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

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

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

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

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

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

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

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-

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

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

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

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-

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

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

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

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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miR-143 modulates 5-FU cytotoxicity P. M. Borralho et al.