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

Increased stable expression of miR-143 was associated with decreased viability and increased cell death after exposure to 5-fluorouracil.. The results obtained demonstrate that miR-143 de

to 5-fluorouracil in HCT116 human colorectal cancer cells Pedro M Borralho1, Betsy T Kren2, Rui E Castro1, Isabel B Moreira da Silva1, Clifford J Steer2,3 and Cecı´lia M P Rodrigues1

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: cmprodrigues@ff.ul.pt

(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 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

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

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, 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.

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 DCt values were 12.10 ± 0.35 in SW480 cells compared to 5.45 ± 0.08 in HCT116 cells (P < 0.001), whereas RNU6B Ct 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.

(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 l M 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

1 mgÆmL)1G418, 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.

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 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.

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 mirVana 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 DDCtmethod, 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.

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 n M 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 l M 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 l M 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 l M 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-trea5-FU-trea-ted mock).

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.,

antimy-cotic solution (Sigma Chemical Co., St Louis, MO, USA)

CO2 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 P < 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.

cells All cell lines were grown under identical conditions.

Cells were seeded at 0.75· 105cellsÆmL)1 for pre-miR-143

tranfections; 1· 105cellsÆmL)1 for morphological

assess-ment of apoptosis; and at 2· 105cellsÆmL)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· 105

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 extracaddi-tion 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, 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 143 or

anti-miR-control, at a final concentration of 100 nm ERK5 silencing

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

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

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-Luciferase 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⁄ Pi(pH 7.4) and incubated with Hoechst dye 33258 (Sigma Chemical Co.) at 5 lgÆmL)1 in NaCl⁄ Pifor 3 min Fluorescent nuclei were categorized according to the con-densation and staining characteristics of chromatin Three

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 MgCl2, 1.5 mm potassium acetate, 2 mm dithiothreitol and