Protein tyrosine phosphatase PTPRJ is negativelyregulated by microRNA-328Francesco Paduano1, Vincenzo Dattilo1, Domenico Narciso1, Anna Bilotta1, Eugenio Gaudio1,Miranda Menniti2, Valter Agosti1, Camillo Palmieri1, Nicola Perrotti2, Alfredo Fusco3, FrancescoTrapasso1 and Rodolfo Iuliano1

1 Dipartimento di Medicina Sperimentale e Clinica, Universita ‘Magna Graecia’, Catanzaro, Italy

2 Dipartimento di Scienze della Salute, Universita ‘Magna Graecia’, Catanzaro, Italy

3 Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita ‘Federico II’, Napoli, Italy

Keywords

microRNA; miR-328; protein tyrosine

phosphatase; PTPRJ; tumour suppressor

gene

Correspondence

R. Iuliano, Dipartimento di Medicina

Sperimentale e Clinica, Universita ‘Magna

Graecia’, viale Europa, 88100 Catanzaro,

Italy

Fax: +39 0961 3694090

Tel: +39 0961 3695182

E-mail: iuliano@unicz.it

Website: http://www.unicz.it

F. Paduano, Dipartimento di Medicina

Sperimentale e Clinica, Universita ‘Magna

Graecia’, viale Europa, 88100 Catanzaro,

Italy

Fax: +39 0961 3694090

Tel: +39 0961 3694106

E-mail: francesco.paduano@unicz.it

Website: http://www.unicz.it

(Received 22 December 2011, revised 31

March 2012, accepted 3 May 2012)

doi:10.1111/j.1742-4658.2012.08624.x

Expression of PTPRJ, which is a ubiquitous receptor-type protein tyrosine

phosphatase, is significantly reduced in a vast majority of human epithelial

cancers and cancer cell lines (i.e. colon, lung, thyroid, mammary and pan-

creatic tumours). A possible role for microRNAs (miRNAs) in the negative

regulation of PTPRJ expression has never been investigated. In this study,

we show that overexpression of microRNA-328 (miR-328) decreases

PTPRJ expression in HeLa and SKBr3 cells. Further investigations demon-

strate that miR-328 acts directly on the 3¢UTR of PTPRJ, resulting in

reduced mRNA levels. Luciferase assay and site-specific mutagenesis were

used to identify a functional miRNA response element in the 3¢UTR of

PTPRJ. Expression of miR-328 significantly enhances cell proliferation in

HeLa and SKBr3 cells, similar to the effects of downregulation of PTPRJ

with small interfering RNA. Additionally, in HeLa cells, the proliferative

effect of miR-328 was not observed when PTPRJ was silenced with small

interfering RNA; conversely, restoration of PTPRJ expression in miR-328-

overexpressing cells abolished the proliferative activity of miR-328. In con-

clusion, we report the identification of miR-328 as an important player in

the regulation of PTPRJ expression, and we propose that the interaction of

miR-328 with PTPRJ is responsible for miR-328-dependent increase of epi-

thelial cell proliferation.

Introduction

The tyrosine phosphatase PTPRJ (also called DEP-1,

HPTPg or CD148) is a widely expressed transmem-

brane protein that plays a role in multiple signalling

pathways [1]. Substrates of PTPRJ include proteins

that belong to the family of tyrosine kinase receptors

(EGFR, HGFR, PDGFR, RET and VEGFR-2) [2–6]

as well as those involved in cell adhesion (c-Src, p120-

catenin and ZO-1) [7–9]. Consequently, in several epi-

thelium-derived cell lines, PTPRJ inhibits cell prolifera-

tion and promotes cell adhesion [10–13]. PTPRJ has

been proposed to be a tumour suppressor owing to

downregulation of its expression in cancer cell lines

[10,12–14] and inhibition of tumorous growth upon

restoration of normal expression levels [12–14].

Abbreviations

CFSE, carboxyfluorescein succinimidyl ester; hsa-miR, Homo sapiens microRNA; LOH, loss of heterozygosity; miR-328, microRNA-328;

miRNA, microRNA; MRE, microRNA response element; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small

interfering RNA.

FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 1

Although expression of PTPRJ decreases significantly

during the progression of human tumours [15,16], little

is known about the mechanisms involved in the pro-

cess. Loss of heterozygosity (LOH) was detected at the

PTPRJ locus (11p11.2) in lung, breast, colon and thy-

roid carcinomas [17,18]; however, LOH and promoter

methylation of PTPRJ [19,20] can only partly explain

the association between tumour progression and

reduced PTPRJ expression.

MicroRNAs (miRNAs) are tiny non-coding RNAs

that downregulate gene expression by binding to the

3¢UTRs of their targets resulting in the inhibition of

protein translation or mRNA degradation [21]. Given

their varied effects on gene regulation, miRNAs have

been implicated in several diseases, including cancer.

MiRNA signatures have been used in the classification

of human tumours and in cancer prognosis predictions

[22–24]. MiRNA expression levels are not only useful as

biomarkers of disease progression, but it is now clear

that they also play a causative role in cancer progres-

sion. Genetic and epigenetic variations of miRNAs have

been detected in tumours [25,26], and polymorphisms in

miRNA coding regions have been associated with can-

cer susceptibility [27]. Probing their mechanistic role in

cancer, it appears that miRNAs can target either onco-

genes or tumour suppressor genes, and act by blocking

or favouring tumour progression as a consequence. The

balance in targets between oncogenes and tumour sup-

pressors by a certain miRNA defines its role in cancer

development [24]. Therefore, it is crucial to identify can-

cer-related genes targeted by miRNAs.

In this study, we report that microRNA-328 (miR-328)

targets the 3¢UTR of PTPRJ resulting in its decreased

expression. Furthermore, PTPRJ downregulation could

Table 1. Computational analysis with MIRTAR, FINDTAR and RNAHYBRID of microRNA recognition elements (MREs) for miR-328 in the 3¢UTR of

PTPRJ mRNA. MREs identified by RNAHYBRID are indicated as MRE_1 to MRE_5 in the text.

PTPRJ expression is negatively regulated by microRNA-328 F. Paduano et al.

2 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

explain the proliferative effects observed in HeLa cells

withmiR-328 overexpression. Therefore, we propose that

miR-328 is a negative regulator of PTPRJ expression,

with a possible role in the development of human

tumours.

Results

The 3¢UTR of PTPRJ was analysed by the bioinfor-

matics algorithm mirtar [28] to identify possible inter-

acting miRNAs. Several miRNAs were selected as

likely interactors and were considered for further bio-

informatic analyses with other computational algo-

rithms (findtar and rnahybrid) [29,30]. Among the

selected miRNAs, for miR-328, two miRNA response

elements (MREs) were found with mirtar; four with

findtar; and five with rnahybrid in the 3¢UTR of

PTPRJ (Table 1). For further analyses on MREs, we

considered the sites picked with rnahybrid.

In order to evaluate the reliability of computer pre-

diction, we performed the transfection of synthetic

miR-328 in two different cell lines (HeLa and SKBr3),

and we then detected PTPRJ by western blot. We

found a significant decrease of PTPRJ protein levels in

miR-328 transfected cells compared with the control

(Fig. 1A). As a positive control, a PTPRJ-specific

small interfering RNA (siRNA) was used. To evaluate

whether miR-328 affects PTPRJ expression by decreas-

ing mRNA levels, we quantified, by qRT-PCR,

mRNA levels of PTPRJ and its alternatively spliced

shorter isoform, encoding an mRNA with a different

3¢UTR (Fig. 1B). We found that miR-328 decreased

mRNA levels of PTPRJ but not of the shorter iso-

form, indicating that miR-328 probably acts on the

3¢UTR of PTPRJ mRNA. In addition, the PTPRJ

siRNA oligonucleotide that binds a region on exon 6

shared by the two PTPRJ isoforms decreased mRNA

levels of both isoforms (Fig. 1C). Thus, we demon-

strated that miR-328 downregulates endogenous

PTPRJ at both the mRNA and the protein levels.

We hypothesized that miR-328 interacts directly with

the 3¢UTR of PTPRJ mRNA to suppress PTPRJ

expression. To test this hypothesis, the ability of miR-

328 to regulate the 3¢UTR of PTPRJ was evaluated by

luciferase reporter assays. The region starting from

nucleotide +6259 to nucleotide +7761 of the PTPRJ

sequence (NM_002843.3) was cloned downstream of a

reporter luciferase gene (pmirGLO 3¢-WT) (Fig. 2A), as

described in Materials and methods. Co-transfection of

synthetic miR-328 and pmirGLO 3¢-WT reduced the

luciferase activity significantly in comparison with Lipo-

fectamine and miR-342 (Fig. 2B), an miRNA that did

not influence endogenous PTPRJ expression in HeLa

cells (data not shown). However, co-transfection of

miR-328 in cells transfected with pmirGLO empty vec-

tor did not influence luciferase activity significantly.

These results prompted us to study recognition elements

of miR-328 in the PTPRJ 3¢UTR. Five possible MREs

(MRE_1 to MRE_5, Fig. 2A) containing an miR-328

seeding region were predicted by computational

*

*

*

Rel

- m

RN

A le

vel

1.2

0.8

0.6

0.4

0.2

0

1.4

1

1.2

0.8

0.6

0.4

0.2

0

1.4

1

* *

*

HeLa SKBr3

PTPRJ long form

PTPRJ short form

SKBr3 HeLa

PTPRJ

γ-tubulin

220 kDa

50 kDa

PTPRJ transcript long form

PTPRJ transcript short form miR-328

siRNA-PTPRJ

3 UTR exon 6

A

C

BFig. 1. PTPRJ expression is downregulated

by miR-328. (A) Western blot analysis for

PTPRJ detection in protein extracts from

cells transfected transiently for 24 h with

siRNA-PTPRJ, Lipofectamine or miR-328.

Expression is normalized on c-tubulin. (B)

Schematic representation of the PTPRJ

pre-mRNA (long and short forms) and loca-

tion of miRNA and siRNA target sites on

PTPRJ mRNA (Genbank accession no.

NM_002843.3). (C) qRT-PCR with specific

primers amplifying the two isoforms of the

PTPRJ gene from cell lines transfected

transiently for 24 h with Lipofectamine,

siRNA-PTPRJ or miR-328. Values were

normalized to HPRT RNA levels. *P < 0.01.

F. Paduano et al. PTPRJ expression is negatively regulated by microRNA-328

FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 3

analyses, as reported before (Table 1). To map interac-

tion sites of miR-328 in the 3¢UTR of PTPRJ, we gener-

ated a deletion mutant that includes the 3¢UTR

(pmirGLO 3¢D) downstream of the SacI site and con-

tains just two possible miR-328 binding sites (MRE_4

and MRE_5). Luciferase activity of that mutant was sig-

nificantly affected by the co-transfection with miR-328,

and the amount of luciferase reduction was not lower

than that detected by the pmirGLO 3¢UTR. Therefore,

deletion of nucleotides +6259 to +7082 of the human

PTPRJ 3¢UTR containing three MREs (MRE_1 to

MRE_3) did not decrease susceptibility to miR-328

inhibition (Fig. 2C), indicating that MRE_1 to MRE_3

were not functional. To better characterize the interac-

tion, we generated two site-specific mutations in

MRE_4 and MRE_5 (3¢D-4 mut and 3¢D-5 mut)

affecting the possible remaining miR-328 binding sites

in the 3¢UTR of PTPRJ mRNA (Fig. 2D). When the

MRE_5 site is mutated (pmirGLO 3¢D-5 mut), only a

slight decrease in the luciferase activity was observed.

Mutation of the MRE_4 site (pmirGLO 3¢D-4 mut) sig-

nificantly abolished the repression of luciferase activity

operated by miR-328. The same results were obtained

with the double mutant 3¢UTR (pmirGLO 3¢D-4 ⁄ 5 mut),

indicating that MRE_4 in position from +7174 to

+7203 plays a key role in the miR-328 mediated repres-

sion (Fig. 2E). Taken together, these data indicate that

miR-328 directly regulates PTPRJ expression mainly

through interaction with the predicted MRE_4 in the

3¢UTR of PTPRJ.

Proliferation of HeLa and SKBr3 cells is sensitive to

PTPRJ levels modulated by vector transfection [10,31].

Fig. 2. PTPRJ is a direct target of miR-328. (A) Reporter plasmid containing five potential miR-328 binding sites within the 3¢UTR of human

PTPRJ gene (MRE_1 to MRE_5). (B) Luciferase assay of HeLa cells co-transfected with PTPRJ WT 3¢UTR reporter plasmid (pmirGLO 3¢-WT)

or empty vector (pmirGLO) and synthetic miR-328 or miR-342. (C) Luciferase assay of HeLa cells co-transfected with 3¢UTR reporter plasmid

(pmirGLO 3¢-WT) or deleted 3¢UTR (pmirGLO 3¢D) and synthetic miR-328. (D) Base pairing for comparison between mature miR-328 and

wild-type (PTPRJ WT MRE) or mutant (PTPRJ mut MRE) putative target sites (MRE_4 and MRE_5) in the 3¢UTR of PTPRJ mRNA. Mutations

were generated on the potential target sequence of PTPRJ (bold letters with underlining) by site-directed mutagenesis. (E) Reporter repres-

sion of miR-328 in transfected HeLa cells with vector is indicated. Luciferase values were normalized to the double mutant (pmirGLO 3¢D-4 ⁄ 5 mut). Significant differences are indicated by an asterisk, P < 0.01. All the experiments were performed in quadruplicate and repeated

twice.

PTPRJ expression is negatively regulated by microRNA-328 F. Paduano et al.

4 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

Therefore, to functionally link the PTPRJ ⁄miR-328

interaction to cell proliferation, we tested the effects of

miR-328 overexpression on HeLa cells by using the

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT) assay. We generated an expression vector

containing the genomic region flanking miR-328

(pcDNA3.1-miR-328) (Materials and methods). Upon

transient transfection in HeLa cells, significant expres-

sion of miR-328 was detected by qRT-PCR (data not

shown). Transfection with pcDNA3.1-miR-328

enhanced proliferation of HeLa cells in a dose-depen-

dent manner (Fig. 3A). We also found that knock-

down of PTPRJ expression with siRNA induced a

significant increase in cell proliferation (Fig. 3B),

similar to the effects of miR-328 overexpression.

Importantly, miR-328 transfection in siRNA-treated

cells did not further increase cell proliferation

(Fig. 3B), indicating that the effect of miR-328 on pro-

liferation was due, at least in part, to downregulation

of PTPRJ expression. In addition, PTPRJ overexpres-

sion (Fig. S1) rescued the cell proliferation effect

observed in HeLa cells treated with the miR-328

construct (Fig. 3C). To confirm the effect of miR-328

on cell proliferation, an in vitro cell proliferation assay

was performed in HeLa cells by using the fluorescent

dye carboxyfluorescein succinimidyl ester (CFSE),

which labels the intracellular protein content. With

each cell division, intracellular fluorescence decreases

with increasing cell number, the intensity of which can

be assayed by flow cytometry. Proliferation in HeLa

cells increased with both pcDNA3.1-miR-328 and

pcDNA3.1 + PTPRJ-siRNA compared with empty

00.20.40.60.8

11.21.41.61.8

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

00.20.40.60.8

11.21.41.61.8

Rel

ativ

e ce

ll pr

olife

ratio

n

pcDNA3.1 (ng) pcDNA3.1-miR-328 (ng)

250 210 40

100 150 250 –

–

**

*

pCEFL miR-328 pCEFL-PTPRJ

+ ––

+ + –

–+ +

–––

Rel

ativ

e ce

ll pr

olife

ratio

n

* **

siRNA-PTPRJ pcDNA3.1 pcDNA3.1-miR-328

+ + –

+ –+

–– –

–– +

Rel

ativ

e ce

ll pr

olife

ratio

n

Lipo + + + +

Lipo + + + +

CFSE fluorescence

Num

ber o

f cel

ls

0 h

24 h

100 101 102 103 104

100 101 102 103 104

020406080100

020406080

100

pcDNA3.1 pcDNA3.1-miR-328 pcDNA3.1+ siRNA PTPRJ

0

10

20

30

Geo

met

ric m

ean

CFS

E

**

A B

C D E

Fig. 3. MiR-328 increases proliferation in HeLa cells by repressing PTPRJ. (A) Proliferation of HeLa cells transfected with vectors is indi-

cated. Cell proliferation was measured 48 h after seeding with the MTT assay and values were normalized to the empty vector. *P < 0.05

compared with pcDNA3.1 (250 ng), analysed by ANOVA followed by Dunnett’s test. (B) Proliferation of HeLa cells co-transfected with

pcDNA3.1 (250 ng) + siRNA-PTPRJ (100 nM), pcDNA3.1-miR-328 (250 ng) and pcDNA3.1-miR-328 (250 ng) + siRNA-PTPRJ (100 nM). Cell

proliferation was measured after 48 h by MTT assay and values were normalized to the Lipofectamine. *P < 0.05 compared with Lipofecta-

mine, analysed by ANOVA followed by Dunnett’s test. (C) Proliferation of HeLa cells co-transfected with synthetic miR-328

(100 nM) + pCEFL (250 ng) and synthetic miR-328 (100 nM) + pCEFL-PTPRJ (250 ng). Cell proliferation was measured after 48 h by MTT

assay, and values were normalized to the Lipofectamine. *P < 0.05 compared with Lipofectamine, analysed by ANOVA followed by Dun-

nett’s test. (D) Representative cell cycle progression of HeLa cells transiently transfected with pcDNA3.1, pcDNA3.1-miR-328 and

pcDNA3.1 + siRNA-PTPRJ using CFSE. Cells were analysed at 0 and 24 h by flow cytometry. (E) Geometric mean intensities of CFSE stain-

ing in HeLa cells transiently transfected with pcDNA3.1, pcDNA3.1-miR-328 and pcDNA3.1 + siRNA-PTPRJ at 24 h. P values were gener-

ated by ANOVA followed by Bonferroni’s test for multiple comparisons (significant differences are indicated by an asterisk, P < 0.05).

F. Paduano et al. PTPRJ expression is negatively regulated by microRNA-328

FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 5

vector, confirming that miR-328-dependent induction

of cell proliferation can also be achieved by repressing

PTPRJ levels (Fig. 3D,E). The proliferative effect of

miR-328 was also evaluated in SKBr3 cells. MiR-328

increased cell proliferation in transiently transfected

SKBr3 cells as demonstrated by MTT and CFSE

assays (Fig. S2).

To further investigate the link between PTPRJ and

miR-328, we generated stable HeLa cell lines overex-

pressing miR-328. Expression of miR-328 was quanti-

fied by qRT-PCR and two clones, miR-328 clone-1

and miR-328 clone-2, showed 50- and 80-fold

increases, respectively, in miR-328 expression com-

pared with empty vector control (Fig. 4A). Western

blot analysis of the HeLa miR-328 clones confirmed

PTPRJ downregulation (Fig. 4B). Additionally, flow

cytometry showed a reduction in membrane PTPRJ

expression in the miR-328 stable clones compared with

empty vector control (Fig. 4C). Finally, we aimed to

elucidate the effects of miR-328 overexpression on pro-

liferation rates in HeLa cells. This was performed by

using the MTT assay, and the miR-328 stable clones

showed an increase in cell proliferation compared with

empty vector control (Fig. 4D). This was also con-

firmed by the CFSE assay: clearly, the two miR-328

clones displayed an increase in cell proliferation com-

pared with empty vector control (Fig. 4E,F). PTPRJ is

a negative regulator of cell migration [11]. To investi-

gate whether miR-328 affects HeLa cell migration, we

used the wound-healing assay. We found that cell

migration was increased in both miR-328 stable clones

compared with empty vector control (Fig. S3).

Fig. 4. HeLa clones stably expressing miR-328 decrease PTPRJ expression and induce cell proliferation. (A) Relative miR-328 expression in

two HeLa stable clones (miR-328 clone-1 and miR-328 clone-2) quantified by qRT-PCR. Values were normalized to pcDNA3.1 vector.

*P < 0.01. (B) Western blot analysis of PTPRJ expression in HeLa stable clones. Expression is normalized to c-tubulin. Relative levels of

PTPRJ were quantified by QUANTITY ONE software (BioRad Laboratories). (C) Fluorescence-activated cell sorting analysis of membrane-bound

PTPRJ expression in HeLa clones stably expressing miR-328 or empty vector. Grey peaks represent unlabelled cells; open peaks represent

cells labelled with PTPRJ antibody. (D) Proliferation of HeLa stable clones measured after 24 and 48 h with the MTT assay. Values were nor-

malized to the empty vector. *P < 0.05 compared with pcDNA3.1 at 24 h, analysed by ANOVA followed by Dunnett’s test. (E) Representa-

tive cell cycle progression of HeLa stable clones using CFSE. Cells were analysed at 0, 24 and 48 h by flow cytometry. (F) Geometric mean

intensities of CFSE staining in HeLa stable clones at 24 and 48 h. *P < 0.05 compared with pcDNA3.1, analysed by ANOVA followed by

Bonferroni’s test.

PTPRJ expression is negatively regulated by microRNA-328 F. Paduano et al.

6 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

Discussion

Several studies have addressed the function of PTPRJ in

the regulation of cellular pathways and a number of

PTPRJ-interacting proteins or substrates have been iden-

tified [1], underlining the role ofPTPRJ as a negative reg-

ulator of cell proliferation in epithelial [10,12–14] and

endothelial [32] cells. PTPRJ acts by downregulating

receptor-type tyrosine kinase activity [2–6] or by inhibit-

ing the pro-proliferative PI3K and Ras-dependent path-

ways [33,34]. To clarify the role of PTPRJ in vivo,

knockout mice were generated resulting in viable mice

that do not develop tumours spontaneously [35]. How-

ever, significant downregulation of PTPRJ expression

has been observed in tumours [15,16]. Thus, it is impor-

tant to study the mechanisms involved in the regulation

of PTPRJ expression that is currently not verywell under-

stood. In general, regulation of the expression of protein

tyrosine phosphatases is poorly characterized [36].

Post-transcriptional regulation of PTPRJ has been

studied by Karagyozov et al. [37], who found a novel

mechanism of translational regulation that was based

on the use of different translational start sites. Our

study provides an independent mechanism of control of

PTPRJ expression which involves the 3¢UTR. We used

three independent bioinformatics algorithms to identify

MREs in the 3¢UTR of PTPRJ. Interestingly, MRE_4,

which was the only MRE to be detected by all three

algorithms, appeared to interact with miR-328.

Recently, a novel mechanism of action has been

attributed to miR-328. In fact, miR-328 acts through a

decoy activity that interferes with the function of regula-

tory proteins [38]. However, miR-328 is also able to

repress gene expression through the typical miRNA

mechanism of acting on the 3¢UTR of target genes [38],

as shown here.

In our study, miR-328 increased proliferation of

HeLa and SKBr3 cells by means of a mechanism

dependent, at least in part, on the downregulation of

PTPRJ expression. The number of possible miR-328

gene targets in the cell lines analysed probably influ-

enced the extent of PTPRJ downregulation. However,

effects on cell proliferation would not necessarily have

to correlate with the extent of PTPRJ downregulation

as different cell lines could have varied sensitivities to

expression of PTPRJ, or other targets of miR-328.

The list of validated targets of miR-328 includes

CD44, ABCG2 and PIM-1 [38–40]. CD44 is involved

in cell adhesion and could act synergistically with

PTPRJ. Given that CD44 and PIM-1 are considered

pro-oncogenic proteins, this could appear to contrast

with the tumour suppressor role of PTPRJ. However,

the oncogenic activity of PIM-1 is mainly restricted to

haematological malignancies [41] in which, it should be

emphasized, the role of PTPRJ is still unclear. Indeed,

CD148 ⁄PTPRJ has been proposed to be a valuable

marker of mantle cell lymphomas in an unbiased pro-

teomic study [42].

Loss of expression of miR-328 is strongly correlated

with blast crisis in patients afflicted with chronic mye-

logenous leukaemia [38]. In glioblastoma multiforme,

miR-328 levels are lower compared with normal brain

tissues [43]. Conversely, miR-328 levels are upregulated

in patients with brain metastasis of lung cancer [44].

Expression levels of miR-328 are significantly higher in

blood samples of melanoma patients compared with

healthy individuals [45].

Studies on miR-328 discussed thus far reflect the

well understood feature of miRNAs: they can act as

onco-miRs (favouring tumour progression) or onco-

suppressor miRs (blocking tumour progression)

depending on the cellular context, as exemplified by

miR-221 and miR-222 that behave as tumour suppres-

sors in haematological malignancies and as oncogenes

in other tumours [46]. In fact, the role of an miRNA

in a pathological context is also dependent on the rela-

tive expression levels of its targets in the tissue under

examination [47]. However, in epithelium-derived cells,

miR-328 increases cell proliferation (our results) and

cell migration [44], and this is in line with the opposing

role played by PTPRJ in these processes [10–15,48,49].

In conclusion, we have identified a novel mechanism

of regulation of PTPRJ expression in which the

3¢UTR of PTPRJ mRNA is targeted by miR-328. This

interaction is important for proliferation of epithelium-

derived cells. It would be of interest to investigate in

future studies the correlation between expression of

PTPRJ and miR-328 in human tumours.

Materials and methods

Bioinformatic analysis

The algorithms used were mirtar, rnahybrid and find-

tar. We first screened the possible miRNAs interacting with

the 3¢UTR of PTPRJ by using mirtar [28], a bioinformat-

ics algorithm that allows a selection of potential interacting

miRNAs with a given 3¢UTR sequence. Every miRNA can-

didate resulting from that analysis was further investigated

with findtar [29] and rnahybrid [30] to determine the best

possible miRNA candidate for the interaction with the

3¢UTR of PTPRJ. The threshold value of minimum free

energy for the identification of MREs located on PTPRJ

3¢UTR was set at the default value ()16 kcalÆmol)1) in the

screening with mirtar and at )25 kcalÆmol)1 in the analyses

with findtar and rnahybrid.

F. Paduano et al. PTPRJ expression is negatively regulated by microRNA-328

FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 7

Cells and transfections

HeLa (human cervix adenocarcinoma) and SKBr3 (human

breast adenocarcinoma) cell lines were maintained in culture

at 37 �C and 5% CO2 in DMEM supplemented with 10%

fetal bovine serum, 100 U penicillin and 100 lgÆmL)1 strepto-

mycin sulfate. Transfections were made with Lipofectamine�

2000, following the manufacturer’s instructions (Invitrogen,

Carlsbad, CA, USA). For western blot and qRT-PCR experi-

ments, 4 · 105 cells were seeded in six-well plates and trans-

fected with 50 pmol Pre-miRTM (Ambion, Life Technologies,

Paisley, UK) or 100 pmol siRNA. For luciferase assays, 2 ·05 cells were seeded in 24-well plates and co-transfected with

10 pmolÆmL)1 Pre-miRTM (Ambion) and 25 ngÆmL)1 repor-

ter plasmid (pmirGLO vector; Promega,Madison,WI, USA).

Western blot analysis

Cells were harvested after 48 h, washed with ice-cold

NaCl ⁄Pi and lysed in a buffer containing 50 mm Tris ⁄HCl

pH 7.5, 1% Nonidet P40, 150 mm NaCl, 25 mm NaF, 1 mm

Na3VO4 and a complete mixture of protease inhibitors

(Roche, Basel, Switzerland). Lysates were clarified by centri-

fugation at 10 000 g for 15 min at 4 �C. Protein concentra-

tion was determined by a modified Bradford assay (BioRad,

Hercules, CA, USA). Fifty micrograms of proteins were sub-

jected to electrophoresis carried out in an 8%–12% gradient

gel (Invitrogen) and then transferred to nitrocellulose mem-

branes (Hybond-C; Amersham Biosciences, Piscataway, NJ,

USA). The membranes were blocked with 5% non-fat dry

milk and then probed for 2 h with the appropriate primary

antibodies. After incubation with specific horseradish peroxi-

dase conjugated secondary antibodies, protein bands were

revealed with an enhanced chemiluminescence system (Santa

Cruz Biotechnology, Santa Cruz, CA, USA). Primary anti-

bodies used were anti-human PTPRJ and anti-tubulin pur-

chased from R&D Systems (Minneapolis, MN, USA) and

Santa Cruz Biotechnology, respectively.

Quantitative real-time PCR

RNA extraction was performed with miRNeasy Mini Kit�(Qiagen, Valencia, CA, USA), following the manufacturer’s

instructions, and total RNA was quantified with a spectro-

photometer. Total RNA samples (250 ng) were subjected to

the reaction of reverse-transcription using the High Capa-

city RNA-to-cDNA Kit (Applied Biosystems, Foster City,

CA, USA), following the manufacturer’s instructions. Five

hundred nanolitres of cDNAs were amplified by real-time

PCR with Promega SYBR green kit and 5 pmol of primers

in a total volume of 25 lL.The primers used were as follows: 5¢-GTATTAT

CATTGGTGGCTTGTTC-3¢ (forward for long and short

form of PTPRJ), 5¢-CATCTCCGTGGTGGTGAC-3¢ (reversefor long form of PTPRJ) and 5¢-AGGCAGGTGTTCAA

ATCATCC-3¢ (reverse for short form ofPTPRJ). Specific oligo-

nucleotides used for hypoxanthine phosphoribosyltransferase

(normalization control) amplification were reported by Vande-

sompele et al. [50].

Real-time PCR reactions were carried out in a BioRad

iQ�5 apparatus with the following conditions: initial dena-

turation step at 95 �C for 3 min, followed by 40 cycles of

10 s at 95 �C and 1 min at 57 �C. Specificity of PCR prod-

ucts was checked by melting curve analysis and gel electro-

phoresis. Efficiencies of real-time PCRs were calculated by

constructing curves with cycle threshold (Ct) obtained from

amplifications of serial tenfold dilutions of a cDNA sam-

ple, resulting in higher than 90%. For miR-328 quantifica-

tion, 50 ng of total RNA was subjected to reverse-

transcription with miRCURY LNA� Universal cDNA

synthesis kit (Exiqon, Vedbaek, Denmark), following the

manufacturer’s instructions. One hundredth of the cDNA

was amplified by real-time PCR with SYBR� Green master

mix 2X (Exiqon) and miRCURY LNA� hsa-miR-328 spe-

cific PCR primer set (Exiqon) or miRCURY LNA� U6

snRNA specific PCR primer set (Exiqon), following the

manufacturer’s instructions. Reactions were performed in a

total volume of 20 lL in a BioRad IQ�5 apparatus.

Plasmids and mutagenesis

The 3¢UTR of PTPRJ containing the predicted sites for

miR-328 ⁄PTPRJ interaction (the region from +6259 to

+7761 of PTPRJ) was amplified by PCR using genomic

DNA as the template, Pfu polymerase and the following

primers: forward 5¢-CATGTTTAAACCTTCTCAAATG

GAAATTGCA-3¢ and reverse 5¢- GCCCTCGAGCAACC

AACAGCAATACTCTGTA-3¢. The amplified product was

purified and then cloned in pmirGLO vector (Promega) at

PmeI and XhoI restriction sites, downstream of the firefly

luciferase reporter gene.

A deletion mutant (Delta) was generated by exciding from

pmirGLO-PTPRJ the PmeI-SacI fragment and re-ligating

the plasmid after blunting of the SacI digested end. Site-

specific mutants of Delta plasmid were generated by PCR

mutagenesis. Primers used were forward PmeI, 5¢-CATGTTTAAACCTCAAATTGGAAGAGTCC-3¢; reverseXhoI, 5¢- GCCCTCGAGCAACCAACAGCAATACTCT-

GTA-3¢; forward mut7440, 5¢-GAGGGGCGGAAATAAG-

CTATGCAGC-3¢; reverse mut7440, 5¢-GCTGCATAGCT-

TATTTCCGCCCCTC-3¢; forward mut7174: 5¢-GGTGT

CAATGAAAAAACAAAAGACTGGTG-3¢; reverse mut71

74, 5¢-CACCAGTCTTTTGTTTTTTCATTGACACC-3¢.PCR amplification was performed using Pfu polymerase

(Promega) and pmirGLO-Delta mutant as the template.

Mutants were all cloned at PmeI and XhoI sites of pmirGLO.

The 75 nucleotide precursor hairpin sequences of miR-328

and 100 nucleotides of genomic sequences flanking each side

of the hairpin sequence were amplified by PCR using the

following primers: forward, 5¢-CACGAATTCCCTTGTC

PTPRJ expression is negatively regulated by microRNA-328 F. Paduano et al.

8 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

GAAGTCCTCCTGTGTAG-3¢; reverse, 5¢- CACCTCGAG

GACTCCTTGCCCCATTCTCTGCG-3¢.To generate the miR-328 expression construct, the 467-

nucleotide DNA fragment obtained was digested with

EcoRI and XhoI and inserted into the EcoRI-XhoI sites of

the pCDNA3.1 expression vector (Invitrogen). All plasmids

were checked by sequence analysis.

Luciferase assay

HeLa transfected cells were lysed in a passive lysis buffer

(Promega). One hundred microlitres of luciferase assay

reagent 2 were added to 20 lL of lysate, and samples were

analysed in a luminometer (firefly reading). After the addi-

tion of Stop and Glo reagent (Promega), the samples were

again analysed in the luminometer (Renilla reading). Values

were expressed as the ratio of firefly ⁄Renilla readings.

Cell proliferation assay

HeLa cells were plated in 96-well plates at a density of

2 · 103 cells per well in 100 lL of medium and were trans-

fected with siRNA and ⁄or plasmid DNA. Each condition

was plated in octuplicate. Cell proliferation rates were deter-

mined 48 h post-transfection with the MTT assay (Sigma-

Aldrich, St Louis, MO, USA). Absorbance of the formazan

dye that is formed was measured at a wavelength of 570 nm,

using a Microplate Reader (BioRad); absorbance is propor-

tional to the number of metabolically active cells in culture.

For CFSE staining, cells were harvested and washed twice

with NaCl ⁄Pi, followed by labelling with 5 mm CFSE for

5 min at room temperature. Residual CFSE was removed by

washing twice with NaCl ⁄Pi, and cells were seeded in six-well

plates containing growth medium. CFSE fluorescence was

measured by fluorescence-activated cell sorting analysis, and

data were analysed with flowjo software (Tree Star,

Ashland, OR, USA).

Flow cytometry analysis

Cells were centrifuged at 400 g for 3 min, the supernatant

was discarded and the cells were re-suspended in 500 lL of

NaCl ⁄Pi. Briefly, cells were blocked with 1% BSA in

NaCl ⁄Pi, washed once and incubated for 20 min with

PTPRJ primary antibody (R&D), followed by incubation

with phycoerythrin-conjugated secondary antibody. Samples

were analysed with FACSCan instruments (Becton Dickinson,

Franklin Lakes, NJ, USA). Data were analysed with flow-

jo software (Tree Star).

Generation of stable cell clones

HeLa cells were transfected using Lipofectamine 2000 (Invi-

trogen). Cells were plated into selection medium containing

0.4 mgÆmL)1 G418 (Sigma Aldrich) 1 day post-transfection.

After 25 days of selection, individual G418-resistant colo-

nies were isolated, expanded and analysed for miR-328

expression levels by qRT-PCR.

Cell migration assay

Cell migration assay was performed using the scratch

wound-healing method on HeLa cells that were 80–90%

confluent in a six-well plate format. Cell migration was

monitored by microscopy. Images acquired for each sample

were then quantitatively analysed. For each image, dis-

tances between the points of the scratch were measured. By

comparing the images from t = 0 to t = 12 h, which was

the last time point, the distance of each scratch closure was

obtained by measuring the distances using imagej software

(NIH Image, Bethesda, MD, USA).

Statistical analyses

Results are expressed as the mean ± standard deviation of

three separate experiments. The significance of differences

between groups was evaluated by using Student’s t test or one-

way analysis of variance (ANOVA), followed by Dunnett’s

test ⁄Bonferroni’s test for multiple comparisons. Analysis was

conducted using the graphpad prism software (San Diego, CA,

USA), and differences were considered significant if P < 0.05.

Acknowledgements

The present study was supported by a grant from Min-

istero dell’Istruzione, dell’Universita e della Ricerca

(MIUR) to CP and RI (No. 2007F7T537_003) and a

grant from the Associazione Italiana Ricerca Cancro

(AIRC) to FT. FP was a recipient of an FIRC (Fond-

azione Italiana Ricerca Cancro) fellowship.

References

1 Ostman A, Hellberg C & Bohmer FD (2006)

Protein-tyrosine phosphatases and cancer. Nat Rev

Cancer 6, 307–320.

2 Tarcic G, Boguslavsky SK, Wakim J, Kiuchi T, Liu A,

Reinitz F, Nathanson D, Takahashi T, Mischel PS, Ng

T et al. (2009) An unbiased screen identifies DEP-1

tumor suppressor as a phosphatase controlling EGFR

endocytosis. Curr Biol 19, 1788–1798.

3 Palka HL, Park M & Tonks NK (2003) Hepatocyte

growth factor receptor tyrosine kinase met is a substrate

of the receptor protein-tyrosine phosphatase DEP-1.

J Biol Chem 278, 5728–5735.

4 Kovalenko M, Denner K, Sandstrom J, Persson C,

Gross S, Jandt E, Vilella R, Bohmer F & Ostman A

(2000) Site selective dephosphorylation of the platelet-

F. Paduano et al. PTPRJ expression is negatively regulated by microRNA-328

FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 9

derived growth factor beta-receptor by the receptor-like

protein tyrosine phosphatase DEP-1. J Biol Chem 275,

16219–16226.

5 Iervolino A, Iuliano R, Trapasso F, Viglietto G, Melillo

RM, Carlomagno F, Santoro M & Fusco A (2006) The

receptor-type protein tyrosine phosphatase J

antagonizes the biochemical and biological effects of

RET-derived oncoproteins. Cancer Res 66, 6280–6287.

6 Lampugnani MG, Zanetti A, Corada M, Takahashi T,

Balconi G, Breviario F, Orsenigo F, Cattelino A,

Kemler R, Daniel TO et al. (2003) Contact inhibition

of VEGF-induced proliferation requires vascular

endothelial cadherin, beta-catenin, and the phosphatase

DEP-1 ⁄CD148. J Cell Biol 161, 793–804.

7 Le Pera I, Iuliano R, Florio T, Susini C, Trapasso F,

Santoro M, Chiariotti L, Schettini G, Viglietto G &

Fusco A (2005) The rat tyrosine phosphatase eta

increases cell adhesion by activating c-Src through

dephosphorylation of its inhibitory phosphotyrosine

residue. Oncogene 24, 3187–3195.

8 Holsinger LJ, Ward K, Duffield B, Zachwieja J & Jallal

B (2002) The transmembrane receptor protein tyrosine

phosphatase DEP1 interacts with p120(ctn). Oncogene

21, 7067–7076.

9 Sallee JL & Burridge K (2009) Density-enhanced phos-

phatase 1 regulates phosphorylation of tight junction

proteins and enhances barrier function of epithelial

cells. J Biol Chem 284, 14997–15006.

10 Keane MM, Lowrey GA, Ettenberg SA, Dayton MA &

Lipkowitz S (1996) The protein tyrosine phosphatase

DEP-1 is induced during differentiation and inhibits

growth of breast cancer cells. Cancer Res 56,

4236–4243.

11 Jandt E, Denner K, Kovalenko M, Ostman A &

Bohmer FD (2003) The protein-tyrosine phosphatase

DEP-1 modulates growth factor-stimulated cell migra-

tion and cell–matrix adhesion. Oncogene 22, 4175–4185.

12 Trapasso F, Yendamuri S, Dumon KR, Iuliano R,

Cesari R, Feig B, Seto R, Infante L, Ishii H, Vecchione

A et al. (2004) Restoration of receptor-type protein

tyrosine phosphatase eta function inhibits human

pancreatic carcinoma cell growth in vitro and in vivo.

Carcinogenesis 25, 2107–2114.

13 Balavenkatraman KK, Jandt E, Friedrich K, Kauten-

burger T, Pool-Zobel BL, Ostman A & Bohmer FD

(2006) DEP-1 protein tyrosine phosphatase inhibits pro-

liferation and migration of colon carcinoma cells and is

upregulated by protective nutrients. Oncogene 25,

6319–6324.

14 Iuliano R, Trapasso F, Le Pera I, Schepis F, Sama I,

Clodomiro A, Dumon KR, Santoro M, Chiariotti L,

Viglietto G et al. (2003) An adenovirus carrying the rat

protein tyrosine phosphatase eta suppresses the growth

of human thyroid carcinoma cell lines in vitro and

in vivo. Cancer Res 63, 882–886.

15 Trapasso F, Iuliano R, Boccia A, Stella A, Visconti R,

Bruni P, Baldassarre G, Santoro M, Viglietto G &

Fusco A (2000) Rat protein tyrosine phosphatase eta

suppresses the neoplastic phenotype of retrovirally

transformed thyroid cells through the stabilization of

p27(Kip1). Mol Cell Biol 20, 9236–9246.

16 Massa A, Barbieri F, Aiello C, Arena S, Pattarozzi A,

Pirani P, Corsaro A, Iuliano R, Fusco A, Zona G et al.

(2004) The expression of the phosphotyrosine phospha-

tase DEP-1 ⁄PTPeta dictates the responsivity of glioma

cells to somatostatin inhibition of cell proliferation.

J Biol Chem 279, 29004–29012.

17 Ruivenkamp CA, van Wezel T, Zanon C, Stassen AP,

Vlcek C, Csikos T, Klous AM, Tripodis N, Perrakis A,

Boerrigter L et al. (2002) Ptprj is a candidate for the

mouse colon-cancer susceptibility locus Scc1 and is

frequently deleted in human cancers. Nat Genet 31,

295–300.

18 Iuliano R, Le Pera I, Cristofaro C, Baudi F, Arturi F, Pal-

lante P, Martelli ML, Trapasso F, Chiariotti L & Fusco A

(2004) The tyrosine phosphatase PTPRJ ⁄DEP-1 genotype

affects thyroid carcinogenesis.Oncogene 23, 8432–8438.

19 Cuozzo C, Porcellini A, Angrisano T, Morano A, Lee

B, Di Pardo A, Messina S, Iuliano R, Fusco A, Santillo

MR et al. (2007) DNA damage, homology-directed

repair, and DNA methylation. PLoS Genet 3, e110.

20 Venkatachalam R, Ligtenberg MJ, Hoogerbrugge N,

Schackert HK, Gorgens H, Hahn MM, Kamping EJ,

Vreede L, Hoenselaar E, van der Looij E et al. (2010)

Germline epigenetic silencing of the tumor suppressor

gene PTPRJ in early-onset familial colorectal cancer.

Gastroenterology 139, 2221–2224.

21 Bartel DP (2009) MicroRNAs: target recognition and

regulatory functions. Cell 136, 215–233.

22 Calin GA, Ferracin M, Cimmino A, Di Leva G, Shi-

mizu M, Wojcik SE, Iorio MV, Visone R, Sever NI,

Fabbri M et al. (2005) A microRNA signature associ-

ated with prognosis and progression in chronic lym-

phocytic leukemia. N Engl J Med 353, 1793–1801.

23 Visone R, Pallante P, Vecchione A, Cirombella R,

Ferracin M, Ferraro A, Volinia S, Coluzzi S, Leone V,

Borbone E et al. (2007) Specific microRNAs are down-

regulated in human thyroid anaplastic carcinomas.

Oncogene 26, 7590–7595.

24 Garzon R, Calin GA & Croce CM (2009) MicroRNAs

in cancer. Annu Rev Med 60, 167–179.

25 Calin GA & Croce CM (2006) MicroRNAs and chro-

mosomal abnormalities in cancer cells. Oncogene 25,

6202–6210.

26 Bueno MJ, Perez de Castro I, Gomez de Cedron M,

Santos J, Calin GA, Cigudosa JC, Croce CM, Fern-

andez-Piqueras J & Malumbres M (2008) Genetic and

epigenetic silencing of microRNA-203 enhances ABL1

and BCR-ABL1 oncogene expression. Cancer Cell 13,

496–506.

PTPRJ expression is negatively regulated by microRNA-328 F. Paduano et al.

10 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

27 Jazdzewski K, Murray EL, Franssila K, Jarzab B,

Schoenberg DR & de la Chapelle A (2008) Common

SNP in pre-miR-146a decreases mature miR expression

and predisposes to papillary thyroid carcinoma. Proc

Natl Acad Sci USA 105, 7269–7274.

28 Hsu JB, Chiu CM, Hsu SD, Huang WY, Chien CH,

Lee TY & Huang HD (2011) miRTar: an integrated

system for identifying miRNA-target interactions in

human. BMC Bioinformatics 12, 300.

29 Ye W, Lv Q, Wong CK, Wu S, Fu C, Hua Z, Cai G,

Li G, Yang BB & Zhang Y (2008) The effect of central

loops in miRNA:MRE duplexes on the efficiency of

miRNA-mediated gene regulation. PLoS One 3, e1719.

30 Rehmsmeier M, Steffen P, Hochsmann M & Giegerich

R (2004) Fast and effective prediction of microR-

NA ⁄ target duplexes. RNA 10, 1507–1517.

31 Iuliano R, Raso C, Quintiero A, Pera IL, Pichiorri F,

Palumbo T, Palmieri D, Pattarozzi A, Florio T, Vigli-

etto G et al. (2009) The eighth fibronectin type III

domain of protein tyrosine phosphatase receptor

J influences the formation of protein complexes and cell

localization. J Biochem 145, 377–385.

32 Takahashi T, Takahashi K, Mernaugh RL, Tsuboi N,

Liu H & Daniel TO (2006) A monoclonal antibody

against CD148, a receptor-like tyrosine phosphatase,

inhibits endothelial-cell growth and angiogenesis. Blood

108, 1234–1242.

33 Tsuboi N, Utsunomiya T, Roberts RL, Ito H, Takah-

ashi K, Noda M & Takahashi T (2008) The tyrosine

phosphatase CD148 interacts with p85 regulatory

subunit of PI 3-kinase. Biochem J 413, 193–200.

34 Sacco F, Tinti M, Palma A, Ferrari E, Nardozza AP,

Hooft van Huijsduijnen R, Takahashi T, Castagnoli L

& Cesareni G (2009) Tumor suppressor density-

enhanced phosphatase-1 (DEP-1) inhibits the RAS

pathway by direct dephosphorylation of ERK1 ⁄ 2kinases. J Biol Chem 284, 22048–22058.

35 Trapasso F, Drusco A, Costinean S, Alder H, Aqeilan

RI, Iuliano R, Gaudio E, Raso C, Zanesi N, Croce CM

et al. (2006) Genetic ablation of Ptprj, a mouse cancer

susceptibility gene, results in normal growth and devel-

opment and does not predispose to spontaneous tumor-

igenesis. DNA Cell Biol 25, 376–382.

36 Den Hertog J, Ostman A & Bohmer FD (2008) Protein

tyrosine phosphatises: regulatory mechanisms. FEBS J

275, 831–847.

37 Karagyozov L, Godfrey R, Bohmer SA, Petermann A,

Holters S, Ostman A & Bohmer FD (2008) The struc-

ture of the 5¢-end of the protein-tyrosine phosphatase

PTPRJ mRNA reveals a novel mechanism for

translation attenuation. Nucleic Acids Res 36, 4443–

4453.

38 Eiring AM, Harb JG, Neviani P, Garton C, Oaks JJ,

Spizzo R, Liu S, Schwind S, Santhanam R, Hickey CJ

et al. (2010) miR-328 functions as an RNA decoy to

modulate hnRNP E2 regulation of mRNA translation

in leukemic blasts. Cell 140, 652–665.

39 Wang CH, Lee DY, Deng Z, Jeyapalan Z, Lee SC,

Kahai S, Lu WY, Zhang Y & Yang BB (2008)

MicroRNA miR-328 regulates zonation morphogenesis

by targeting CD44 expression. PLoS One 3, e2420.

40 Pan YZ, Morris ME & Yu AM (2009) MicroRNA-328

negatively regulates the expression of breast cancer

resistance protein (BCRP ⁄ABCG2) in human cancer

cells. Mol Pharmacol 75, 1374–1379.

41 Brault L, Gasser C, Bracher F, Huber K, Knapp S

& Schwaller J (2010) PIM serine ⁄ threonine kinases in

the pathogenesis and therapy of hematologic

malignancies and solid cancers. Haematologica 95,

1004–1015.

42 Miguet L, Bechade G, Fornecker L, Zink E, Felden

C, Gervais C, Herbrecht R, Van Dorsselaer A, Mau-

vieux L & Sanglier-Cianferani S (2009) Proteomic

analysis of malignant B-cell derived microparticles

reveals CD148 as a potentially useful antigenic biomar-

ker for mantle cell lymphoma diagnosis. J Proteome

Res 8, 3346–3354.

43 Malzkorn B, Wolter M, Liesenberg F, Grzendowski M,

Stuhler K, Meyer HE & Reifenberger G (2010) Identifi-

cation and functional characterization of microRNAs

involved in the malignant progression of gliomas. Brain

Pathol 20, 539–550.

44 Arora S, Ranade AR, Tran NL, Nasser S, Sridhar S,

Korn RL, Ross JT, Dhruv H, Foss KM, Sibenaller Z

et al. (2011) MicroRNA-328 is associated with (non-

small) cell lung cancer (NSCLC) brain metastasis and

mediates NSCLCmigration. Int J Cancer 129, 2621–2631.

45 Leidinger P, Keller A, Borries A, Reichrath J, Rass K,

Jager SU, Lenhof HP & Meese E (2010) High-through-

put miRNA profiling of human melanoma blood

samples. BMC Cancer 10, 262.

46 Di Leva G & Croce CM (2010) Roles of small RNAs

in tumor formation. Trends Mol Med 16, 257–267.

47 Leung AK & Sharp PA (2010) MicroRNA functions in

stress responses. Mol Cell 40, 205–215.

48 Brunner PM, Heier PC, Mihaly-Bison J, Priglinger U,

Binder BR & Prager GW (2011) Density enhanced

phosphatase-1 down-regulates urokinase receptor

surface expression in confluent endothelial cells. Blood

117, 4154–4161.

49 Petermann A, Haase D, Wetzel A, Balavenkatraman

KK, Tenev T, Guhrs KH, Friedrich S, Nakamura M,

Mawrin C & Bohmer FD (2011) Loss of the protein-

tyrosine phosphatase DEP-1 ⁄PTPRJ drives meningioma

cell motility. Brain Pathol 21, 405–418.

50 Vandesompele J, De Preter K, Pattyn F, Poppe B, Van

Roy N, De Paepe A & Speleman F (2002) Accurate

normalization of real-time quantitative RT-PCR data

by geometric averaging of multiple internal control

genes. Genome Biol 3, 0034.

F. Paduano et al. PTPRJ expression is negatively regulated by microRNA-328

FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 11

Supporting information

The following supplementary material is available:

Fig. S1. PTPRJ protein expression in HeLa transfected

cells.

Fig. S2. miR-328 increases cell proliferation of SKBr3

cells.

Fig. S3. Stable transfection of miR-328 in HeLa cells

increased the cell migration.

This supplementary material can be found in the

online version of this article.

Please note: As a service to our authors and readers,

this journal provides supporting information supplied

by the authors. Such materials are peer-reviewed and

may be reorganized for online delivery, but are not

copy-edited or typeset. Technical support issues arising

from supporting information (other than missing files)

should be addressed to the authors.

PTPRJ expression is negatively regulated by microRNA-328 F. Paduano et al.

12 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS