Fyn, a member of the Src family kinases (SFKs), has a pivotal role in cell adhesion, proliferation, migration and survival, and its overexpression 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-suppressing miRNA that is downregulated in several cancer cell types and on our bioinformatics studies presented here, we chose to examine the post-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, paxillin and Akt (proteins known to be overexpressed in various tumors), is also reduced. On a broader level, we show that miR-125a-3p causes an 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 cancer cell lines show a significant downregulation of Fyn after mir-125a-3p overexpression. Collectively, we conclude that miR-125a-3p has an 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.
The Src-family kinase (SFK) is one of the most studied protein families in cancer biology. Since the identification and characterization of pp60c-SRC, eight other proteins, which have a homologous structure, have been identified. Each member is characterized by a unique region that specifies its respective binding partners and, hence, its functions (Kefalas et al., 1995; Thomas and Brugge, 1997). Although it is well established that the functions of SFKs are partially overlapping and thus that they might compensate for one another, this notion is contradicted by studies identifying large non-overlapping signaling mechanisms among them (Nakayama et al., 2012). In general, SFKs play crucial roles in the regulation of cell functions, including proliferation, differentiation, adhesion, motility and survival (Parsons and Parsons, 2004), by mediating extracellular interactions driven by various molecules, as Met, epidermal growth factor receptor (EGFR) and integrins (Kim et al., 2009). Many of these interactions are highly dependent on the kinase function of the SFK. Overexpression and/or elevated activation of SFKs occur frequently in tumor tissues, leading to alterations in cellular growth, shape and function, and making the SFKs attractive targets 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 broad range of tissues and is downstream of important cell surface receptors. Thus, it is involved in cellular functions, such as the cell 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 of the organization of microtubules and actin-dependent processes (Thomas et al., 1995; Martín-Cófreces et al., 2006; Talmor-Cohen et 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 of a complex of Fyn, neural cell adhesion molecule (NCAM) and FAK leads to activation of mitogen-stimulated protein kinases Erk1/2 (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 regulate integrin-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 variety of pathologies, such as Alzheimer's disease and hypo-degranulation of mast cells in allergic responses (Rivera and Olivera, 2007). Overexpressed Fyn is involved in progression of several types of cancer, including the epithelial to mesenchymal transition 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 Fyn expression 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 are initially transcribed as large primary (pri) transcripts; these are then processed to precursor (pre) transcripts and then to the functional single-stranded mature miRNAs (Kiezun et al., 2012). A base-pairing complement between the seed sequence of miRNAs and the 3′ untranslated region (3′UTR) of their target mRNAs is the key determinant of miRNA–target recognition. It is estimated that ∼500–1000 miRNAs are expressed from the mammalian genome (Kiezun et al., 2012), many of which participate in regulation of a large variety of cellular processes including proliferation, differentiation and cell death (Inui et al., 2010; Chitwood and Timmermans, 2010). Furthermore, the majority of miRNAs show tissue or developmental stage-specific expression, suggesting an intricate role in these pathways (Chitwood and Timmermans, 2010). It has been reported that several SFKs are regulated by miRNAs. For example, Src, Lyn and Yes have been shown to be controlled by miR-205 in a renal cancer cell line (Majid et al., 2011). However, despite its crucial and 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 studies demonstrating 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 of bioinformatics studies presented here, we investigated the role of miRNA-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 its 3′UTR. Elevated levels of miR-125a-3p resulted in an arrest at the G2/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 of miR-125a-3p were mediated at least in part through a Fyn-directed pathway. 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 overexpressed Fyn-related diseases.
Fyn is a direct miR-125a-3p target
miRNAs regulate their targets via binding to their 3′UTR. Based on 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 3′UTR (nucleotides 359–365) is highly conserved among mammals for miR-125a-3p (Fig. 1A). miR-125a-3p and miR-125a-5p are two isoforms derived, respectively, from the 3′ and 5′ arms of pre-miR-125a. Given that only the miR-125a-3p isoform seed-sequence was complementary to the 3′UTR 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 the miR-125a precursor resulted in overexpression of the miR-125-3p isoform alone (300 fold, P<0.01; Fig. 1B, miR-125a) and thus overexpression of miR-125a precursor will be referred to hereafter as overexpression of miR-125a-3p. Transfection with a plasmid 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 a transfection control in the following experiments.
To demonstrate the specific interaction of Fyn transcript and miR-125a-3p, we used the luciferase assay to measure the level of a reporter gene fused to the 3′UTR of Fyn (psiCHECK-Fyn). HEK 293T cells were co-transfected with the reporter plasmid and with either miR-125a-3p or control vector (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 to validate the binding specificity of miR-125a-3p and Fyn 3′UTR, we co-transfected HEK 293T cells with psiCHECK-2 vector 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, light gray columns). These results indicate a specific binding of miR-125a-3p and Fyn 3′UTR that led to the reduced luciferase signal expressed by the psiCHECK-Fyn plasmid, suggesting that Fyn is targeted by miR-125a-3p.
miR-125a-3p regulates both the expression level and activity 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 sequence was replaced by a guanine nucleotide (miR-125a-AG; Fig. 3A). We then transfected the cells with miR-125a-3p, miR-125a-AG mutant or empty miR-Vec (control) and examined the level of Fyn protein 48 hours later. Overexpression of miR-125a-3p led to a dramatic decrease in the amount of Fyn mRNA (40%, P = 0.03; Fig. 3B), as well as a decrease in the amount of Fyn protein (59%, P<0.02; Fig. 3C) compared with that in cells transfected with an empty vector (control). As expected, overexpression of miR-125a-AG did not change significantly (P>0.05) the amount of either Fyn mRNA (Fig. 3B) or Fyn protein (Fig. 3C); probably because it enables the binding of miRNA and Fyn but prevents miRNA from functioning. Interestingly, a miR-125a-3p mutant in which nucleotides 1–3 of the seed sequence were mutated (miR-125a-3M; supplementary material Fig. S1A) increased the level of Fyn protein (supplementary material Fig. S1B) probably because the mutations hampered the ability of miR-125a-3p to bind Fyn. Immunofluorescence assays reinforced our findings that Fyn is regulated by miR-125a-3p, as there was a weaker staining of Fyn in cells transfected with miR-125a-3p than in control cells (Fig. 3D).
The activity of the SFK members is regulated by the level of phosphorylation at two tyrosine residues. In the active form, the Src-Y527-equivalent site (Y527) is dephosphorylated, whereas the Src-Y416-equivalent site (Y416) that determines the activity level of the SFK is phosphorylated (Hardwick and Sefton, 1995). We therefore examined whether the miR-125a-3p-induced reduction in Fyn protein amount is reflected also in its level of activity. For that, lysates of cells either transfected with miR-125a-3p or with empty miR-Vec (control), were immunoprecipitated with anti-Fyn antibody, blotted and reacted with an antibody directed against the phosphorylated-Y416 (p-Y416). We found that the phosphorylation of Y416 in miR-125a-3p-overexpressing cells was significantly lower than that in control cells (P<0.05; Fig. 3E), indicating a decreased Fyn activity. As the amount of total Fyn was also reduced in miR-125a-3p-overexpressing cells, the ratio between p-Y416-Fyn and total Fyn remained almost constant. Overall, these results indicate that Fyn mRNA is controlled post-transcriptionally by miR-125a-3p, leading to a reduction in the amount of Fyn protein with a concomitant reduction in the fraction of the active form of the protein.
Fyn inhibition by siRNA induces cell cycle arrest and impairs 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 and migration of the cells. Indeed, by using Fyn-siRNA (si-Fyn) we were able to obtain an 80% decrease in the expression of Fyn mRNA (Fig. 4A). For analyzing the cell cycle, we used propidium iodide 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 performed an MTT assay on Fyn-under-expressing cells and found a 27% decrease in cell viability (P<0.05, Fig. 4C). The migratory ability of 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 cell viability 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 that overexpression of anti-miR-125a-3p (Anti-miR) (Gambari et al., 2011), an inhibitor directed against the mature miR-125a-3p sequence, led to an 80% reduction in the expression of miR-125a-3p (P<0.05; Fig. 5A). As expected, the cellular effects exerted by miR-125a-3p were similar to those caused by si-Fyn, whereas those exerted by Anti-miR were opposite to the effects caused by si-Fyn; miR-125a-3p caused a 57% increase in the proportion of cells 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 in cells transfected with Anti-miR. We found a 56% decrease in viability of miR-125a-3p-overexpressing cells (P = 0.05) and a 26% increase in viability of miR-125a-3p-under-expressing cells (Anti-miR, P = 0.07; 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 results indicate 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 proteins downstream 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 are known to participate in its signaling pathway. We selected three key proteins, FAK and paxillin, which play a role in cell morphology and motility, and Akt, which is involved in viability and proliferation of cells.
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 the activation 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 respectively similar 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 proteins is 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, probably through the regulation of Fyn and its downstream proteins.
miR-125a-3p regulates the expression of Fyn mRNA in cancer 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 </emph>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.
In this study, we have shown that miR-125a-3p acts as a regulator of 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 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 3′UTR, 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 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 (Llić 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 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 et al., 1995). Moreover, there is ample evidence pointing at the connection of unregulated Fyn with cancer biology.
Studies show that, although the level of expression of Fyn in normal tissues is low, it is significantly overexpressed in primary and 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; Ban et al., 2008). Fyn has been implicated as a mediator of EGF and TGF-β-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 in breast cancer patients (Garcia et al., 2007) and in patients with pancreatic cancer metastasis (Chen et al., 2010) or with solid tumors (Saito et al., 2010; Huang et al., 2007). Collectively, our results might suggest that an miR-125a-3p-exerted mechanism might regulate aberrant expression of Fyn during cancer progression. The ability of miR-125a-3p to regulate FAK, paxillin and Akt proteins located downstream of Fyn and known to be overexpressed in tumors, strengthens the need to study its therapeutic potential.
Some crucial genes, such as those encoding lin-28 (Wu and Belasco, 2005), ERBB2 and ERBB3 (Scott et al., 2007), which are involved in cancer progression, are validated miR-125a target genes. 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 recently identified as a new member of the miR-125a family, with a function that is not yet well understood. Our findings, as well as a study showing that miR-125a-3p is downregulated in non-small cell lung cancer cells and that its expression is negatively correlated 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 be attributed to downregulation of miR-125a-3p. Other genes suspected of being regulated by miR-125a-3p are those encoding chemokine (C-C motif) ligand 4 and IGF-2 (Jiang et al., 2010), both known to have a role in migration of tumor cells (Menten et al., 2002; Nussbaum et al., 2008).
Our study is the first report indicating that miR-125a-3p interacts directly with Fyn and regulates its expression and activity. 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 potential in regulating aberrantly overexpressed Fyn, especially in overexpressed Fyn-related tumors.
Materials and Methods
The primary antibodies used were: anti-Fyn antibody (sc-16), anti-Fyn antibody conjugated 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). The secondary 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 transfection
Adherent cultures of HEK 293T, MNT-1, U2OS, MDA-MB-231, MCF-7 SKBR-3 and 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 cells were incubated in a humidified atmosphere of 5% CO2 in air at 37°C. Cells were seeded onto 6-well plates (35 mm; Nunc, Copenhagen, Denmark) at a density of 8×105 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 transfection reagent (Invitrogen; MNT-1, MD-MBA-231, MCF-7, SKBR-3 and PC3 cell lines), according to manufacturer's instructions. Fyn-siRNA (sc-29321; Santa Cruz Biotechnology), was transfected with polyethylenimine transfection reagent (Sigma Chemical Company, St. Louis, MO, USA) according to the manufacturer's instructions, using 0.5 µg siRNA duplex and 4 µl siRNA transfection reagent. Anti-miR-125a-3p (assay ID: MH12378; Ambion, Austin, TX, USA) transfection was performed using 90 pmol of anti-miR-125a-3p and 5 µl of Lipofectamine 2000 transfection reagent. Complete medium was added 24 hours after transfection, for an additional 24 hours, before subjecting the cells to subsequent analysis.
Immunoblotting (western blot)
Cells were lysed for 20 minutes in ice-cold radio-immuno-precipitation assay buffer (RIPA; 20 mM Tris-HCl pH 7.4, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA pH 8, 2 mM vanadate, 1 mM PMSF and a cocktail of protease inhibitors; Boehringer, Mannheim, Germany). Cell lysate was cleared by centrifugation and an appropriate 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 enhanced chemiluminescence assay (ECL; Thermo Scientific, Rockford, IL, USA). The intensity of the bands was analyzed using the Image J software.
Cells were lysed in buffer A (50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 1 mM PMSF and cocktail of protease inhibitors) at 4°C for 10 minutes and cleared by centrifugation at 12,000 rpm for 15 minutes at 4°C. Aliquots of cleared supernatants containing 500 µg protein were incubated for 2 hours at 4°C with 20 µl of anti-Fyn antibody conjugated to agarose beads at a constant rotation. The mixture was cleared by centrifugation. The supernatant after the first centrifugation was used to estimate the immunoprecipitation efficacy. The pellet was washed five times with lysis buffer A devoid of Triton X-100. Proteins were removed from the beads by an appropriate sample buffer, boiled for 10 minutes, resolved by 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose paper (Whatman GmbH, Dassel, Germany) for western blotting with anti p-Y416 and anti Fyn antibodies.
RNA isolation, reverse transcription and real-time polymerase chain reaction
Total RNA was extracted using TriZOL (Invitrogen) according to the manufacturer's instructions. Reverse transcription (RT) for gene expression or miRNA expression was carried out by using the Maxima Reverse transcriptase (Fermentas, Burlington, Ontario, Canada; 1 µg total RNA) or the high-capacity cDNA RT kit (Applied Biosystems, Foster City, CA, USA; 10 ng RNA fractions), respectively. All RT reactions were carried out by a StepOnePlus real-time PCR system (Applied Biosystems).
For measuring gene expression, the quantitative real-time PCR (qPCR) was conducted using SYBR Green dye (Applied Biosystems) according to the manufacturer's protocol. The following primers were used for the analysis: Fyn, forward primer, 5′-GGACATGGCAGCACAGGTG-3′, reverse primer, 5′-TTTGCTGATCGCAGATCTCTATG-3′; hypoxanthine phosphoribosyltransferase 1 (HPRT1, as an endogenous control), forward primer, 5′-TGACACTGGCAAAACAATGCA-3′, reverse primer, 5′-GGTCCTTTTCACCAGCAAGCT-3′.
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. Mature miRNAs were normalized to U6-snRNA. Relative expression was calculated using the comparative Ct.
The 3′UTR region of Fyn mRNA, flanking the miR-125a-3p-binding site, was extracted from mouse brain genomic DNA and amplified by PCR using Phusion high-fidelity DNA polymerase (Fermentas) with forward primer, 5′-ATACTCGAGAGCCTGCGCTTCAG-3′ and reverse primer, 5′-GATGCGGCCGCAATCTATAGGTATGATTAG-3′. The PCR product was cloned into psiCHECK-2 vector (Promega, Madison, WI, USA) using the NotI and XhoI restriction enzymes (Fermentas). Empty miR-Vec was provided by R. Agami (Voorhoeve et al., 2006).
miR-125a-3p mutants were created by using the QuikChange site-directed mutagenesis kit (Stratagene, Cedar Creek, TX, USA) with the following primers: miR-125a-AG, forward primer, 5′-GGACATCCAGGGTCGCAGGTGAGGTTCTTGGGAGCCTGGC-3′, reverse primer: 5′-GCCAGGCTCCCAAGAACCTCACCTGCGACCCTGGATGTCC-3′; miR-125a-3M, forward primer, 5′-GGACATCCAGGGTCGACGGTGAGGTTCTTGGGAGCCTGG-3′, reverse primer, 5′-GCCAGGCTCCCAAGAACCTCACAGTTGACCCTGGATGTCC-3′.
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-Glo luciferase assay system (Promega) in accordance with the manufacturer's instructions. Luminescence readings were acquired using a TD 20/20 luminometer (Turner Design Inc., Sunnyvale, CA, USA). The Renilla luciferase signal was normalized to the firefly luciferase signal to adjust for variations in transfection efficiency. Sample values were compared to the reference value of cells expressing psiCHECK-2 empty vector and empty miR-Vec.
Cell cycle analysis
Following the desired treatments, cells were subjected to trypsin treatment, washed three times in cold PBS, re-suspended in 1.0 ml hypotonic buffer (50 µg/ml propidium iodide, 0.1% sodium citrate and 0.1% Triton X-100) and incubated for at least 1 hour at 4°C in the dark. The DNA content of the cells was measured by a fluorescence-activated sorter (Becton Dickinson FACSort Flow Cytometer, San Jose, CA, USA) and analyzed using the WinMDI 2.8 software.
MTT cell proliferation assay
Transfected cells were seeded at 2×104cells per well onto 24-well plates to a final volume of 100 µl and incubated for 48 hours. 10 µl of MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; 5 mg/ml] were added to each well before an additional 2–4 hours incubation period at 37°C. The reaction was terminated by addition of 110 µl HCl (0.07 M in isopropanol) and OD560 nm was measured by the SpectraMax 190 microplate reader (Molecular Probes, Eugene, Oregon, USA).
HEK 293T cells were cultured on 13-mm round glass coverslips (Marienfeld GmbH, Germany). After the desired treatment, culture medium was aspirated, cells were washed three times with cold PBS, fixed for 30 minutes in 3% paraformaldehyde and permeabilized for an additional 30 minutes with a permeabilization solution (0.1% TritonX-100, 5% FCS and 2% BSA [BSA] in PBS). Cells were subsequently incubated 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 goat anti-rabbit antibody (1∶400; Invitrogen). Coverslips were washed five times in PBS, stained with Hoechst 33342 (1 µg/ml; Sigma) for 10 minutes and mounted with Gel Mount (Sigma). Cells samples were analyzed using an LSM 510, Zeiss laser confocal scanning microscope (Carl Zeiss, Oberkochen, Germany).
HEK 293T cells (2×105 cells) were pre-incubated in FCS-free DMEM in the upper wells of Transwell plates (24 wells, 8 µm pore size membranes, Corning 3422; Corning, NY, USA) for the migration assay. After 6 hours, 350 µl of DMEM with 20% FCS, as a chemoattractant, was loaded into the lower well and cells were allowed 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 the migrating cells at the bottom of the membrane were visualized by using a fluorescence microscope, photographed and counted using the Image J software.
Data are expressed as means±s.d. Individual comparisons were made using a one-sample Student's t-test. P<0.05 was considered statistically significant.
The 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 would affect the impartiality of this scientific work. This work was performed in partial fulfillment of the requirements for a PhD degree of L. N.-M., Sackler Faculty of medicine, Tel Aviv University, Israel.
L.N.-M. developed the concept, designed experiments and wrote the manuscript. L.N.-M. and H.G. carried out the experiments, data organization and statistical analyses. N.S. and D.C participated in the study design and discussed the manuscript. R.S. conceived the study, participated in its design and coordination, helped draft the manuscript and supervised the study. All authors read and approved the final manuscript.
This work was supported by a grant from the Israel Science Foundation [grant number 261/09 to R.S.].