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microRNA-125a-3p reduces cell proliferation andmigration by targeting Fyn

Lihi Ninio-Many, Hadas Grossman, Noam Shomron, Dana Chuderland and Ruth Shalgi*Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel

*Author for correspondence (shalgir@post.tau.ac.il)

Accepted 18 March 2013Journal of Cell Science 126, 2867–2876� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.123414

SummaryFyn, a member of the Src family kinases (SFKs), has a pivotal role in cell adhesion, proliferation, migration and survival, and itsoverexpression is associated with several types of cancer. MicroRNAs (miRNAs) play a major role in post-transcriptional repression of

protein expression. In light of the significant functions of Fyn, together with studies demonstrating miR-125a as a tumor-suppressingmiRNA that is downregulated in several cancer cell types and on our bioinformatics studies presented here, we chose to examine thepost-transcription regulation of Fyn by miR-125a-3p in the HEK 293T cell line. We show that Fyn expression can be dramatically

reduced by elevated levels of miR-125a-3p. Following this reduction, the activity of proteins downstream of Fyn, such as FAK, paxillinand Akt (proteins known to be overexpressed in various tumors), is also reduced. On a broader level, we show that miR-125a-3p causesan arrest of the cell cycle at the G2/M stage and decreases cell viability and migration, probably in a Fyn-directed manner. The results

are reinforced by control experiments conducted using Fyn siRNA and anti-miR-125a-3p, as well as by the fact that numerous cancercell lines show a significant downregulation of Fyn after mir-125a-3p overexpression. Collectively, we conclude that miR-125a-3p hasan important role in the regulation of Fyn expression and of its signaling pathway, which implies that it has a therapeutic potential in

overexpressed Fyn-related diseases.

Key words: Fyn, miR-125a-3p, miRNA, Migration, Proliferation

IntroductionThe Src-family kinase (SFK) is one of the most studied proteinfamilies in cancer biology. Since the identification and

characterization of pp60c-SRC, eight other proteins, whichhave a homologous structure, have been identified. Eachmember is characterized by a unique region that specifies itsrespective binding partners and, hence, its functions (Kefalas

et al., 1995; Thomas and Brugge, 1997). Although it is wellestablished that the functions of SFKs are partially overlappingand thus that they might compensate for one another, this notion

is contradicted by studies identifying large non-overlappingsignaling mechanisms among them (Nakayama et al., 2012). Ingeneral, SFKs play crucial roles in the regulation of cell

functions, including proliferation, differentiation, adhesion,motility and survival (Parsons and Parsons, 2004), bymediating extracellular interactions driven by various

molecules, as Met, epidermal growth factor receptor (EGFR)and integrins (Kim et al., 2009). Many of these interactions arehighly dependent on the kinase function of the SFK.Overexpression and/or elevated activation of SFKs occur

frequently in tumor tissues, leading to alterations in cellulargrowth, shape and function, and making the SFKs attractivetargets for anti-cancer treatment in several solid tumors and

leukemia (Kim et al., 2009; Edwards, 2010).

Fyn, a widely studied, 59 kDa SFK, is expressed in a broadrange of tissues and is downstream of important cell surface

receptors. Thus, it is involved in cellular functions, such as thecell cycle (Thomas and Brugge, 1997), apoptosis (Tang et al.,2007), integrin-mediated interactions (Wang et al., 2010), growth

factor-induced mitogenesis, cell–cell adhesion (Prag et al., 2002;Volberg et al., 2001), migration and oocyte maturation (Levi et al.,

2010). Several studies suggest that Fyn is involved in regulation ofthe organization of microtubules and actin-dependent processes

(Thomas et al., 1995; Martın-Cofreces et al., 2006; Talmor-Cohenet al., 2004). Fyn interacts with and activates a number of cellular

factors including focal adhesion kinase (FAK) and paxillin, both of

which have a key role in cell spreading and motility. Formation ofa complex of Fyn, neural cell adhesion molecule (NCAM) and

FAK leads to activation of mitogen-stimulated protein kinasesErk1/2 (Schmid et al., 1999). Fyn has also been shown to be

involved in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway,to be directly deactivated by tensin homologue deleted on

chromosome 10 (PTEN), an inhibitor of PI3K, and to regulateintegrin-stimulated signaling in glioma cells through the Rho

family of GTPases (Yadav and Denning, 2010; Dey et al., 2008).

Aberrant regulation of Fyn expression is correlated with a varietyof pathologies, such as Alzheimer’s disease and hypo-

degranulation of mast cells in allergic responses (Rivera andOlivera, 2007). Overexpressed Fyn is involved in progression of

several types of cancer, including the epithelial to mesenchymaltransition and metastasis (Lewin et al., 2010; Lehembre et al.,

2008). Overall, owing to its numerous functions and its

involvement in key signaling pathways, it is anticipated that Fynexpression and activity should be tightly regulated.

MicroRNAs (miRNAs) are a group of naturally occurring

small non-coding RNAs, that have a major role in post-

transcriptional repression of protein expression. They areinitially transcribed as large primary (pri) transcripts; these are

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then processed to precursor (pre) transcripts and then to thefunctional single-stranded mature miRNAs (Kiezun et al., 2012).

A base-pairing complement between the seed sequence ofmiRNAs and the 39 untranslated region (39UTR) of their target

mRNAs is the key determinant of miRNA–target recognition. Itis estimated that ,500–1000 miRNAs are expressed from themammalian genome (Kiezun et al., 2012), many of which

participate in regulation of a large variety of cellular processesincluding proliferation, differentiation and cell death (Inui et al.,2010; Chitwood and Timmermans, 2010). Furthermore, the

majority of miRNAs show tissue or developmental stage-specificexpression, suggesting an intricate role in these pathways

(Chitwood and Timmermans, 2010). It has been reported thatseveral SFKs are regulated by miRNAs. For example, Src, Lynand Yes have been shown to be controlled by miR-205 in a renal

cancer cell line (Majid et al., 2011). However, despite its crucialand functional roles in cellular processes, regulation of Fyn by

miRNAs had not been previously established.

In light of the crucial functions of Fyn, together with studiesdemonstrating miR-125a as a tumor suppressing miRNA (Zhang

et al., 2009; Cowden Dahl et al., 2009; Guo et al., 2009; Kim et al.,2012) that is downregulated in several cancer cell types (O’Day

and Lal, 2010; Laneve et al., 2007), and on the basis ofbioinformatics studies presented here, we investigated the role ofmiRNA-125a-3p (miR-125a-3p) in the inhibition of Fyn-mediated

pathways in human embryonic kidney 293T cell line (HEK 293T).We report that miR-125a-3p regulates Fyn mRNA, as well as its

protein expression level and activity through interaction with its39UTR. Elevated levels of miR-125a-3p resulted in an arrest at theG2/M phase of the cell cycle, in reduced proliferation and motility

of the cells and in reduced activity of FAK, paxillin and Akt,whereas low levels of miR-125a-3p resulted in an opposite effect.

We validated, by Fyn RNA interference, that the cellular effects ofmiR-125a-3p were mediated at least in part through a Fyn-directedpathway. Finally, we showed that miR-125a-3p also regulates Fyn

expression in other tumor cell lines. Our results suggest that miR-125a-3p could have a role as a therapeutic agent in overexpressedFyn-related diseases.

ResultsFyn is a direct miR-125a-3p target

miRNAs regulate their targets via binding to their 39UTR. Basedon three commonly used algorithms, Target Scan (Lewis et al.,

2005), Miranda (John et al., 2004) and PicTar (Krek et al., 2005),

we found that the Fyn 39UTR (nucleotides 359–365) is highlyconserved among mammals for miR-125a-3p (Fig. 1A). miR-

125a-3p and miR-125a-5p are two isoforms derived, respectively,from the 39 and 59 arms of pre-miR-125a. Given that only the

miR-125a-3p isoform seed-sequence was complementary to the39UTR of the Fyn gene (Fig. 1A), it was selected for further

analyses. Both isoforms of miR-125a are expressed in native

HEK 293T cells (Fig. 1B, naive cells). Ectopic expression of themiR-125a precursor resulted in overexpression of the miR-125-

3p isoform alone (300 fold, P,0.01; Fig. 1B, miR-125a) andthus overexpression of miR-125a precursor will be referred to

hereafter as overexpression of miR-125a-3p. Transfection with aplasmid containing no miRNA (empty miR-Vec; see Materials

and Methods) had no effect on the level of both isoforms

(Fig. 1B, empty miR-Vec) and was therefore used as atransfection control in the following experiments.

To demonstrate the specific interaction of Fyn transcriptand miR-125a-3p, we used the luciferase assay to measure the

level of a reporter gene fused to the 39UTR of Fyn(psiCHECK-Fyn). HEK 293T cells were co-transfected with

the reporter plasmid and with either miR-125a-3p or controlvector (empty miR-Vec). A significant decrease in the

luciferase level was observed 48 hours after transfection

with miR-125a-3p (Fig. 2, dark gray columns). In order tovalidate the binding specificity of miR-125a-3p and Fyn

39UTR, we co-transfected HEK 293T cells with psiCHECK-2vector that contained no Fyn sequence (psiCHECK-2 empty

vector) along with either miR-125a-3p or control empty miR-

Vec, and found no difference between the two (Fig. 2, lightgray columns). These results indicate a specific binding of

miR-125a-3p and Fyn 39UTR that led to the reduced luciferasesignal expressed by the psiCHECK-Fyn plasmid, suggesting

that Fyn is targeted by miR-125a-3p.

miR-125a-3p regulates both the expression level andactivity of Fyn

To determine whether miR-125a-3p regulates the expression of

endogenous Fyn, we first generated a miR-125a-3p mutant, in

which the adenine nucleotide at position 1 of the seed sequencewas replaced by a guanine nucleotide (miR-125a-AG; Fig. 3A).

We then transfected the cells with miR-125a-3p, miR-125a-AGmutant or empty miR-Vec (control) and examined the level of

Fig. 1. Bioinformatics prediction for Fyn-binding

miRNAs. (A) The 39UTR of Fyn mRNA is predicted to

be targeted by miR-125a-3p (nucleotides 359–365).

(B) miR-125a-3p is expressed in HEK 293T cells and is

upregulated after transfection. HEK 293T cells, either

non-transfected (naive cells) or transfected for 48 hours

with miR-125a-3p (a miR-125a precursor) or with empty

miR-Vec, were subjected to qPCR analysis with specific

primers for miR-125a-3p and miR-125a-5p, calibrated to

the endogenous control, U6 snRNA. The experiment was

repeated three times and analyzed by using a one-sample

Student’s t-test. Results are mean6s.d. **P,0.01

compared with naive cells.

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Fyn protein 48 hours later. Overexpression of miR-125a-3p led to

a dramatic decrease in the amount of Fyn mRNA (40%, P50.03;Fig. 3B), as well as a decrease in the amount of Fyn protein

(59%, P,0.02; Fig. 3C) compared with that in cells transfectedwith an empty vector (control). As expected, overexpression ofmiR-125a-AG did not change significantly (P.0.05) the amount

of either Fyn mRNA (Fig. 3B) or Fyn protein (Fig. 3C); probablybecause it enables the binding of miRNA and Fyn but prevents

miRNA from functioning. Interestingly, a miR-125a-3p mutant inwhich nucleotides 1–3 of the seed sequence were mutated (miR-

125a-3M; supplementary material Fig. S1A) increased the levelof Fyn protein (supplementary material Fig. S1B) probablybecause the mutations hampered the ability of miR-125a-3p to

bind Fyn. Immunofluorescence assays reinforced our findingsthat Fyn is regulated by miR-125a-3p, as there was a weaker

staining of Fyn in cells transfected with miR-125a-3p than incontrol cells (Fig. 3D).

The activity of the SFK members is regulated by the level of

phosphorylation at two tyrosine residues. In the active form, theSrc-Y527-equivalent site (Y527) is dephosphorylated, whereas the

Src-Y416-equivalent site (Y416) that determines the activity levelof the SFK is phosphorylated (Hardwick and Sefton, 1995). We

therefore examined whether the miR-125a-3p-induced reductionin Fyn protein amount is reflected also in its level of activity. Forthat, lysates of cells either transfected with miR-125a-3p or with

empty miR-Vec (control), were immunoprecipitated with anti-Fynantibody, blotted and reacted with an antibody directed against the

phosphorylated-Y416 (p-Y416). We found that the phosphorylationof Y416 in miR-125a-3p-overexpressing cells was significantly

lower than that in control cells (P,0.05; Fig. 3E), indicating adecreased Fyn activity. As the amount of total Fyn was alsoreduced in miR-125a-3p-overexpressing cells, the ratio between p-

Y416-Fyn and total Fyn remained almost constant. Overall, theseresults indicate that Fyn mRNA is controlled post-transcriptionallyby miR-125a-3p, leading to a reduction in the amount of Fyn

protein with a concomitant reduction in the fraction of the activeform of the protein.

Fyn inhibition by siRNA induces cell cycle arrest andimpairs cell viability and migratory ability

Because Fyn is considered an important mediator of mitogenic

signaling and a regulator of cell cycle, growth and proliferation(Cowden Dahl et al., 2009; Ban et al., 2008; Posadas et al., 2009),we conducted experiments where Fyn expression was inhibited by

siRNA and assessed the effect on proliferation, viability andmigration of the cells. Indeed, by using Fyn-siRNA (si-Fyn) wewere able to obtain an 80% decrease in the expression of Fyn

mRNA (Fig. 4A). For analyzing the cell cycle, we used propidiumiodide staining, along with FACS analysis, and observed a 23%increase in the proportion of cells at the G2/M phase of the cell

cycle (P,0.05; Fig. 4B). To estimate cell viability, we performedan MTT assay on Fyn-under-expressing cells and found a 27%decrease in cell viability (P,0.05, Fig. 4C). The migratory abilityof the cells, tested by using a transwell migration assay, was

decreased by 23% (P,0.05; Fig. 4D).

miR-125a-3p induces cell cycle arrest and impairs cellviability and migratory ability

As we were able to demonstrate that miR-125a-3p regulates Fyn

expression, we assumed that the cellular functions affected by si-Fyn would be phenocopied by miR-125a-3p. We found thatoverexpression of anti-miR-125a-3p (Anti-miR) (Gambari et al.,

2011), an inhibitor directed against the mature miR-125a-3psequence, led to an 80% reduction in the expression of miR-125a-3p (P,0.05; Fig. 5A). As expected, the cellular effects exertedby miR-125a-3p were similar to those caused by si-Fyn, whereas

those exerted by Anti-miR were opposite to the effects caused bysi-Fyn; miR-125a-3p caused a 57% increase in the proportion ofcells at the G2/M phase of the cell cycle (P,0.02) with a

concomitant decrease in the proportion of cells at the G1 phase(P,0.05; Fig. 5B). No arrest at the G2/M phase was detected incells transfected with Anti-miR. We found a 56% decrease in

viability of miR-125a-3p-overexpressing cells (P50.05) and a26% increase in viability of miR-125a-3p-under-expressing cells(Anti-miR, P50.07; Fig. 5C). The migratory ability of the cells

was decreased in miR-125a-3p-overexpressing cells (67%,P,0.05) and increased in miR-125a-3p-under-expressing cells(Anti-miR, 43% increase, P,0.05; Fig. 5D). Overall, our resultsindicate that miR-125a-3p exerts its cellular effects, at least in

part, by modulating the expression and activity of Fyn.

miR-125a-3p negatively regulates the activity of proteinsdownstream of Fyn

The finding that miR-125a-3p inhibits proliferation, viability and

migration of cells, prompted us to examine the effect of miR-125a-3p on central proteins located downstream of Fyn that areknown to participate in its signaling pathway. We selected three

key proteins, FAK and paxillin, which play a role in cellmorphology and motility, and Akt, which is involved in viabilityand proliferation of cells.

Fig. 2. Fyn is post-transcriptionally regulated by miR-125a-3p. HEK

293T cells were lysed 48 hours after transfection and the luciferase activity

was determined by using a luminometer according to the Dual-Glo luciferase

assay system manual. The firefly reporter cassette served as a transfection

normalization reporter. The results represent the effect of miR-125a-3p or

empty miR-Vec on the recombinant plasmid, psiCHECK-Fyn (dark grey bars)

and on the psiCHECK-2 empty vector (light grey bars) and are means6s.d. of

the relative Rennila/firefly luciferase ratio, normalized to control samples of

cells transfected with psiCHECK-2 empty vector and empty miR-Vec. The

experiment was performed three times and analyzed by using a one-sample

Student’s t-test. *P,0.05 compared with control.

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First, we showed that Fyn protein expression was significantly

decreased (,50%) by either si-Fyn or miR-125a-3p overexpression

(decrease of 44% or 59%, respectively) and was significantly

increased by Anti-miR (39.5%; Fig. 6A). To validate the pivotal role

of Fyn in activating FAK, paxillin and Akt, we examined theactivation state of these proteins, as reflected by their phosphorylation

state in si-Fyn-knocked-down cells. A significant decrease was

observed in the activity of the three proteins (p-FAK, 25%; p-paxillin,

35%; p-Akt, 19%; P,0.05; Fig. 6). The next step was to examine the

activity of the proteins in cells that either over- or under-express miR-

125a-3p. Cells transfected with miR-125a-3p exhibited a respectivelysimilar decrease (p-FAK, 35%; p-paxillin, 21%; p-Akt, 28%;

P,0.05; Fig. 6), whereas those transfected with Anti-miR showed

a significant increase in the activity of these proteins (p-FAK, 39%; p-

paxillin, 27%; p-Akt, 50%; P,0.05; Fig. 6). These results imply that

the miR-125a-3p-induced-decrease in the activity of the three proteinsis mediated by a decrease in the amount of Fyn.

Taken together, our results indicate a role for miR-125a-3p in

the regulation of cell proliferation, viability and motility, probablythrough the regulation of Fyn and its downstream proteins.

miR-125a-3p regulates the expression of Fyn mRNA incancer cell lines

Fyn, which is known to be activated in some human cancers and

to play a key role in cell invasion and metastasis, has been

suggested as a potential unique target for anti-cancer therapy

(Yadav and Denning, 2011). To assess the feasibility of the use of

miR-125a-3p as a regulator of Fyn expression in various

cancerous cells, we examined the expression of Fyn mRNA

in miR-125a-3p-overexpressing cancer cell lines: MNT-1(melanoma cancer), U2OS (osteosarcoma), MDA-MB-231,

MCF-7 and SKBR3 (breast cancers), PC3 (prostate cancer) and

HeLa (cervical cancer). The role of Fyn in tumor progression or

its overexpression has already been illustrated in these cancer cell

lines, with the exception of osteosarcoma (Teutschbein et al.,

2009; Huang et al., 2003; Zhao et al., 2011; Posadas et al., 2009;Bilal et al., 2010; Yadav and Denning, 2011). We found that the

expression of Fyn mRNA was reduced in all the examined cell

lines, except HeLa cells (Fig. 7). The results imply that, on top of

its physiological role of regulating Fyn expression, miR-125a-3p

might also have a similar role in cancerous cells.

DiscussionIn this study, we have shown that miR-125a-3p acts as a regulatorof Fyn expression and activity. We demonstrated, by the use of

functional assays, that overexpression of miR-125a-3p, as well as

of si-Fyn, reduced the activity of FAK, paxillin and Akt, blocked

the cell cycle, and impaired the viability and motility of the cells.

The post-translational modifications of SFK proteins that

determine their activity, localization and protein–protein

Fig. 3. Fyn protein expression is regulated by

miR-125a-3p. (A) A schematic representation

indicating the position of the single base

mutation (black square) in the miR-125a-3p

sequence (miR-125a-AG). (B,C) HEK 293T

cells, transfected with miR-125a-3p, miR-125a-

3p mutant (miR-125a-AG) or empty miR-Vec

(control), were cultured for 48 hours and

subjected to (B) qPCR analysis with specific

primers for Fyn and for HPRT1 as an

endogenous control and (C) western blot

analysis, using anti Fyn antibody and anti-actin

as a loading calibrator. The lower panel in C

shows a representative western blot and the

upper panel a graphic representation of the

densitometry analysis of three independent

experiments. Results are mean6s.d. analyzed by

one-sample Student’s t-test. *P,0.05 compared

with control. (D,E) HEK 293T cells were

transfected with miR-125a-3p or empty miR-Vec

(control) and were cultured for 48 hours. (D)

Cells were stained with anti-Fyn antibody

(green) and visualized by a confocal microscope.

(E) Pellets (P) and supernatants (S) of whole-

cells lysates were incubated with anti-Fyn

antibody (IP: anti Fyn). The immunoprecipitated

complexes were immunoblotted with antibodies

against the Src p-Y416-equivalent site (IB: anti

p-Y416 antibody) and against Fyn (IB: anti Fyn

antibody). All experiments were performed

three times.

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interactions are well known (Saito et al., 2010), although their

post-transcriptional regulation has not yet been elucidated.

Regulation of some SFK members by miRNAs had already

been demonstrated; specifically, miR-205 was shown to regulate

the expression of Src, Lyn and Yes in renal cancer cell lines

(Majid et al., 2011). In the current study, we focused on Fyn,

aiming to reveal its post-transcriptional regulation by miRNAs.

We showed, by luciferase assay, a direct interaction between

miR-125a-3p and the Fyn 39UTR, as overexpression of miR-

125a-3p was associated with suppression of luciferase activity.

Furthermore, we demonstrated that overexpression of miR-125a-

3p caused a significant downregulation of both Fyn mRNA and

protein.

We further examined whether miR-125a-3p also regulates Fyn

activity. As an SFK member, Fyn possesses two key sites of

tyrosine phosphorylation; the Src-Y416- and Src-Y527-

equivalent sites. In its active form, Y527 is dephosphorylated

and that allows the phosphorylation of Y416. The latter

determines level of activity of the SFK (Thomas and Brugge,

1997). Given that the phosphorylation at Y416 is important for

the full activation of SFKs (Thomas and Brugge, 1997), we

examined the level of phosphorylation at this site. We showed that

Fyn activity was inhibited in miR-125a-3p-overexpressing cells,

which exhibited a decreased phosphorylation at Y416; this implys

that there is a role for miR-125a-3p in the regulation of Fyn activity.

Fyn participates in signaling pathways that control a diverse

spectrum of biological activities. We initially showed that

inhibition of Fyn either by siRNA or by miR-125a-3p led to a

decrease in the proliferation of HEK 293T cells and to their arrest

at the G2/M phase of the cell cycle. These findings are in

agreement with lines of evidence indicating the role of SFKs in

cell proliferation and in the G2/M transition of the cell cycle

(Zhao et al., 2011; Arai et al., 2012; Hitosugi et al., 2007) as well

as with the finding that SU6656, an SFK inhibitor, induces a

defective cleavage of mouse oocyte (Levi et al., 2010) and a

defective cleavage furrow formation in synovial sarcoma cells,

resulting in their arrest at the G2/M phase and their subsequent

apoptosis (Arai et al., 2012). Furthermore, inhibition of Fyn by

microinjection of dominant-negative Fyn into mouse oocytes

caused inhibition of the second polar body extrusion and of the

Fig. 4. Reduced Fyn level regulates cell

cycle arrest and impairs cell viability and

migration. (A) Manipulation of Fyn mRNA

by Fyn-siRNA (si-Fyn). HEK 293T cells

transfected with si-Fyn were subjected

48 hours later to qPCR with specific primers

for Fyn and HPRT1, as a calibrator.

(B–D) Fyn-siRNA triggers the accumulation

of cells at the G2/M stage of the cell cycle,

reduces cell growth and impairs the

migratory ability of the cells. HEK 293T

cells were transfected with si-Fyn or control

vector (control), and subjected, 48 hours

later, to (B) fluorescence activated cell

sorting (FACS), (C) a cell growth MTT

assay, and (D) a cell migration assay using

the trans-well system. The upper panel in B

shows a representative cell cycle analysis;

the lower panel shows a graphic

representation of three independent

experiments. The upper panel in D shows

representative culture micrographs; the lower

panel shows a graphic representation of three

independent experiments. Thick arrows

indicate the migrating cells. The thin arrow

points at the membrane pores. All

experiments were performed three times.

Results are mean+s.d. and were analyzed by

using a one-sample Student’s t-test. *P,0.05

compared with control. Scale bar: 50 mm (D).

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first mitotic cleavage (Levi et al., 2011; Gallo et al., 2012). In the

current paper, we showed that inhibition of Fyn, either by siRNA

or by miR-125a-3p, inhibited cell migration, which had already

been demonstrated to be regulated by SFK members in general

and by Fyn in particular (Roche, 1998; Yadav and Denning,

2011; Miyamoto et al., 2008).

The observed effects on cell proliferation, viability and

motility prompted us to examine whether miR-125a-3p affects

the expression of Fyn downstream proteins that are involved in

these pathways. We focused on three regulatory Fyn downstream

proteins: Akt, a regulator of cell growth and proliferation (Testa

and Tsichlis, 2005), and FAK and paxillin, regulators of cell

motility (Llic et al., 1995; Zhao et al., 2011; Deakin and Turner,

2008). We demonstrated that their activation was inhibited

once Fyn was knocked-down either by Fyn siRNA or by

overexpressing miR-125a-3p. An opposite effect was obtained in

Fig. 5. miR-125a-3p regulates cell cycle arrest and impairs viability and migratory ability. (A) Manipulation of miR-125a-3p by Anti-miR. HEK 293T cells

transfected with Anti-miR or with empty miR-Vec (control) were subjected, 48 hours later, to qPCR with specific primers for miR-125a-3p and U6-snRNA as a

calibrator. (B–D) miR-125a-3p triggers the accumulation of cells at the G2/M stage of the cell cycle, reduces cell growth and impairs the migratory ability of the

cells. HEK 293T cells were transfected with miR-125a-3p, Anti-miR or empty vector (control) and subjected 48 hours later to (B) FACS analysis, (C) MTT

assay and (D) a migration assay as described in Fig. 4. Results were analyzed using one-sample Student’s t-test and are presented as the relative expression

(mean+s.d.) of mRNA normalized to control. *P,0.05 compared with control. Scale bar: 50 mm.

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cells under-expressing miR-125a-3p. Given that bioinformatics

does not predict these proteins to be miR-125a-3p targets, it

implies that miR-125a-3p regulates their activity via regulation of

Fyn expression. However, since a single miRNA can have

multiple targets, it is possible that miR-125a-3p regulates these

proteins simultaneously, in a non-Fyn-dependent fashion,

although further analysis is needed.

Although it is not accurate to directly compare the effects of si-

Fyn and miR-125a-3p, because the method of their transfection

was different, we found them both to be almost equally effective

in downregulating the activity of FAK, paxillin and Akt.

However, we found that miR-125a-3p exerts a greater effect

than si-Fyn on cell viability and migration. This could be

explained by a compensation of the activity of Fyn by other

SFKs, and will be difficult to examine because the mode of

action of a particular kinase is dependent not only on its

activation but also on its relative distribution at the plasma

membrane and the cellular micro-domains (Oneyama et al.,

2009). Another possible explanation is that the miRNA

simultaneously regulates many proteins, which makes it

reasonable to believe that the ability of a single miRNA (e.g.

miR-125a-3p) to target a few proteins belonging to a single

signaling pathway could be stronger than inhibition exerted by a

single regulator.

The notion that Fyn plays a central role in diverse cellular

functions implies that its expression has to be tightly regulated. It

has been reported that non-regulated expression of Fyn is

correlated with a variety of pathologies. For example,

unbalanced expression of Fyn, a protein essential for normal

neural development (Beggs et al., 1994), myelination (Umemori

et al., 1994) and synaptic plasticity (Kojima et al., 1997;

Miyakawa et al., 1995), is associated with Alzheimer’s disease

(Chin et al., 2005) as well as with bipolar disorder (Miyakawa

Fig. 6. Proteins downstream of Fyn are regulated by miR-125a-3p. (A–D) HEK 293T cells, transfected with Fyn-siRNA (si-Fyn), miR-125a-3p, Anti-miR or

with empty vector (control), were cultured for 48 hours. Cells were lysed and their proteins were analyzed by western blotting with specific antibodies

against (A) Fyn, (B) phospho-Akt (p-Akt), (C) phospho-FAK (p-FAK) and (D) phospho-paxillin (p-paxillin), as well as against their loading controls (actin,

general Akt, general FAK and general paxillin, respectively). The experiment was repeated three times. The intensity of bands was analyzed using the Image J

software and the ratio between each protein and its control is plotted as mean6s.d. *P,0.05, **P,0.01 compared with control.

Fig. 7. miR-125a-3p regulates Fyn expression in cancerous cells. MNT-1,

U2OS, MDA-MB-231, MCF-7, SKBR-3, PC3 and HeLa cell lines,

transfected with miR-125a-3p or with empty vector (control cells), were

cultured for 48 hours and subjected to qPCR analysis with specific primers

for Fyn and for HPRT1, as an endogenous control. The expression of Fyn

mRNA in each cell line was normalized to Fyn expression in the

corresponding control cells and is shown as mean6s.d. The experiment was

repeated three times and analyzed by a one-sample Student’s t-test. *P,0.05

compared with control.

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et al., 1995). Moreover, there is ample evidence pointing at theconnection of unregulated Fyn with cancer biology.

Studies show that, although the level of expression of Fyn innormal tissues is low, it is significantly overexpressed in primaryand metastatic tumors such as those of the prostate and pancreas,as well melanoma, glioma and chronic myelogenous leukemia

(Saito et al., 2010; Posadas et al., 2009; Huang et al., 2003; Banet al., 2008). Fyn has been implicated as a mediator of EGF andTGF-b-driven transformations in murine epidermal cells and

human lung cancer cells, respectively (He et al., 2008; Kim et al.,2011). Its expression is correlated with a lower survival rate inbreast cancer patients (Garcia et al., 2007) and in patients with

pancreatic cancer metastasis (Chen et al., 2010) or with solidtumors (Saito et al., 2010; Huang et al., 2007). Collectively, ourresults might suggest that an miR-125a-3p-exerted mechanism

might regulate aberrant expression of Fyn during cancerprogression. The ability of miR-125a-3p to regulate FAK,paxillin and Akt proteins located downstream of Fyn andknown to be overexpressed in tumors, strengthens the need to

study its therapeutic potential.

Some crucial genes, such as those encoding lin-28 (Wu andBelasco, 2005), ERBB2 and ERBB3 (Scott et al., 2007), which

are involved in cancer progression, are validated miR-125a targetgenes. It should be noted that these studies refer to the miR-125a-5p isoform, an isoform that does not share the same seed-

sequence as the miR-125a-3p isoform. The latter was recentlyidentified as a new member of the miR-125a family, with afunction that is not yet well understood. Our findings, as well as a

study showing that miR-125a-3p is downregulated in non-smallcell lung cancer cells and that its expression is negativelycorrelated with the pathological stage or metastasis (Jiang et al.,2010), both imply a role for miR-125a-3p as a tumor suppressing

miRNA and raise the possibility that Fyn overexpression can beattributed to downregulation of miR-125a-3p. Other genessuspected of being regulated by miR-125a-3p are those

encoding chemokine (C-C motif) ligand 4 and IGF-2 (Jianget al., 2010), both known to have a role in migration of tumorcells (Menten et al., 2002; Nussbaum et al., 2008).

Our study is the first report indicating that miR-125a-3pinteracts directly with Fyn and regulates its expression andactivity. More research is required to examine the effect of miR-125a-3p on cellular growth, shape and function, especially in

cancerous cells, in order to determine its therapeutic potentialin regulating aberrantly overexpressed Fyn, especially inoverexpressed Fyn-related tumors.

Materials and MethodsAntibodiesThe primary antibodies used were: anti-Fyn antibody (sc-16), anti-Fyn antibodyconjugated to agarose beads (sc-16AC) and anti-phospho-paxillin antibody (sc-101774) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-phospho-416-Src antibody (#2101), anti-phospho and anti-general Akt antibody (#9721 and#2938, respectively) from Cell Signaling Technology (Danvers, MA, USA); anti-FAK (#AHO0502) and anti-phospho-FAK (#44625G) from Invitrogen (Carlsbad,CA, USA); anti-actin (#MAB150) from Millipore (Temecula, CA, USA); and anti-paxillin antibody (#610052) from BD Transduction (San Diego, CA, USA). Thesecondary antibodies were monoclonal and polyclonal HRP-conjugated antibodies(Jackson Immunoresearch, West Grove, PA, USA) and Cy-2-conjugated goat anti-rabbit Ig antibody (Invitrogen).

Cell culture and transfectionAdherent cultures of HEK 293T, MNT-1, U2OS, MDA-MB-231, MCF-7 SKBR-3and HeLa cell lines were maintained in Dulbecco’s modified Eagle’s medium(DMEM; Biological Industries, Beit-Ha’emek, Israel) supplemented with 10%fetal calf serum (FCS; Biological Industries), 2 mM L-glutamine and antibiotics

(complete medium). The PC3 cell line was maintained in RPMI medium(Biological Industries) supplemented with 10% FCS and antibiotics. All the cellswere incubated in a humidified atmosphere of 5% CO2 in air at 37 C. Cells wereseeded onto 6-well plates (35 mm; Nunc, Copenhagen, Denmark) at a density of86105 cells per well and transfected 24 hours later.

Plasmid transfection was performed using polyethylenimine transfection reagent(HEK 293T, U2OS and HeLa cell lines) or with Lipofectamine 2000 transfectionreagent (Invitrogen; MNT-1, MD-MBA-231, MCF-7, SKBR-3 and PC3 cell lines),according to manufacturer’s instructions. Fyn-siRNA (sc-29321; Santa CruzBiotechnology), was transfected with polyethylenimine transfection reagent(Sigma Chemical Company, St. Louis, MO, USA) according to themanufacturer’s instructions, using 0.5 mg siRNA duplex and 4 ml siRNAtransfection reagent. Anti-miR-125a-3p (assay ID: MH12378; Ambion, Austin,TX, USA) transfection was performed using 90 pmol of anti-miR-125a-3p and5 ml of Lipofectamine 2000 transfection reagent. Complete medium was added24 hours after transfection, for an additional 24 hours, before subjecting the cellsto subsequent analysis.

Immunoblotting (western blot)

Cells were lysed for 20 minutes in ice-cold radio-immuno-precipitation assaybuffer (RIPA; 20 mM Tris-HCl pH 7.4, 137 mM NaCl, 10% glycerol, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA pH 8, 2 mMvanadate, 1 mM PMSF and a cocktail of protease inhibitors; Boehringer,Mannheim, Germany). Cell lysate was cleared by centrifugation and anappropriate sample buffer was added. Samples were subjected to SDS-PAGE,immunoblotted with the appropriate primary antibodies (anti-Fyn, 1:300; anti-phospho-FAK 1:1000; anti-FAK, 1:100; anti-phospho-paxillin, 1:1000; anti-paxillin, 1:1000; anti-phospho-Akt, 1:1000; anti-phospho-Y416-Src antibody,1:1000; or anti-actin, 1:10,000), incubated with the corresponding horseradish-peroxidase-conjugated secondary antibodies and subjected to enhancedchemiluminescence assay (ECL; Thermo Scientific, Rockford, IL, USA). Theintensity of the bands was analyzed using the Image J software.

Immunoprecipitation

Cells were lysed in buffer A (50 mM HEPES pH 7.4, 150 mM NaCl, 5 mMMgCl2, 1% Triton X-100, 1 mM PMSF and cocktail of protease inhibitors) at 4 Cfor 10 minutes and cleared by centrifugation at 12,000 rpm for 15 minutes at 4 C.Aliquots of cleared supernatants containing 500 mg protein were incubated for2 hours at 4 C with 20 ml of anti-Fyn antibody conjugated to agarose beads at aconstant rotation. The mixture was cleared by centrifugation. The supernatant afterthe first centrifugation was used to estimate the immunoprecipitation efficacy. Thepellet was washed five times with lysis buffer A devoid of Triton X-100. Proteinswere removed from the beads by an appropriate sample buffer, boiled for10 minutes, resolved by 10% SDS-PAGE under reducing conditions andtransferred to nitrocellulose paper (Whatman GmbH, Dassel, Germany) forwestern blotting with anti p-Y416 and anti Fyn antibodies.

RNA isolation, reverse transcription and real-time polymerase chainreaction

Total RNA was extracted using TriZOL (Invitrogen) according to themanufacturer’s instructions. Reverse transcription (RT) for gene expression ormiRNA expression was carried out by using the Maxima Reverse transcriptase(Fermentas, Burlington, Ontario, Canada; 1 mg total RNA) or the high-capacitycDNA RT kit (Applied Biosystems, Foster City, CA, USA; 10 ng RNA fractions),respectively. All RT reactions were carried out by a StepOnePlus real-time PCRsystem (Applied Biosystems).

For measuring gene expression, the quantitative real-time PCR (qPCR) wasconducted using SYBR Green dye (Applied Biosystems) according to themanufacturer’s protocol. The following primers were used for the analysis: Fyn,forward primer, 59-GGACATGGCAGCACAGGTG-39, reverse primer, 59-TTTGCTGATCGCAGATCTCTATG-39; hypoxanthine phosphoribosyltransfer-ase 1 (HPRT1, as an endogenous control), forward primer, 59-TGACACTGGCA-AAACAATGCA-39, reverse primer, 59-GGTCCTTTTCACCAGCAAGCT-39.

Expression and miR-125a-3p (assay ID: 2199), miR-125a-5p (assay ID: 2198)and U6-snRNA (assayID: 001973) were measured using the TaqMan miRNA kit(Applied Biosystems) according to the manufacturer’s instructions. MaturemiRNAs were normalized to U6-snRNA. Relative expression was calculatedusing the comparative Ct.

Cloning

The 39UTR region of Fyn mRNA, flanking the miR-125a-3p-binding site, wasextracted from mouse brain genomic DNA and amplified by PCR using Phusionhigh-fidelity DNA polymerase (Fermentas) with forward primer, 59-ATACT-CGAGAGCCTGCGCTTCAG-39 and reverse primer, 59-GATGCGGCCGCAAT-CTATAGGTATGATTAG-39. The PCR product was cloned into psiCHECK-2vector (Promega, Madison, WI, USA) using the NotI and XhoI restriction enzymes(Fermentas). Empty miR-Vec was provided by R. Agami (Voorhoeve et al., 2006).

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miR-125a-3p mutants were created by using the QuikChange site-directedmutagenesis kit (Stratagene, Cedar Creek, TX, USA) with the following primers:miR-125a-AG, forward primer, 59-GGACATCCAGGGTCGCAGGTGAGGTTC-TTGGGAGCCTGGC-39, reverse primer: 59-GCCAGGCTCCCAAGAACCTCA-CCTGCGACCCTGGATGTCC-39; miR-125a-3M, forward primer, 59-GGACAT-CCAGGGTCGACGGTGAGGTTCTTGGGAGCCTGG-39, reverse primer, 59-GCCAGGCTCCCAAGAACCTCACAGTTGACCCTGGATGTCC-39.

In vitro luciferase assay

HEK 293T cells were transfected with miR-125a-3p or with empty miR-Vec(50 ng) along with psiCHECK-Fyn construct or with psiCHECK-2 empty vector(10 ng). Firefly and Renilla luciferase activities were assessed using the Dual-Gloluciferase assay system (Promega) in accordance with the manufacturer’sinstructions. Luminescence readings were acquired using a TD 20/20luminometer (Turner Design Inc., Sunnyvale, CA, USA). The Renilla luciferasesignal was normalized to the firefly luciferase signal to adjust for variations intransfection efficiency. Sample values were compared to the reference value ofcells expressing psiCHECK-2 empty vector and empty miR-Vec.

Cell cycle analysis

Following the desired treatments, cells were subjected to trypsin treatment, washedthree times in cold PBS, re-suspended in 1.0 ml hypotonic buffer (50 mg/mlpropidium iodide, 0.1% sodium citrate and 0.1% Triton X-100) and incubated forat least 1 hour at 4 C in the dark. The DNA content of the cells was measured by afluorescence-activated sorter (Becton Dickinson FACSort Flow Cytometer, SanJose, CA, USA) and analyzed using the WinMDI 2.8 software.

MTT cell proliferation assay

Transfected cells were seeded at 26104cells per well onto 24-well plates to a finalvolume of 100 ml and incubated for 48 hours. 10 ml of MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; 5 mg/ml] were added to each well before anadditional 2–4 hours incubation period at 37 C. The reaction was terminated byaddition of 110 ml HCl (0.07 M in isopropanol) and OD560 nm was measured by theSpectraMax 190 microplate reader (Molecular Probes, Eugene, Oregon, USA).

Immunofluorescence staining

HEK 293T cells were cultured on 13-mm round glass coverslips (Marienfeld GmbH,Germany). After the desired treatment, culture medium was aspirated, cells werewashed three times with cold PBS, fixed for 30 minutes in 3% paraformaldehydeand permeabilized for an additional 30 minutes with a permeabilization solution(0.1% TritonX-100, 5% FCS and 2% BSA [BSA] in PBS). Cells were subsequentlyincubated for 1 hour at room temperature in the presence of anti-Fyn antibody(1:100), washed three times and incubated for 1 hour with Cy-2-conjugated goatanti-rabbit antibody (1:400; Invitrogen). Coverslips were washed five times in PBS,stained with Hoechst 33342 (1 mg/ml; Sigma) for 10 minutes and mounted with GelMount (Sigma). Cells samples were analyzed using an LSM 510, Zeiss laserconfocal scanning microscope (Carl Zeiss, Oberkochen, Germany).

Migration assay

HEK 293T cells (26105 cells) were pre-incubated in FCS-free DMEM in the upperwells of Transwell plates (24 wells, 8 mm pore size membranes, Corning 3422;Corning, NY, USA) for the migration assay. After 6 hours, 350 ml of DMEM with20% FCS, as a chemoattractant, was loaded into the lower well and cells wereallowed to migrate during a 24-hour period of incubation at 37 C and with 5% CO2

in air. Cells attached to both sides of the membrane were washed twice with PBS.Cells on the upper side of the membrane were removed by cotton swabs, and themigrating cells at the bottom of the membrane were visualized by using afluorescence microscope, photographed and counted using the Image J software.

Statistical analysis

Data are expressed as means6s.d. Individual comparisons were made using a one-sample Student’s t-test. P,0.05 was considered statistically significant.

AcknowledgementThe authors are grateful to Gorzalczany Yaara (Tel-Aviv University)for her valuable help with some of the techniques used in this study.The authors declare that there is no conflict of interest that wouldaffect the impartiality of this scientific work. This work was performedin partial fulfillment of the requirements for a PhD degree of L. N.-M.,Sackler Faculty of medicine, Tel Aviv University, Israel.

Author contributionsL.N.-M. developed the concept, designed experiments and wrote themanuscript. L.N.-M. and H.G. carried out the experiments, data

organization and statistical analyses. N.S. and D.C participated in thestudy design and discussed the manuscript. R.S. conceived the study,participated in its design and coordination, helped draft themanuscript and supervised the study. All authors read andapproved the final manuscript.

FundingThis work was supported by a grant from the Israel ScienceFoundation [grant number 261/09 to R.S.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.123414/-/DC1

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