the human ATM gene for
microRNA-binding motifs using the MicroCosm Targets
program(EMBL-EBI). Nine nucleotides at the 5′-end of
hsa-miR-421(miR-421)were perfectly complementary to the target
sequence inthe 3′UTR ofATM (including the “seed sequence” from
positions2–8) (Fig. 1A). This suggested thatATMmight be a target
formiR-421. To validate this in silico prediction, we cloned the
ATM 3′UTR portion containing the miR-421 target site into a
Renillaluciferase reporter construct (Fig. 1B) and established a
luciferasereporter assay following cotransfection of reporter
constructs withprecursor miR-421 (pre-miR-421) into HeLa cells. A
significantreduction (30%) in the luciferase activity of the
reporter constructcontaining the ATM 3′UTRwas observed in the
presence of miR-421, whereas no changes were noted in the
luciferase activity of theunmodified construct (pRL) with miR-421
expression (Fig. 1C).Deletion of six nucleotides of seed sequence
(Δ6) led to the loss ofreduction in miR-421-mediated luciferase
activity (Fig. 1C). Tofurther confirm thatATMis a target
formiR-421, we examined theendogenous ATM protein level by
immunoblot after transientlytransfecting pre-miR-421 intoHeLa
cells. As shown in Fig. 1D, theATM expression level decreased as
the concentration of trans-fected pre-miR-421 was increased. As an
indication of ATM kin-ase activity (16), phosphorylation of SMC1 at
the serine-966residue (pS966-SMC1) was measured following DNA
damage by10-Gy irradiation (IR).A significant reduction in the
pS966-SMC1was observed when pre-miR-421 was introduced into HeLa
cellsfollowed by IR, as compared with the introduction of a
non-relevant control pre-miR precursor (Fig. 1E). ATMmRNA
levelswere measured by quantitative real-time PCR and were
notdecreased in the presence of miR-421 (Fig. 1F), suggesting
thatmiR-421 down-regulates ATM at a translational rather
thantranscriptional level.
MiR-421 Regulates Cell Cycle S-Phase Checkpoint and
CellularRadiosensitivity. To determine the cellular functions of
miR-421,we created an miR421-overexpressing HeLa stable cell line
byinfecting the cells with an miR421-containing lentivirus
andselecting a stable infectant with blasticidin (HeLa/miR-421)
(Fig.2A). We also created a control stable infectant cell line
withscrambled shRNA (HeLa/scram) (17). Real-time PCR detectedan
∼120-fold increase in the expression of mature miR-421 in
theHeLa/miR-421 cells compared with the HeLa/scram control
cells(Fig. 2B). Both ATMprotein expression andATM kinase
activity,as indicated by the level of post-IR pS966-SMC1, were
sig-nificantly reduced in the HeLa/miR-421 cells (Fig. 2C).
Author contributions: H.H. and R.A.G. designed research; H.H.,
L.D., and G.N. performedresearch; R.C.S. contributed new
reagents/analytic tools; H.H. and R.A.G. analyzed data;and H.H.,
R.C.S., and R.A.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.1To whom correspondence
may be addressed at: Department of Pathology and Labora-tory
Medicine, David Geffen School of Medicine at UCLA, 675 Charles
Young Drive, LosAngeles, CA 90095. E-mail: [email protected] or
[email protected].
This article contains supporting information online at
www.pnas.org/cgi/content/full/0907763107/DCSupplemental.
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ATM regulates DNA damage-induced cell cycle checkpoints atG1-S
and intra-S phase (18, 19). A hallmark of A-T cells is thefailure
to arrest DNA synthesis in S-phase following DNA dam-age and
continuous incorporation of nucleotides into DNAdespite damage
(i.e., radioresistant DNA synthesis) (20, 21).Thus, we anticipated
that miR-421 overexpression might regulateDNA damage-induced cell
cycle S-phase checkpoints. To assessthis, HeLa/scram and
HeLa/miR-421 cells were irradiated (10Gy) to introduce DNA damage
and BrdU was used to followDNA incorporation. As expected, a
reduction in the percentageof BrdU-positive cells in S-phase was
observed for the HeLa/scram control cells (14.88% pre-IR vs. 12.56%
post-IR), indi-cating a normal block in the DNA synthesis (Fig. 2D,
Upper Leftand Right); in contrast, an increase in the percentage of
BrdU-positive cells in S-phase was observed for HeLa/miR-421
cells(11.67% pre-IR vs. 15.38% post-IR) (Fig. 2D, Lower Left
andRight), indicating that miR-421 overexpression overcomes
theIR-induced DNA synthesis block and mimics the radioresistantDNA
synthesis of A-T cells. The miR421-induced continuous
DNA synthesis was also seen with lower doses of IR at 2 and 5
Gy(Fig. S1A). We noticed that the pre-IR percentage of HeLa/miR-421
cells in S-phase was lower than that of control HeLa/scramcells,
suggesting that miR-421 might regulate this cell cyclecheckpoint
independent of DNA damage. Similar results wereobserved using a
human breast cancer cell line, MDA-MB-231,when miR-421 was
overexpressed (Fig. S2 A and B).A clonogenic assay was used to
determine whether over-
expression ofmiR-421 affects cellular radiosensitivity. As shown
in
Fig. 1. miR-421 suppresses ATM expression by targeting ATM
3′UTR. (A)Mature miR-421 sequences and recognition sites within
3′UTR of ATM. Theseed sequence of miR-421 is shown in the box. WT
and del6 (Δ6) ATM-3′UTRtargets are also shown. (B) Constructs of
Renilla luciferase [Luc; unmodifiedconstruct (pRL)-CMV] containing
WT or 6-nt deleted (Δ6) ATM 3′UTR. (C)Luciferase (Luc.) activity of
pRL and modified constructs containing WT ormutant (Δ6) 3′UTR.
Luciferase constructs were cotransfected with pre-miR-CTL (control,
50 nM) or pre-miR-421 (50 nM) into HeLa cells. Renilla
luciferaseactivity was measured 36 h after incubation and
normalized to firefly luci-ferase. Asterisk indicates significant
down-regulation of pre-miR-421 againstconstruct containing WT ATM
3′UTR. (D) Immunoblot of endogenous ATMexpression in HeLa cells 96
h after transfection of increasing amounts of pre-miR-421 (using
pre-miR-CTL to compensate for equal amounts of total miRs).SMC1
served as a loading control for the blot. (E) Immunoblot of
pS966-SMC1 in HeLa cells that were transiently transfected with
pre-miR-CTL (100nM) or pre-miR-421 (100 nM) after 10-Gy IR to
activate the DNA damageresponse. A WT lymphoblastoid cell line
(LCL) served as a positive control forSMC1 and ATM protein. (F)
Real-time PCR of ATM mRNA from HeLa cellstransfected with
pre-miR-CTL (100 nM) or pre-miR-421 (100n M). Data werenormalized
to the level of GAPDH mRNA, and the ratio of ATM/GAPDH inHeLa
control cells was set to 1.
Fig. 2. miR-421 regulates cell cycle S-phase checkpoint and
cellular radio-sensitivity. (A) Scheme of a U6 promoter-driven
miR-421 cloned into a len-tiviral vector with two LTRs and a
selection marker for blasticidin driven bySV40 promoter. (B)
Real-time PCR of miR-421 expression in HeLa cells
stablyoverexpressing scrambled shRNA (HeLa/scram) or
miR-421(HeLa/miR-421).Data were normalized to an internal control
RNU66, and the ratio of miR-421/RNU66 in HeLa/scram cells was set
to 1. (C) Immunoblot of ATM andpSMC1 in HeLa/scram and HeLa/miR-421
cells with or without 10-Gy IR. Notethe reduction of both ATM and
pSMC1 in miR421-overexpressing cells. (D)Analysis of IR-induced
cell cycle S-phase checkpoint by FC. Stably over-expressing
HeLa/scram and HeLa/miR-421 cells were treated with or without10
Gy. DNA synthesis at S-phase was labeled with BrdU. (Left Upper
andLower) Results of one experiment representative of three
independentexperiments. Box R5 indicates the percentage of BrdU+
S-phase cells pre- orpost-IR. (Right) Summary of change of BrdU+
cells pre- and post-IR for HeLa/scram and HeLa/miR-421 cells from
three independent experiments, usingthe algorithm
(R5+IR−R5−IR)/R5−IR × 100%. (E) HeLa/scram and HeLa/miR-421cells
were irradiated at the indicated doses, and colony survival was
meas-ured after 2 weeks. (F) Effect of miR-421 on proliferation of
HeLa cells, asmeasured by cell population doublings with culture
time. (G) Effect ofmiR-421 on IR-induced cell death, as measured by
propidium iodide stainingFC. The percentage of propidium
iodide-positive cells was normalized to theunirradiated cells in
each group.
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Fig. 2E, the survival fractions of HeLa/miR-421 cells post-IR
(1and 2 Gy) were significantly reduced relative to those of
HeLa/scram control cells. MiR-421 overexpression did not alter
theproliferation rate ofHeLa cells (Fig. 2F) but increased post-IR
celldeath (Fig. 2G), which is consistent with the decreased
survivalfraction in the clonogenic assay. A similar effect of
miR-421 onradiosensitivity was observed withMDA-MB-231 cells (Fig.
S2C).
Effects of miR-421 on S-Phase Checkpoint and Radiosensitivity
AreATM Dependent. A singlemicroRNA is predicted
tomodulate>200targets of protein expression (22).To further
determinewhether theeffects ofmiR-421 on cell cycle checkpoints and
radiosensitivity aremediated through ATM, we used an antisense
morpholino oligo-nucleotide (AMO) to block the recognition sequence
of ATM 3′UTR (Fig. 3A). Treatment of HeLa/miR-421 cells with ATM
3′UTR target site-specific AMO (AMO-ATM) resulted in the
abro-gationofmiR421-mediateddown-regulationofATMexpression, asshown
by both Western blot and ELISA (Fig. 3B). This effect wasnot
observed when cells were treated with scrambled control
AMO(AMO-scram) (Fig. S1B). Following IR, AMO-ATM treatment
also resulted in an increase of pS966-SMC1 in HeLa/miR-421
cells(Fig. 3C). Blocking with AMO-ATM further restored the
S-phasecell cycle checkpoint and radiosensitivity of
theHeLa/miR-421cells,as shown by radioresistant DNA synthesis assay
and clonogenicsurvival assay (Fig. 3 D and E). Taken together,
these AMO-ATMexperiments suggest that the effect of miR-421 on the
cell cycle S-phase checkpoint and radiosensitivity is mediated
through ATM.
Transcription Factor N-Myc Up-Regulates miR-421
Expression.HumanmiR-421 is located intergenically at chromosome
Xq13. Inter-estingly, another microRNA, miR-374b, is located just
85 bpproximal to miR-421, forming a microRNA cluster that is
drivenby a single promoter (Fig. 4A). The function of miR-374b is
stillunknown. To determine which transcription factors might
influ-ence miR-421 expression, we performed in silico analysis of
the
Fig. 3. ATMmediates the effect of miR-421 on cell cycle S-phase
checkpointand radiosensitivity. (A) Schematic working model of ATM
3′UTR thatwas targeted by an antisense AMO. AMO-ATM was designed to
match themiR-421 recognition site of ATM 3′UTR and specifically
block the down-regulation of ATM by impeding the binding of mature
miR-421. (B) (Left)Immunoblot of ATM expression in HeLa/scram and
HeLa/miR-421 cells trea-ted with or without AMO-ATM (2 μM) for 5
days. The fold change in ATMexpression is shown below the
immunoblot. (Right) ELISA was also used todetermine ATM
concentration. (C) Immunoblot of pSMC1 in HeLa/scram
andHeLa/miR-421 cells treated with AMO-scram (2 μM) or AMO-ATM (2
μM) for 5days, followed by 10-Gy IR. The fold change in pSMC1 level
is shown belowthe immunoblot. Note the increase of pSMC1 in
HeLa/miR-421 cells treatedwith AMO-ATM. (D) Analysis of cell cycle
S-phase checkpoint after treatmentof AMO. HeLa/scram and
HeLa/miR-421 cells were treated with AMO-scram(2 μM) or AMO-ATM (2
μM) for 5 days and irradiated with increasing doses ofradiation (2,
5, and 10 Gy). DNA synthesis was monitored by BrdU incorpo-ration
and analyzed by FC. The percentage of BrdU+ S-phase cells at the
startpoint (unirradiated) was arbitrarily set to 50%, and all other
data werenormalized to this point. This plot is representative of
three independentexperiments. The arrow indicates that AMO-ATM
treatment rescues thedefect of HeLa/miR-421 cells. (E) Colony
survival fraction with exposureto AMO. HeLa/scram and HeLa/miR-421
cells were treated with AMO-scram(2 μM) or AMO-ATM (2 μM) for 5
days, and 500 cells were plated in triplicate;cells were irradiated
with increasing doses of radiation, and surviving colo-nies were
scored after 2 weeks. The survival fraction at each radiation
dosewas normalized to that of the nonirradiated control. The arrow
indicatesthat the AMO rescued the radiosensitivity of HeLa/miR-421
cells.
Fig. 4. miR-421 is up-regulated by N-Myc overexpression in HeLa
cells. (A)Chromosomal location of miR-374b/miR-421 cluster on
chromosome Xq13,sharing the same promoter. The promoter region (1
kb), containing an E-box(5′-CACGTG-3′), was cloned into luciferase
construct pGL3-basic to createpGL3-PR421 and drives the
transcription of firefly luciferase (Luc). (B) Luci-ferase activity
of the miR-421 promoter. Luciferase constructs [pGL3-PR421and
unmodified construct (pRL)-CMV] were cotransfected, with
vector(Vec) or N-Myc, into HeLa cells. Firefly luciferase activity
was measured 24 hand 48 h after incubation and normalized to
Renilla luciferase activity. (C)Real-time PCR of endogenous
miR-421expression in HeLa cells transientlytransfected with vector
or N-Myc. Data were normalized to RNU66. (D)Immunoblot of ATM
expression in HeLa cells transiently transfected withincreasing
amounts of N-Myc (using vector to compensate for equal amountsof
total DNA). The fold change in ATM protein expression is shown
belowthe blot. (E) ELISA measurement of ATM concentrations in HeLa
cells tran-siently transfected with N-Myc. The asterisk indicates
significant inhibition ofATM by N-Myc overexpression. (F) ELISA to
determine ATM concentration inHeLa cells transiently transfected
with the indicated DNA constructs (vectoror N-Myc) and anti-miR-CTL
(50 nM) or anti-miR-421(50 nM) inhibitors. (G)Immunoblot of ATM
expression in HeLa cells transiently transfected withindicated DNA
constructs (vector or N-Myc) and anti-miR-CTL (50 nM) or
anti-miR-421(50 nM) inhibitor. Only the top band corresponds to
ATM.
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promoter region (including 2 kb upstream of the miR-374b
stemloop) using the transcription factor binding site program
CON-SITE (Materials and Methods). This identified a binding site
forN-Myc (an E-box) at −85 nucleotides relative to the miR-374bstem
loop (Fig. 4A). To validate this prediction, we cloned a 1-kbDNA
fragment of the promoter region into a firefly luciferasereporter
construct and examined the effect of N-Myc on
miR-421promoter-driven luciferase activity. Overexpression of N-Myc
inHeLa cells activated miR-421 promoter-driven luciferase
activity24 and 48 h after transfection (Fig. 4B). Consistent with
theluciferase assay, the expression level of endogenous mature
miR-421 was also increased by overexpression of N-Myc in the
HeLacells, as measured by microRNA real-time PCR (Fig. 4C).
Mostinterestingly, ATM protein expression was reduced in HeLa
cellsthat were transiently transfected with N-Myc, as detected
byimmunoblotting and ELISA (Fig. 4 D and E), strongly
suggestingthat N-Myc stimulates miR-421 expression, which, in turn,
down-regulates ATMexpression. Anti-miR-421 inhibitor is a
chemicallymodified antisense oligonucleotide designed specifically
to bindto and inhibit endogenous miR-421 molecule. Cotransfection
ofanti-miR-421 inhibitor into HeLa cells along with N-Myc
con-struct restored the ATM expression that was suppressed
inN-Myc-transfected cells, further confirming that miR-421
medi-ates N-Myc-induced ATM down-regulation (Fig. 4 F and G).
Wealso noticed that anti-miR-CTL relieved the ATM expression tosome
extent, which might be caused by the nonspecific binding
ofanti-miR-CTL to the endogenous miR-421 (Fig. 4 F and G).
N-Myc/miR-421/ATM Pathway in Neuroblastoma Cells. The N-Mycgene
is frequently amplified in human neuroblastoma cells andis used as
a prognostic marker for neuroblastoma (23, 24). Tofurther explore
the N-Myc/ATM relation, we examined ATMexpression in seven human
neuroblastoma cell lines: Four celllines (CHLA-134, CHLA-136,
LA-N-1, and LA-N-5) are N-Mycamplified, whereas the other three
(CHLA-15, CHLA-90, andCHLA-255) are not N-Myc amplified. We noted a
low level of N-Myc expression in CHLA-90 cell lines compared with
the othertwo cell lines, CHLA-15 and CHLA-255, with undetectable
N-Myc expression (Fig. 5A). We found that the ATM expressionlevels
were significantly lower in the four N-Myc-amplified celllines
compared with those of three N-Myc-nonamplified cell lines(Fig.
5A), suggesting that N-Myc might negatively regulate ATMexpression
through miR-421. To confirm the interaction of N-Myc, miR-421, and
ATM in neuroblastoma cells, we selected LA-N-1 (N-Myc+) and
CHLA-255(N-Myc−) for the followingexperiments. i) Chromatin
immunoprecipitation (ChIP) showedthat the in vivo binding of N-Myc
to miR-421 promoter onlyoccurred in the N-Myc-amplified LA-N-1
cells and not in the N-Myc-nonamplified CHLA-255 cells (Fig. 5B).
ii) Consistent withthe in vivo binding of N-Myc, endogenous miR-421
expressionwas up-regulated (∼2-fold) in LA-N-1 cells (Fig. 5C). We
alsoexaminedmiR-421 expression in the other five neuroblastoma
celllines. The miR-421 levels were significantly higher in the four
N-Myc-amplified cell lines (Fig. S3A). We noticed that the
miR-421level in CHLA-90 was higher than that in the other two
N-Myc-nonamplified cell lines CHLA-15 and CHLA-255 (Fig. S3A).
Thismight be caused by the low-level expression of N-Myc in CHLA-90
(Fig. 5A). A similar expression pattern for miR-374b wasobserved in
these neuroblastoma cell lines (Fig. S3B), supportinga model that
the miR-421 and miR-374b cluster is driven by thesame promoter
(Fig. 4A). iii) Treatment of LA-N-1 cells withAMO-ATM, which is
complementary to the miR-421 bindingsites at ATM 3′UTR, and with
anti-miR-421 inhibitor, which iscomplementary to miR-421, led to an
increase in ATM expression(Fig. 5D). As expected, AMO-ATM treatment
did not change themiR-421 expression level, whereas anti-miR-421
inhibitor down-regulated miR-421 expression (Fig. 5E), suggesting
two differentmechanisms for AMO-ATM and anti-miR-421 on the
abrogation
of miR-421-mediated down-regulation of ATM expression.Finally,
the increase of ATM expression by AMO-ATM wasfurther confirmed by
the flow cytometry phospho-SMC1 (FC-pSMC1) assay, which was
recently developed to measure ATM
Fig. 5. N-Myc negatively regulates ATM via miR-421 in
neuroblastoma cells.(A) Immunoblot of ATM expression in
N-Myc-amplified (CHLA-134, CHLA136,LA-N-5, and LA-N-1) or
-nonamplified (CHLA-15, CHLA-90, and CHLA-255)neuroblastoma cell
lines. We observed some N-Myc expression in CHLA-90,although it is
an N-Myc-nonamplified cell line; ATM expression was relativelylower
in this cell line when compared with CHLA-15 or CHLA-255. A-T
lym-phoblastoid cells (AT-LCL) and WT lymphoblastoid cells (WT-LCL)
are neg-ative and positive controls, respectively, for ATM
expression. In total, 100 μgof total protein for all neuroblastoma
cells and only 25 μg of total proteinfor AT-LCL and WT-LCL were
loaded; SMC1 served as a loading control.(B) ChIP PCR assay detects
the in vivo binding of N-Myc protein to the miR-421 promoter DNA. A
PCR fragment of expected size (246 bp) was seenin the
N-Myc-amplified (amp.) LA-N-1 cells immunoprecipitated with
thespecific anti-N-Myc antibody (lane 5) but not without antibody
or withnonspecific mouse IgG (lanes 2 and 3). No signal was seen in
the N-Myc-nonamplified CHLA-255 cells immunoprecipitated with no
antibody, non-specific mouse IgG, or specific anti-Myc antibody
(lanes 7–9). PCR with inputDNA was used as a positive control. (C)
Real-time PCR of endogenous miR-421 in the N-Myc-amplified LA-N-1
cells and the N-Myc-nonamplified CHLA-255 cells. RNU66 was used as
an internal control. (D) Immunoblot of ATMexpression in CHLA-255
and LA-N-1 cells treated with AMO-scram (4 μM) orAMO-ATM (4 μM) for
5 days or in L-AN-1 cells transfected with anti-miR-CTL(100 nM) or
anti-miR-421 inhibitor (100 nM) for 96 h. The fold change inATM
expression is shown below. (E) Real-time PCR of miR-421 expression
inLA-N-1 cells treated with AMO-scram (4 μM) or AMO-ATM (4 μM) for
5 daysor transfected with anti-miR-CTL (100 nM) or anti-miR-421
(100 nM) inhibitorfor 4 days. RNU66 was used as an internal
control. (F) FC-SMC1 detection ofIR-induced ATM-dependent
phosphorylation of SMC1 in AMO-treated neu-roblastoma cells. LA-N-1
and CHLA-255 cells were treated with AMO-scram(4 μM) or AMO-ATM (4
μM) for 5 days and subjected to 10-Gy IR. The pSMC1level is
indicated by the fluorescence intensity. The filled peaks represent
thecells without IR, and unfilled peaks represent post-IR cells.
This panel isrepresentative of three independent experiments. (G)
Linear signalingpathway in which N-Myc up-regulates miR-421
expression and miR-421, inturn, down-regulates ATM expression by
targeting its 3′UTR.
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kinase activity in A-T patients and carriers (25). As shown in
Fig.5F, treatment of LA-N-1 cells with AMO-ATM caused a
post-IRshift in pSMC1, whereas treatment of CHLA-255 cells showed
nochange in the post-IR pSMC1 shift. These observations
arecompatible with the model that AMO-ATM could increase
ATMexpression in N-Myc-amplified neuroblastoma cells and are
alsoconsistent with the immunoblot results of ATM expression
asshown in Fig. 5D.Because c-Myc shares a conserved E-box binding
site (5′-CA-
CGTG-3′) with N-Myc (Fig. 4A), we were prompted to
determinewhether c-Myc functions in a manner similar to N-Myc in
up-regulating miR-421 expression. As shown in Fig. S4A,
cotrans-fection of c-Myc with the miR-421 promoter construct into
HeLacells resulted in a significant increase in miR-421
promoter-drivenluciferase activity, as did cotransfection of N-Myc.
EndogenousmiR-421 expression in HeLa cells was similarly increased
∼1.5-fold after transfection of c-Myc (Fig. S4B).
DiscussionTaken together, our experiments suggest a previously
undescribedmechanism of ATM regulation in which a noncoding small
RNA,miR-421, down-regulates ATM expression through targetingATM
3′UTR. This substantially expands our understanding ofATM functions
in cellular physiology, such as cell cycle check-point,
radiosensitivity, and other ATM-mediated cellular func-tions. For
example, microRNA profiling study has revealed thatmiR-421 is
up-regulated in germinal center centroblast B cells(26), where
physiological DNAdamage occurs frequently becauseof somatic
hypermutation and class switch recombination (27).The
miR421-mediated ATM down-regulation in centroblastsmight contribute
to the escape of centroblast B cells from DNAdamage-induced cell
cycle checkpoints and allow centroblasts todevelop into memory B
cells or plasma cells. A recent reportcorroborates this concept in
whichATR (ATMandRad3-related)kinase is transiently silenced by a
transcription repressor Bcl-6 ingerminal center B cells (28).
Interestingly, miR-421 expression isalso up-regulated in diffuse
large B-cell lymphoma cell lines (29),suggesting that this newly
identified miR421–ATM interactionmight be involved in the
progression of diffuse large B-cell lym-phoma. It is known that
about 10% of cases have overexpressionof c-Myc, a result of the
c-myc translocation into the Ig locus (27).We have established that
miR-421 expression is up-regulated by
the transcription factor N-Myc, establishing a linear
signalingpathway (N-Myc → miR-421 → ATM) in such a manner that
theoncogene N-Myc negatively regulates the tumor suppressor
ATM(Fig. 5G). Because the ATM-driven DNA damage response isthought
to be a physiological barrier in early human tumorigenesis(30–33),
our findings add that miR421-mediated ATM down-reg-ulation may
contribute to N-Myc-induced tumorigenesis in neuro-blastoma. The
finding that the up-regulation of miR-421 can alterthe cellular
radiosensitivity suggests that treatment of proliferatingcancer
cells with miR-421-inducing agents might sensitize them
forradiotherapy. Conversely the finding that exposure of
neuro-blastoma cells to AMO-ATM increases ATM expression
impliesthatAMO-ATMholds therapeutical potential
forN-Myc-amplifiedneuroblastomas, perhaps by enhancing
ATM-dependent apoptosisin response to DNA damage (34, 35) or
driving nondividing dif-ferentiated neuronal cells to reenter
S-phase (36). Lastly, the sup-pression of ATM bymiR-421 introduces
two possible pathogeneticmechanisms forA-T:Amutation in
theATM3′UTRmight enhancethe binding of miR-421, or a mutation of
miR-421 might result inmiR-421 overexpression, both leading to the
down-regulation ofATM expression. Such disease-causing mutations of
microRNA-binding sites in the 3′UTR of the target genes have been
reported(37). However, no suchmutations have been observed to date
in A-T patients. Our findings also suggest thatmiR-421 could
function asa modifier gene, contributing to the A-T phenotype and
perhaps tothe variability of disease onset and progression.
Materials and MethodsCell Culture, miRNA Precursors, miRNA
Inhibitors, AMO and Transfection.Neuroblastoma cell lines LA-N-1
and LA-N-5 were cultured in RPMI 1640with 15% (vol/vol) FBS and
streptomycin/penicillin, and CHLA-15, CHLA-90,CHLA-134, CHLA-136,
and CHLA-255 were cultured in Iscove’s Modified Dul-becco’s Medium
with 15% (vol/vol) FBS and streptomycin/penicillin. Theprecursor
miR-421, pre-miR-CTL, anti-miR negative control 1, and anti-miR-421
inhibitor were purchased from Applied Biosystems. Antisense AMO
wassynthesized based on the ATM 3′UTR target sequence and
conjugated withnonpeptide chemicals that are used to deliver AMO to
cells (Gene-Tools). Thesequence of AMO-ATM is
5′-ATCAACAGATATAAACAGCAGG. A standardcontrol AMO (AMO-scram)was
also purchased fromGene-Tools. N-Myc and c-Myc plasmidswere
obtained fromOrigene andOpen Biosystems, respectively.All
transfectionsweredonewith Lipofectamine2000 (Invitrogen) according
tothe provided protocols. The M4 lentiviral vector expressing
miR-421 wasgenerated by standard methods as detailed in SI
Text.
RNA Extraction and Real-Time Quantitative PCR. Total RNA from
cultured cellswas extracted by the mirVana miRNA isolation kit
(Applied Biosystems). Taq-Man microRNA expression assays (Applied
Biosystems) were usedto quantitate mature miR-421 expression
according to the provided protocol.RNU66 or U6 expression assay was
used as an internal control for
miR-421expression.ATMmRNAquantificationweremeasured by real-time
PCR basedon TaqMan Gene Expression Assays (Applied Biosystems), as
previouslydescribed (38). Glyceraldehyde-3-phosphate-dehydrogenase
(GAPDH) mRNAwas used as an internal control to normalize ATMmRNA
level. Real-time PCRquantitation of transcripts was expressed as
ATM/GAPDH ratios.
Luciferase Reporter Assays and Transcription Factor Analysis.
Cells were trans-fected with appropriate reporter vectors and then
harvested and lysed forluciferase assays by a Dual-Glo assay kit
(Promega) according to the manu-facturer’s protocol. The detailed
protocol is described in SI Text.
The transcription factor binding site analysis was done using
the CONSITEdatabase. A 2-kb genomic sequence upstream of miR-421
was used as theanalyzing template, and the cutoff value for
transcription factors was set to99%; N-Myc transcription factor was
the top candidate.
ATM-ELISA. An ELISA was used to determine the relative ATM
expression incells and performed as previously described (39). More
details on the ATM-ELISA are provided in SI Text.
BrdU Incorporation Assay. To analyze the S-phase checkpoint,
cells takenat 70% confluence were irradiated with the indicated
dose and incubated for20 h. BrdU was added to cells and incubated
for 2 h. Cells were collected bytrypsinization and centrifugation.
Cells were subject to the BrdUflow stainingaccording to
themanufacturer’s protocol for BrdU FlowKits (BD Pharmingen).Three
independent experiments were performed.
Clonogenic Survival Assay and Propidium Iodide-Staining Cell
Death. MiR-421-expressing stable and control shRNA cells were
plated at 500 cells per wellonto a six-well dish in triplicate and
then incubated for 24 h to allow settling.Cells were treated with a
series of IR doses (0, 1, 2, and 5 Gy) and grown for 2weeks before
stainingwith 1% crystal violet. Clumps containingmore than 50cells
were scored as “colony-positive”wells and counted by the Quantify
Oneprogram in the VersaDoc Imaging System (Bio-Rad). To generate a
radiationsurvival curve, the surviving fraction at each radiation
dose was normalizedto that of a nonirradiated control. All
experiments were repeated at leasttwice. For IR-induced cell death,
cells were treated with 10-Gy radiation andstained with propidium
iodide after 48 h of incubation to assess the numberof dead cells.
Samples were analyzed by a FACScan Analytic Flow Cytometer(Becton
Dickinson). Three independent experiments were done. Cell deathwas
normalized to nonirradiated control cells.
FC-pSMC1 Assay and ChIP Assay. An FC-pSMC1 assay was performed
as pre-viously described (25), and a ChIP assay was performed as
previouslydescribed (40). More detailed protocols for Fc-pSMC1 and
ChIP assays areprovided in SI Text.
Statistics. The Student’s t -test was used to evaluate the
significant differenceof two groups of data in all the pertinent
experiments. A P value
-
ACKNOWLEDGMENTS. We thank Dr. John Colicelli for M4 lentiviral
vector andDr. Matteo Pellegrini and Aliz Raksi for microRNA target
predictions andanalyses. This work was supported by Grant NS052528
from the National
Institutes of Health, the Ataxia-Telangiectasia Medical Research
Foundation(Los Angeles, CA), and the Ataxia-Telangiectasia Ease
Foundation (NewYork, NY).
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