Báo cáo khoa học: MicroRNA-143 reduces viability and increases sensitivity to 5-fluorouracil in HCT116 human colorectal cancer cells potx

MicroRNA-143 reduces viability and increases sensitivity
to 5-fluorouracil in HCT116 human colorectal cancer cells
Pedro M. Borralho
1
, Betsy T. Kren
2
, Rui E. Castro
1
, Isabel B. Moreira da Silva
1
, Clifford J. Steer
2,3
and Cecı
´
lia M. P. Rodrigues
1
1 Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Portugal
2 Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA
3 Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN, USA
Introduction
MicroRNAs (miRNAs) are a recently discovered and
growing class of small noncoding, single-stranded
RNAs that negatively regulate gene expression.
miRNA biogenesis initiates by RNA polymerase II
transcription of a primary transcript (pri-miRNA).
This pri-miRNA is processed in the nucleus by the
RNase III enzyme Drosha, producing pre-miRNAs,
comprising hairpin structures of approximately 70
nucleotides. Subsequently, pre-miRNAs are exported
to the cytoplasm through an Exportin-5-dependent
mechanism, and further processed by the RNAse III

enzyme Dicer, producing miRNA duplexes. One strand
of the duplex is incorporated into the RNA-induced
silencing complex, whereas the other is usually rapidly
degraded. In mammals, miRNA-directed gene silencing
occurs primarily via incomplete miRNA binding to 3¢-
UTRs of target mRNAs, which represses translation
and, in some cases, leads to mRNA degradation [1].
More than 670 miRNAs have been identified in
humans, but mRNA targets and regulatory pathways
Keywords
5-fluorouracil; apoptosis; chemosensitizer;
ERK5; miR-143
Correspondence
C. M. P. Rodrigues, Research Institute for
Medicines and Pharmaceutical Sciences,
Faculty of Pharmacy, University of Lisbon,
Avenida Professor Gama Pinto, 1649-003
Lisbon, Portugal
Fax: +351 21 794 6491
Tel: +351 21 794 6400
E-mail:
(Received 29 April 2009, revised 2 August
2009, accepted 15 September 2009)
doi:10.1111/j.1742-4658.2009.07383.x
MicroRNAs are aberrantly expressed in cancer; microRNA-143 (miR-143)
is down-regulated in colon cancer. HCT116 human colorectal cancer cells
were used to investigate the biological role of miR-143. Transient miR-
143 overexpression resulted in an approximate 60% reduction in cell via-
bility. In addition, stable miR-143 overexpressing cells were selected with
G418 and exposed to 5-fluorouracil. Increased stable expression of miR-

143 was associated with decreased viability and increased cell death after
exposure to 5-fluorouracil. These changes were associated with increased
nuclear fragmentation and caspase -3, -8 and -9 activities. In addition,
extracellular-regulated protein kinase 5, nuclear factor-jB and Bcl-2 pro-
tein expression was down-regulated by miR-143, and further reduced by
exposure to 5-fluorouracil. In conclusion, miR-143 modulates the expres-
sion of key proteins involved in the regulation of cell proliferation, death
and chemotherapy response. In addition, miR-143 increases the sensitivity
of colon cancer cells to 5-fluorouracil, probably acting through extracellu-
lar-regulated protein kinase 5 ⁄ nuclear factor-jB regulated pathways. Col-
lectively, the data obtained in the present study suggest anti-proliferative,
chemosensitizer and putative pro-apoptotic roles for miR-143 in colon
cancer.
Abbreviations
ERK5, extracellular-regulated protein kinase 5; 5-FU, 5-fluorouracil; LDH, lactate dehydrogenase; miRNA, microRNA; miR-143, miRNA-143;
miR-145, miRNA-145; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NF, nuclear factor; pNA,
p-nitroanilide
FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6689
have only been explored for a handful of miRNAs.
Furthermore, miRNAs are significantly involved in the
regulation of a myriad of biological processes, such as
differentiation, proliferation and apoptosis [2], which
are commonly altered in cancer. It is now apparent
that miRNAs are differentially expressed in a wide
array of human cancers, including colorectal cancer
[3,4]. Nevertheless, the molecular signalling pathways
modulated by miRNAs, which play a role in colorectal
cancer, as well as cancer in general, are only partially
characterized. miRNA-143 and miRNA-145 (miR-143
and miR-145) expression was found reduced in

tumours versus matched normal mucosa, both at the
adenomatous and cancer stages of colorectal neoplasia,
as well as in colon cancer cell lines [3,5]. Furthermore,
miR-143 is a putative cancer biomarker that is also
down-regulated in B-cell malignancies [6], bladder [7]
and cervical cancer [8]. miR-143 targets extracellular-
regulated protein kinase 5 (ERK5) during adipocyte
differentiation [9] and in DLD-1 colon cancer cells [5].
To date, miR-143 has been demonstrated to directly
target the mRNA of KRAS [10,11], ERK5 [12],
DNMT3A [13] and ELK1 [14]. Despite growing evi-
dence for an anti-proliferative and putative pro-apop-
totic role for miR-143, a more detailed understanding
of miR-143 biological function is warranted because
reduced miR-143 expression may deregulate molecular
signalling pathways with direct implications in cancer
onset and ⁄ or progression and response to chemo-
therapy.
ERK5 is a mitogen-activated protein kinase that is
regulated by a wide range of mitogens and cellular
stresses. In addition, activated ERK5 is involved in cell
survival, differentiation and proliferation. Several
ERK5 targets and potential regulators of cell prolifera-
tion have been identified, including c-Myc, cyclin D1
and nuclear factor (NF)-kB [15]. ERK5 activation of
NF-jB promotes cellular transformation [16] and is a
critical factor for G2–M cell cycle progression and
timely mitotic entry [17]. NF-jB is a critical transcrip-
tion factor involved in the suppression of apoptosis,
stimulation of cell growth and the modulation of genes

that appear to be important in tumour promotion,
angiogenesis and metastasis. NF-jB activation is also
associated with increased resistance to chemotherapeu-
tic agents [18]. Therefore, strategies aimed at reducing
ERK5 and NF-jB signalling may modulate tumour
growth and sensitivity to chemotherapeutic agents.
5-Fluorouracil (5-FU) has been the drug of choice
for the treatment of colorectal cancer for more than
four decades. Its limited efficacy as a single agent for
advanced colorectal cancer has been improved by
combination with newer chemotherapeutic agents [19].
5-FU has been shown to induce apoptosis in colon
cancer cell lines [20,21]. The ability of tumour cells to
evade apoptosis is an enormous obstacle for effective
treatment. Consequently, strategies aiming to over-
come tumour cell resistance to chemotherapy and to
increase drug efficacy, thereby minimizing toxic effects,
are critically important. The molecular mechanisms of
5-FU cytotoxicity have been characterized, especially
its ability to incorporate into RNA and DNA and to
inhibit thymidylate synthase. 5-FU has recently been
shown to modulate miRNA expression in colon cancer
cells [22]. However, the relationship between 5-FU and
miRNAs, their potential interactions and their rele-
vance for drug efficacy have not been extensively stud-
ied. Nevertheless, drug function could potentially be
improved via modulation of miRNAs that play a role
in chemoresistance.
In the present study, we evaluated the role of
miR-143 in the response of HCT116 colon cancer cells

to 5-FU. The results obtained demonstrate that
miR-143 decreased colon cancer cell viability and
increased 5-FU sensitivity, suggesting that it may act
as a potential chemosensitizer to 5-FU in colon cancer
cells. In addition, miR-143 overexpression resulted in
the down-regulation of ERK5, NF-jB and Bcl-2 pro-
tein expression, which was further reduced by 5-FU.
Collectively, our data indicate that reduced miR-143
expression in colon cancer may contribute to
unchecked proliferation and decreased sensitivity to
5-FU. Furthermore, increasing miR-143 expression in
colon cancer cells may comprise a promising strategy
for reducing tumour growth and aggressiveness, at the
same time as increasing sensitivity to 5-FU.
Results
miR-143 overexpression reduces colon cancer cell
viability
We first evaluated miR-143 expression in colon cancer
cells lines by semi-quantitative RT-PCR, using total
RNA and miRNA-enriched RNA (providing a higher
abundance of < 200 nucleotides RNA molecules per
lg RNA). The results obtained confirmed that
miR-143 levels were almost undetectable in HCT116,
LoVo, SW480 and SW620 colon cancer cells, using up
to 500 ng of miRNA-enriched RNA and 30 PCR
cycles (data not shown). By contrast, miR-143 expres-
sion was readily detected from total heart RNA, using
lower RNA input, whithout miRNA-enrichement. This
was achieved with as little as 25 ng of total heart
RNA, and at a lower PCR cycle number, thus

reinforcing the notion that mir-143 is expressed at low
miR-143 modulates 5-FU cytotoxicity P. M. Borralho et al.
6690 FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS
levels in colon cancer cells. U6 was used throughout as
normalization control. HCT116 cells were chosen for
further experiments because the molecular pathways of
5-FU cytotoxicity have already been explored in this
cell line [20].
We next confirmed that mature miR-143 is produced
from pCR3-pri-miR-143 vector and that it specifically
binds to miR-143 sensor vector. This was achieved by
co-transfecting HCT116 cells with pCR3-pri-miR-143,
a firefly luciferase miR-143 sensor plasmid (miR-143
sensor) as a reporter for mature miR-143 expression,
and with either miR-143 specific inhibitor (anti-miR-
143) or control (anti-miR-control). pRL-SV40 was also
co-transfected and used as a normalization control.
The results obtained showed that the lower availability
of mature miR-143 after anti-miR-143 co-transfection
led to increased firefly activity (Fig. 1A).
After validating the vectors, we determined the effect
of miR-143 transient overexpression in HCT116 colon
cancer cells. The results obtained demonstrated that
pri-miR-143 overexpression reduced HCT116 cell via-
bility by approximately 60%, at 48 h post-transfection
(P < 0.05) as evaluated by the 3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS) assay (Fig. 1B). In addition,
miR-143 overexpression increased cell death as early as
24 h post-transfection (data not shown). pCR3-empty

transfection resulted in no significant change in cell
viability.
miR-143 increases 5-FU cytotoxicity
We next determined the effects of 5-FU on colon can-
cer cells with endogenous low levels of miR-143.
Therefore, we exposed HCT116 colon cancer cells to
8 lm 5-FU, a clinically relevant concentration [23] that
has been shown to induce cell death and apoptosis
[20]. 5-FU reduced cell viability by approximately 40%
and increased general cell death by approximately
60% after 48 h of exposure (P < 0.01). After miR-143
transient overexpression, 5-FU also reduced cell viabil-
ity and increased cell death compared to pCR3-empty
5-FU exposed cells (data not shown). This prompted
us to create a stable miR-143 overexpression model by
transfecting HCT116 cells with pCR3-pri-miR-143 and
pCR3-empty and selecting with G418.
On the basis of miR-143 expression levels evaluated
by luciferase activity after pGL3-miR-143 sensor and
pGL3-control, we selected a miR-143 overexpression
clone and an empty vector clone, designated HCT116-
OV3 and HCT116-EM1, respectively (Fig. 2A). In
addition, we measured miR-143 expression by TaqMan
real-time PCR, using specific primers for mature
miR-143, and RNU6B for normalization. The results
obtained showed that miR-143 expression is increased
five-fold in HCT116-OV3 cells compared to HCT116
parental cells (P < 0.01) (Fig. 2B).
We also evaluated whether 5-FU directly modulates
the expression of mature miR-143. Total RNA was

extracted from HCT116, HCT116-OV3 and HCT116-
EM1 cells exposed to 8 lm 5-FU for 48 h. Mature
A
B
Fig. 1. Mature miR-143 overexpression decreases HCT116 cell
viability. Cells were transfected with the indicated plasmids and 50
or 100 n
M anti-miR inhibitors, and analyzed at 48 h post-transfec-
tion. (A) Cells were lysed and firefly and renilla luciferase activities
were determined by the dual luciferase assay. (B) Cell viability was
evaluated by MTS metabolism assays. Cells were then lysed and
renilla luciferase activity was determined by the dual luciferase
assay, for normalization of the MTS metabolism assay. The results
are expressed as the mean
± SEM from at least three indepen-
dent experiments. P < 0.01 and *P < 0.001 compared to controls;
§P < 0.05 compared to pCR3-empty + pRL-SV40.
P. M. Borralho et al. miR-143 modulates 5-FU cytotoxicity
FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6691
miR-143 expression was measured by TaqMan real-
time PCR. Interestingly, the results obtained showed
that miR-143 increased almost three-fold in parental
HCT116 and HCT116-EM1 cells, but by only two-fold
in HCT116-OV3 (P < 0.05) (Fig. 2C).
We next evaluated the effect of increased mature
miR-143 expression in colon cancer cells exposed to
5-FU. For this purpose, cells were exposed to 1–10 lm
5-FU for 72 h, and cell viability and cell death were
evaluated by the MTS metabolism and lactate dehy-
drogenase (LDH) release assays. The results obtained

indicated that mature miR-143 enhanced sensitivity to
5-FU. Indeed, cell viability was reduced and cell death
was increased in HCT116-OV3 compared to parental
and HCT116-EM1 cells, after 1–10 lm 5-FU exposure
for 72 h (P < 0.05) (Fig. 3A). In addition, increased
sensitivity to 5-FU was also observed in G418 selected
pCR3-pri-miR-143 transfected mixed populations com-
pared to pCR3-empty transfected mixed populations
after exposure to same doses of 5-FU (Fig. 3B).
Furthermore, 5-FU-induced apoptosis was enhanced in
HCT116-OV3 cells, resulting in increased caspase-3, -8
and -9 activities and subsequent nuclear fragmentation
compared to parental HCT116 and HCT116-EM1
control cells (P < 0.05) (Fig. 4).
Finally, we evaluated miR-143 expression in SW480
cancer cells exposed to 5-FU. SW480 cells were less
sensitive to 5-FU than HCT116 cells (Fig. 5A). In
addition, 5-FU up-regulated miR-143 expression in
both cell lines (Fig. 5B). Nevertheless, miR-143 expres-
sion was significantly lower in SW480 cells. The DC
t
values were 12.10 ± 0.35 in SW480 cells compared to
5.45 ± 0.08 in HCT116 cells (P < 0.001), whereas
RNU6B C
t
values for control and 5-FU-treated
HCT116 and SW480 cells were 24.43 ± 0.22 and
24.42 ± 0.25, respectively (P = 0.954). Collectively,
this represents an increase of almost 100-fold in
miR-143 expression in HCT116 relative to SW480

cells, thereby reinforcing the potential involvement of
miR-143 in 5-FU sensitivity.
miR-143 and 5-FU down-regulate ERK5, NF-jB
and Bcl-2 protein expression
ERK5 targeting by miR-143 has been shown in adipo-
cytes [9] in addition to DLD-1 colon cancer cells [5].
More recently, miR-143 has been demonstrated to
directly target the mRNA of KRAS [10,11], ERK5
[12], DNMT3A [13] and ELK1 [14]. To determine
whether miR-143 modulates ERK5 in HCT116 cells as
well as its relevance to 5-FU cytotoxicity, we evaluated
ERK5 protein expression after transfection of miR-143
precursor molecules (pre-miR-143) and a nonspecific
control (pre-miR-control). The results obtained showed
that miR-143 reduced ERK5 protein expression in a
dose-dependent manner, leading to an approximate
70% reduction at 48 h post-transfection with 80 nm
pre-miR-143 (P < 0.001) (Fig. 6A, upper panel). In
addition, after transfection with 80 nm pre-miR-143,
5-FU further reduced ERK5 protein expression in
a time-dependent manner, reaching a reduction of
50%, 60% and 80% at 24, 48 and 72 h, respectively
A
C
B
Fig. 2. 5-FU increases miR-143 expression in HCT116 cells.
(A) miR-143 expression by luciferase activity assays. Cells were
co-transfected with either pGL3-miR-143 sensor or pGL3-control
and pRL-SV40 and analyzed at 48 h after transfection. Cells were
lysed and luciferase activity was evaluated with the dual luciferase

assay. miR-143 levels were expressed as the luciferase signal ratio
of pGL3-miR-143 sensor to pGL3-control cells. (B, C) Cells were
harvested for total RNA extraction after 48 h in culture with and
without 8 l
M 5-FU exposure. miR-143 expression was evaluated
from 1.33 lL cDNA of 10 ng of total RNA RT reactions, using spe-
cific primers for miR-143 and RNU6B for normalization. miR-143
expression levels were calculated by the DDC
t
method, using
HCT116 control cells as calibrator. The results are expressed as the
mean ± SEM fold-change compared to HCT116 cells from three to
six independent experiments. * P < 0.001 and P < 0.05 compared
to controls.
miR-143 modulates 5-FU cytotoxicity P. M. Borralho et al.
6692 FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS
(P < 0.05) compared to controls (Fig. 6A, lower
panel). In addition, we were also able to regulate
ERK5 protein expression by modulating the availabil-
ity of mature miR-143 (Fig. 6B). At 72 h after
co-transfection of 40 nm pre-miR-143 with 60 nm of
its specific inhibitor anti-miR-143, ERK5 was
increased compared to pre-miR-143 and anti-miR con-
trol co-transfection experiments (Fig. 6B, lane 4 versus
5). Moreover, pre-miR-control and anti-miR-143
co-transfection also increased ERK5 protein expression
compared to co-transfection of pre-miR-control and
anti-miR-control (Fig. 6B, lane 6 versus 7). Exposure
to 5-FU resulted in a further reduction of ERK5 pro-
tein expression (Fig. 6B, lanes 9–14 versus 1–7).

Accordingly, higher miR-143 abundance decreased cell
viability (Fig. 6C). Finally, ERK5 expression was
knocked-down in HCT116 cells by transfecting 80 nm
of specific ERK5 siRNA and then exposing cells to
5-FU. The results obtained show that ERK5 silencing
augmented apoptosis compared to mock-transfected
cells, whereas increasing 5-FU-induced apoptosis
(P < 0.05) (Fig. 6D).
Furthermore, we also found that stable miR-143
overexpressing cells express lower levels of ERK5,
NF-jB and Bcl-2 proteins compared to control and
parental HCT116 cells. Furthermore, 5-FU potentiated
ERK5, NF-jB and Bcl-2 expression knockdown
(Fig. 7). These results further indicate that miR-143 is
a key player in the regulation of cell proliferation and
the response to 5-FU growth inhibition ⁄ cytotoxicity in
HCT116 cells, probably by acting through the
ERK5 ⁄ NF-jB axis.
Discussion
It is now well-established that miRNAs regulate a
plethora of crucial cellular functions, including cell
growth, differentiation and apoptosis, which are
commonly altered in cancer cells. In the present study,
we evaluated the effect of miR-143 overexpression on
HCT116 colon cancer cells. HCT116 cells were trans-
fected with pCR3-pri-miR-143, and the respective con-
trol, pCR3-empty. miR-143 production was assessed
by luciferase assays. The results obtained demonstrate
A
B

Fig. 3. mir-143 overexpressing cells are more sensitive to 5-FU cytotoxicity. Cells were exposed to either 1–10 lM 5-FU or no addition (con-
trol) for 72 h. Mixed populations of miR-143 overexpressing cells (pCR3-pri-miR-143 mix) and respective control cells (pCR3-empty mix) were
generated by transfecting HCT116 cells with pCR3-pri-miR-143 or pCR3-empty vector, respectively. Cells were selected and maintained with
1mgÆmL
)1
G418, under the same conditions as those used for single-clone derived HCT116-OV3 and HCT116-EM1 cells. (A, B) Cells with
higher miR-143 expression (HCT116-OV3 and pCR3-pri-miR-143 mix) displayed decreased viability and increased cell death after exposure to
5-FU. Cell viability and cell death were evaluated by MTS metabolism and LDH activity assays, respectively. In the far right panels of (A) and
(B), LDH normalized to MTS is also plotted for the mean values of each 5-FU concentration used. The results are expressed as the mean ±
SEM fold-change compared to controls from at least three independent experiments. *P < 0.01 and P < 0.05 from HCT116-EM1. No signif-
icant changes were observed between HCT116 and HCT116-EM1 or between HCT116 and pCR3-empty mix for 1–10 l
M 5-FU.
P. M. Borralho et al. miR-143 modulates 5-FU cytotoxicity
FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6693
that increased pri-miR-143 expression reduced cell via-
bility in pCR3-pri-miR-143 transfected cells by 60%
compared to control cells. These results are in accor-
dance with a previous study, in which pre-miR-143
transfection in SW480 and DLD-1 colon cancer cells
reduced cell viability in a dose-dependent manner [5].
We were particularly interested in investigating the
effect of miR-143 on the cellular response to 5-FU.
5-FU is a well-known apoptosis-inducing drug; indeed,
it was shown previously that 5-FU induces apoptosis in
HCT116 cells [20]. 5-FU has been used in the clinic for
several decades, and its metabolic pathways and cyto-
toxic modes of action through the inhibition of thymi-
dylate synthase activity and incorporation into RNA
and DNA are well known. However, the molecular
pathways modulated by 5-FU that lead to cell growth

inhibition and cell death induction are not entirely
understood, nor are the mechanisms of tumour cell
escape from 5-FU cytotoxicity. In addition, little is
known about the role of miRNAs in modulating the
tumour cell response to chemotherapeutic agents, such
as 5-FU. Nevertheless, 5-FU alters the expression of a
set of 22 miRNAs in colon cancer cell lines [22]. Further-
more, S-1, a fourth-generation 5-FU-based oral drug
developed to improve efficacy, also alters the expression
levels of certain miRNAs, as demonstrated in tumour
tissue from patients undergoing S-1 therapy [24].
To evaluate the effect of miR-143 on 5-FU sensitivity,
we created stable miR-143 overexpressing cells
(HCT116-OV3) and the respective control cells
(HCT116-EM1) by pCR3-pri-miR-143 and pCR3-
empty transfection, respectively, and G418 selection.
miR-143 expression was evaluated by luciferase assays
and also by TaqMan real-time PCR with specific primers
for miR-143 and RNU6B. The results obtained showed
that miR-143 expression was increased five-fold in
HCT116-OV3 cells. Cells were then exposed to different
concentrations of 5-FU for 72 h to plot growth inhibi-
tion and cell death dose–response curves. 5-FU at a con-
centration 1–10 lm was significantly more cytotoxic in
HCT116-OV3 cells compared to HCT116-EM1 control
or HCT116 parental cells. These effects were already evi-
dent 48 h after 5-FU exposure but more intense after
72 h of drug exposure. Furthermore, we also observed
higher sensitivity to 5-FU in mixed populations of miR-
143 overexpressing cells compared to controls. Curi-

ously, a chemosensitizer role of miR-143 to 5-FU has
also been reported in gastric cancer cells. Indeed, trans-
fection of pre-miR-143 into MKN-1 cells was shown to
reduce the number of viable cells after exposure to
10 lm 5-FU [25]. Collectively, these data indicate that
miR-143 is an important modulator of 5-FU sensitivity
not only in colon cancer, but also in other types of
cancer, particularly those of the gastrointestinal tract.
Fig. 4. HCT116-OV3 cells are more sensi-
tive to 5-FU-induced apoptosis. Cells were
exposed to 8 l
M 5-FU and processed for
evaluation of nuclear morphology after
Hoechst staining at 24 h, and for caspase
activity assays at 12 h. The results are
expressed as the mean ± SEM fold-change
compared to controls from at least three
independent experiments. *P < 0.01 and
P < 0.05 from HCT116-EM1. No significant
changes were observed between HCT116
and HCT116-EM1.
miR-143 modulates 5-FU cytotoxicity P. M. Borralho et al.
6694 FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS
We next selected 8 lm 5-FU to further explore the
differential sensitivity of our cell model. We have pre-
viously shown that it efficiently triggers apoptosis in
HCT116 cells [20]. Furthermore, the results obtained
in the present study now show that 8 lm 5-FU
induced growth inhibition by 55% in addition to a
two-fold increase in cell death. Notably, 5-FU

significantly increased caspase activation and nuclear
fragmentation in HCT116-OV3 compared to control
and parental cells. We have also evaluated miR-143
expression after 48 h of 5-FU exposure and found it to
be significantly increased. Curiously, 5-FU increased
miR-143 expression to a lesser extent in HCT116-OV3
than in HCT116-EM1 and HCT116 cells, which may
be attributed to the relatively high levels of miR-143 in
HCT116-OV3 cells. Furthermore, up-regulation of
miR-143 by 5-FU also occurs in SW480 cells that are
less sensitive to 5-FU cytotoxicity. However, the basal
expression of miR-143 is significantly lower in SW480
cells than in HCT116, which underlines the relevance
of miR-143 expression on 5-FU sensitivity.
The results obtained in the present study are in agree-
ment with a recent report showing that p53 enhances the
post-transcriptional maturation of miR-143 in response
to DNA damage [10]. Using wild-type p53 HCT116 cells
exposed to the potent p53 inducer doxorubicin, and
despite no significant changes in pri-miR-143 levels,
there was increased processing of pri-miR-143 into pre-
and mature miR-143 [10]. In addition, this DNA-dam-
age-induced up-regulation of pre-miR-143 and mature
miR-143 was diminished in p53 null HCT116 cells,
thereby suggesting that increased mature miR-143
expression may be a result of increased p53 expression.
These data may explain the increased levels of mature
miR-143 that we found in the present study after expo-
sure of wild-type p53 HCT116 cells to 5-FU because it
has previously been shown that 5-FU also strongly

increases p53 expression in this cell line [20].
Interestingly, when mutant p53 (R273H) was intro-
duced into p53 null HCT116 cells, there was a reduc-
tion in pre- and mature miR-143 production,
contrasting with constant levels of pri-miR-143 [10],
which suggests that mutant p53 hampers miR-143 pro-
cessing in a transcription-independent manner. These
results are once again in agreement with the results
obtained in SW480 cells. In these cells expressing the
R273H mutant p53, we observed a dramatic approxi-
mately 100-fold decrease in mature miR-143 expression
compared to wild-type p53 HCT116 cells. However, in
SW480 cells, we also found an increase in mature
miR-143 after 5-FU exposure, which may indicate that
there is some mature miR-143 production that escapes
wild-type p53 control. Nevertheless, the levels of
mature miR-143 in SW480 cells treated with 5-FU are
much lower than those observed in untreated HCT116
cells. This finding may contribute to or be associated
with SW480 lower sensitivity to 5-FU cytotoxicity.
We cannot conclude whether the basal difference in
miR-143 expression between HCT116 and SW480 cells
results from loss of transcriptional or post-transcrip-
tional p53 modulation. p53 has been shown to increase
mature miR-145 expression and bind to a putative
response element located upstream of miR-145 [26]. In
addition, miR-143 and miR-145 are highly conserved
miRNAs in close genomic proximity [14,27], suggesting
A
B

Fig. 5. SW480 cells are less sensitive to 5-FU than HCT116 cells
and express less miR-143. Cells were exposed to either 1–100 l
M
5-FU or no addition (control) for 72 h. (A) HCT116 cells displayed
increased growth inhibition and cell death after exposure to 5-FU,
as evaluated by MTS metabolism and LDH activity assays, respec-
tively. (B) Cells were exposed to 8 l
M 5-FU for 72 h and miRNA-
enriched RNA was extracted with a mirVanaÔ PARIS kit. miR-143
expression was evaluated from 4 lL of cDNA of a 50 ng miRNA-
enriched RNA RT reaction, using specific primers for miR-143, and
RNU6B for normalization. miR-143 expression levels were calcu-
lated by the DDC
t
method, using control cells as calibrator. miR-143
was up-regulated in HCT116 and SW480 cells after 5-FU exposure
by approximately three- and two-fold, respectively. RNU6B C
t
val-
ues for control and 5-FU-treated HCT116 and SW480 cells were
24.43 ± 0.22 and 24.42 ± 0.25, respectively (P = 0.954). The
results are expressed as the mean ± SEM percentage of growth
inhibition, LDH activity or miR-143 expression from at least three
independent experiments. (A) *P < 0.01 and P < 0.05 from
SW480 cells; (B) *P < 0.001 compared to HCT116 and P < 0.05
compared to the respective nontreated cells.
P. M. Borralho et al. miR-143 modulates 5-FU cytotoxicity
FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6695
that their expression is linked to a bicistronic primary
precursor. Indeed, the use of specific primers for

miR-143 and miR-145 resulted in amplicons containing
both miR-143 and miR-145, suggesting that similar
mechanisms are responsibe for the regulation of
miR-143 and miR-145 [14]. Nevertheless, p53 induc-
tion by doxorubicin increases the transcriptional
activation of miR-34a, which is a known p53 transcrip-
tional target, and does not increase pri-miR-143 or
pri-miR-145 [10]. In addition, the increased maturation
of miR-145 after DNA damage was similar to the
reported increase in miR-143 maturation in multiple
cell lines. Further studies are required to clarify the
molecular details of the relationship between p53 and
miR-143 and miR-145.
miR-143 was initially shown to regulate adipocyte
differentiation by targeting ERK5 [9]. Recently,
ERK5 [5], KRAS [10,11] and DNMT3A [13] were
also shown to be negatively modulated by miR-143 in
colon cancer cells. More importantly, these genes
have been proven to be direct targets of miR-143
[10–13]. Our data obtained in HCT116 cells are in
accordance with previous reports and show that
miR-143 overexpression reduces ERK5 protein
expression. ERK5 is involved in cell proliferation
and differentiation [28], as well as the inhibition of
endothelial cell apoptosis [29]. In addition, ERK5 has
been identified as a pro-survival kinase during mito-
sis, whereas ERK5 knockdown by RNA interference
induced apoptosis [30]. ERK5 is also a critical factor
A
B

D
C
Fig. 6. miR-143 and 5-FU down-regulate ERK5 protein expression. (A) HCT116 cells were transfected with 40 or 80 nM pre-miR-143 or pre-
miR-control and ERK5 protein expression was evaluated at 48 h after transfection (upper panel). HCT116 cells were transfected with 80 n
M
pre-miR-143 or pre-miR-control. At 9 h after transfection, cells were exposed to 8 lM 5-FU for 24, 48 or 72 h. ERK5 protein expression was
evaluated at the indicated time-points (lower panel). (B) HCT116 cells were transfected with 40 n
M pre-miR-143 or pre-miR-control, or with
additional 60 n
M anti-miR-control or anti-miR-143. At 9 h after transfection, cells were exposed to 8 lM 5-FU for 72 h, at which time ERK5
protein expression was evaluated. (C) HCT116 cells were transfected with 80 n
M pre-miR-143, pre-miR-control, anti-miR-143 or anti-miR-con-
trol. At 9 h after transfection, cells were exposed to 8 l
M 5-FU for 72 h, at which time cell viability was determined. The results are
expressed as the fold-change of LDH normalized to MTS for pre-miR-143 and anti-miR-143 compared to the respective controls. Black bar,
ratio of pre-miR-143 over pre-miR-control; white bar, ratio of anti-miR-143 over anti-miR-control. (D) HCT116 cells were transfected with
80 n
M ERK5 siRNA or mock transfected. Twenty-four hours after transfection, cells were exposed to 8 lM 5-FU for 48 h and processed for
evaluation of nuclear morphology after Hoechst staining. ERK5 protein expression was also evaluated at the same time. The results are
expressed as the mean ± SEM fold-change compared to controls from at least three independent experiments. Representative blots from at
least three independent experiments are shown. *P < 0.05 compared to control HCT116 cells (mock) and P < 0.05 compared to 5-FU-trea-
ted HCT116 cells (5-FU-treated mock).
miR-143 modulates 5-FU cytotoxicity P. M. Borralho et al.
6696 FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS
in G2–M cell cycle progression and timely mitotic
entry because stimulation of ERK5 activated NF-jB,
whereas inhibition of NF-jB at G2–M significantly
delayed mitotic entry [17]. These results suggest a
potential crosstalk between ERK5 and the apoptotic
machinery. The results obtained in the present study

are in agreement with this notion by demonstrating
that miR-143 overexpression leads to a significant
reduction of ERK5 and NF-jB protein expression.
Because ERK5 directly activates NF- jB to promote
cell cycle progression through G2–M, reduced levels
of these proteins may account for reduced cell growth
as well as an increased response to 5-FU. In addition,
we also see a strong down-regulation of the anti-
apoptotic protein Bcl-2 associated with higher
miR-143 expression, which may contribute to the
putative pro-apoptotic role of this miRNA. Our
results are particularly relevant because they suggest
that miR-143 is an endogenous cell growth ⁄ prolifera-
tion master switch, in which any loss may result in
unchecked cellular proliferation via ERK5 ⁄ NF-jB
signalling. In turn, this may constitute a key event
for colon cancer onset and progression.
miR-143 expression was evaluated in situ in mouse
colonic epithelial crypts [27] and found mainly in the
cytoplasm, where it was more intense in the ascending
more differentiated pre-apoptotic glandular cells than
in the immature, proliferating cells at the bottom of
crypts. Therefore, the extremely low miR-143 expres-
sion levels in cancer and, in particular in colon cancer,
may be an important factor contributing to tumour
growth and ⁄ or escape from apoptosis. In addition,
miR-143 expression has been shown to increase after
a-mangostin exposure in human colon cancer DLD-1
cells, resulting in increased apoptosis [31]. Further-
more, a-mangostin acted synergistically with low dose

5-FU, increasing DLD-1 growth inhibition [31]. Our
data also show that 5-FU increases miR-143 expres-
sion, which may potentiate 5-FU sensitivity, suggesting
a feed-forward mechanism of action.
The results obtained in the present study provide
additional insight into miR-143 regulated pathways
and their impact on 5-FU sensitivity. Exposure of
miR-143 overexpressing cells to 5-FU potentiated a
significant decrease in ERK5, NF-jB and Bcl-2 protein
expression and resulted in increased 5-FU cytotoxicity.
Collectively, our findings emphasize the potential
pivotal relevance of miR-143 in the colon cancer envi-
ronment and suggest that it has a role as chemosensi-
tizer to 5-FU. Further studies are necessary to
elucidate the full extent of the molecular signalling
pathways and players modulated by miR-143.
Materials and methods
Cell culture
HCT116 cells were grown in DMEM supplemented with
10% fetal bovine serum (Invitrogen, Grand Island, NY,
USA), 1% l-glutamine 200 mm (Merck and Co. Inc.,
Whitehouse Station, NJ, USA) and 1% antibiotic ⁄ antimy-
cotic solution (Sigma Chemical Co., St Louis, MO, USA)
and maintained at 37 °C in a humidified atmosphere of 5%
CO
2
. In selected experiments, HCT116 cells were compared
with SW480, LoVo and SW620 human colorectal cancer
Fig. 7. miR-143 and 5-FU down-regulate ERK5, NF-jB and Bcl-2
protein expression. Populations of HCT116 cells, miR-143 over-

expressing cells (HCT116-OV3 and pCR3-pri-miR-143 mix) and
respective control cells (HCT116-EM1 and pCR3-empty mix) were
synchronized to early S-phase by a double thymidine block. Eight-
hours after seeding, 2 m
M thymidine was added and cells were
cultured for 14 h. Cells were then released from the block for 10 h
in media lacking thymidine, followed by an additional 14 h of
culture in the presence of 2 m
M thymidine (second block). Cells
were released from the second thymidine block into media with
5-FU or no addition (control) and harvested for protein extraction
72 h later. Representative blots from at least three independent
experiments are shown. àP < 0.05 compared to HCT116;
P < 0.05 compared to 5-FU-treated HCT116 cells; §P < 0.05
compared to the respective untreated cells; *P < 0.05 compared to
HCT116-EM1 or pCR3-empty mix cells; #P < 0.05 compared to
5-FU-treated HCT116-EM1 or 5-FU-treated pCR3-empty mix.
P. M. Borralho et al. miR-143 modulates 5-FU cytotoxicity
FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6697
cells. All cell lines were grown under identical conditions.
Cells were seeded at 0.75 · 10
5
cellsÆmL
)1
for pre-miR-143
tranfections; 1 · 10
5
cellsÆmL
)1
for morphological assess-

ment of apoptosis; and at 2 · 10
5
cellsÆmL
)1
for RNA and
protein extraction, cell viability and cell death assays.
Cell synchronization
Cell synchronization was performed using a double thymi-
dine block (early S-phase). Eight-hours after seeding
0.75 · 10
5
cellsÆmL
)1
,2mm thymidine (Sigma) was added
and cells were cultured for 14 h. Cells were then released
from block for 10 h in media lacking thymidine, followed
by an additional 14 h culture in the presence of 2 mm
thymidine (second block). Cells were released from second
thymidine block into media with either 5-FU or no addi-
tion (control). Cells were harvested for protein extraction at
the indicated times.
5-FU exposure
5-FU (Sigma) stock solutions of 100 and 8 mm were pre-
pared in dimethyl sulfoxide. Twenty-four hours after plat-
ing, cells were incubated with either 5-FU or no addition
(control) for the indicated times. For the 5-FU dose–
response curves, media was removed 24 h after plating and
replaced with fresh media containing 5-FU. The final
dimethyl sulfoxide concentration was always 0.1%.
Transfection of miR-143 vectors, anti-miR-143

inhibitor, pre-miR-143 and ERK5 siRNA
HCT116 cells were transiently transfected with miR-143
overexpression vector coding for the miR-143 precursor
(pCR3-pri-miR-143) and miR-143 sensor comprising two
sequences complementary to mature miR-143 sequence
(pGL3-miR-143 sensor) [9]. pRL-SV40 (Promega, Madison,
WI, USA) was used for transfection normalization. pGL3-
control plasmid (Promega) and pCR3-empty vector were
used as negative controls. To further validate the experi-
mental model, cells were co-transfected with anti-miR
inhibitors by adding either 50 or 100 nm anti-miR-143 or
anti-miR-control inhibitors to the vector mixture described
above. In addition, HCT116 cells were transfected with
pre-miR-143, pre-miR-control, anti-miR-143 and anti-miR-
control at a final concentration of 40 or 80 nm. We also
co-transfected HCT116 cells with 40 nm pre-miR-143 or
pre-miR-control plus 60 nm anti-miR-143 or anti-miR-
control, at a final concentration of 100 nm. ERK5 silencing
was performed using 80 nm MAPK7 Silencer
Ò
Select
Pre-Designed & Validated siRNA (all from Applied Bio-
systems, Foster City, CA, USA). Transfections were
performed using Lipofectamine 2000 (Invitrogen), accord-
ing to the manufacturer’s instructions.
Generation of HCT116 cells with stable
expression of miR-143
HCT116 cells were selected and maintained with 1 mgÆmL
)1
G418 (Invitrogen), after transfection with pCR3-pri-miR-

143 and pCR3-empty vectors. Isolated single clone-derived
cell foci were picked up by ring cloning strategies, expanded
and propagated in complete media plus G418. In addition,
mixed populations of miR-143 overexpressing and control
cells were obtained in a similar manner, without the single-
cell selection step. miR-143 expression was evaluated by
luciferase assay after co-transfection and normalization
with pGL3-miR-143 sensor and pRL-SV40. This assay was
performed regularly to control cell clone consistency. In
addition, TaqMan real-time PCR (Applied Biosystems) was
used to confirm the expression of mature miR-143 in cell
clones.
Evaluation of cell death and viability
At the indicated times, general cell death was evaluated by
the LDH assay kit (Sigma). LDH activity was evaluated in
cell culture media, using a Bio-Rad microplate reader
Model 680 (Bio-Rad, Hercules, CA, USA). Cell viability
was evaluated with CellTiter96Ò AQ
ueous
Non-Radioactive
Cell Proliferation Assay (Promega), using MTS inner salt.
Finally, cells were processed for luciferase assay and trans-
fection efficiency normalization.
Luciferase activity
At the indicated times, firefly and renilla luciferase activities
were measured using the Dual-LuciferaseÒ Reporter Assay
System (Promega). Renilla luciferase activity was used as a
transfection normalization control.
Nuclear morphology
Hoechst labelling of cells was used to detect apoptotic

nuclei. Attached cells were fixed with 4% paraformaldehyde
in NaCl ⁄ P
i
(pH 7.4) and incubated with Hoechst dye 33258
(Sigma Chemical Co.) at 5 lgÆmL
)1
in NaCl ⁄ P
i
for 3 min.
Fluorescent nuclei were categorized according to the con-
densation and staining characteristics of chromatin. Three
random microscopic fields per sample of $ 100 nuclei were
counted and mean values expressed as the percentage of
apoptotic nuclei.
Caspase activity
Caspase activity was determined in cytosolic protein extracts
after harvesting and homogenization of cells in isolation buf-
fer, containing 10 mm Tris-HCl buffer (pH 7.6), 5 mm
MgCl
2
, 1.5 mm potassium acetate, 2 mm dithiothreitol and
miR-143 modulates 5-FU cytotoxicity P. M. Borralho et al.
6698 FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS
protease inhibitor mixture tablets (Complete; Roche Applied
Science, Mannheim, Germany). General caspase-3, -8 and
-9-like activities were determined by enzymatic cleavage of
chromophore p-nitroanilide (pNA) from the substrates
N-acetyl-Asp-Glu-Val-Asp-pNA (DEVD-pNA), N-acetyl-
Ile-Glu-pro-Asp-pNA (IEPD-pNA) and N-acetyl-Leu-Glu-
His-Asp-p-NA (LEHD-pNA) (Sigma), respectively. The

proteolytic reaction was carried out in isolation buffer con-
taining 50 lg of cytosolic protein and 50 lm specific caspase
substrate. The reaction mixtures were incubated at 37 °C for
1 h, and the formation of pNA was measured by monitoring
A
405
using a 96-well plate reader.
miR-143 expression
Total and miRNA-enriched RNA was extracted from cells
using the mirVanaÔ PARIS kit from Ambion (Austin, TX,
USA), recovering either small-RNA containing total RNA
(total RNA) or small RNA species of less than 200 nucleo-
tides (miRNA-enriched RNA). In addition, total RNA was
also extracted using Trizol reagent (Invitrogen). RNA
concentration was determined by monitoring A
260
.
For real-time PCR, RT reactions were performed using a
TaqMan MicroRNA Reverse Transcription Kit and TaqMan
MicroRNA assays for hsa-miR-143 and human RNU6B for
normalization to endogenous control. Data were collected
with 7000 System Sequence Detection Software, version 1.2.3
(Applied Biosystems). Triplicate reactions were run per sam-
ple. The comparative threshold cycle method was used to cal-
culate the amplification factor where the threshold cycle (C
t
)
is defined as the cycle number at which the fluorescence passes
the fixed threshold intensity level. miR-143 expression levels
in different samples were calculated on the basis of DDC

t
method. HCT116 or SW480 parental untreated (control) cells
were used as calibrator; the n-fold change in miR-143 expres-
sion was obtained using the formula: 2
ÀDDC
t
.
Immunoblotting
Steady-state levels of ERK5, NF-jB (p65) and Bcl-2 pro-
tein were determined by immunoblot analysis. Briefly,
50 lg of total protein extracts were separated on 8% or
12% SDS-polyacrylamide electrophoresis gels. After elec-
trophoretic transfer onto nitrocellulose membranes, immu-
noblots were incubated with 15% H
2
O
2
for 15 min at room
temperature. After blocking with 5% milk solution, the
blots were incubated overnight at 4 °C with primary rabbit
polyclonal antibody reactive to ERK5 (#3372; Cell Signal-
ing, Beverly, MA, USA) and a mouse monoclonal antibody
reactive to NF-jB (p65) or to Bcl-2 (#sc-8008 and #sc-
7382, respectively; Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA). Finally, membranes were incubated with
secondary anti-mouse ⁄ -rabbit sera conjugated with horse-
radish peroxidase (Bio-Rad) for 3 h at room temperature.
The membranes were processed for protein detection using
Super SignalÔ substrate (Pierce, Rockford, IL, USA).
b-actin (Sigma) was used as a loading control. Protein

concentrations were determined using the Bio-Rad protein
assay kit according to the manufacturer’s instructions.
Statistical analysis
All data are expressed as the mean ± SEM from at least
three independent experiments. Statistical significance was
evaluated using the Student’s t-test. P < 0.05 was consid-
ered statistically significant.
Acknowledgements
The authors thank Dr Christine Esau, ISIS Pharma-
ceuticals Inc., for the kind gift of miR-143 overexpres-
sion and sensor plasmids. The study was supported by
grants FCG 68796 ⁄ 2004 from Fundac¸ a
˜
o Calouste
Gulbenkian and PTDC⁄ SAU-GMG ⁄ 099161 ⁄ 2008 from
Fundac¸ a
˜
o para a Cieˆ ncia e a Tecnologia (FCT),
Lisbon, Portugal (to C.M.P.R.); by PhD fellowship
SFRH ⁄ BD ⁄ 24165 ⁄ 2005 (to P.M.B.) from FCT; and by
postdoctoral fellowship SFRH ⁄ BPD ⁄ 30257 ⁄ 2006 (to
R.E.C) from FCT.
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