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Loss of Git2 induces epithelial–mesenchymaltransition by miR146a-Cnot6L-controlled expressionof Zeb1
Wu Zhou1 and Jean Paul Thiery1,2,3,*1Institute of Molecular and Cell Biology, A*STAR, Singapore 1386732Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 1175973Cancer Science Institute, National University of Singapore, Singapore 117599
*Author for correspondence ([email protected])
Accepted 11 March 2013Journal of Cell Science 126, 2740–2746� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.126367
SummaryEpithelial–mesenchymal transition (EMT) can be induced by several pleiotropically activated transcription factors, including the zinc-
finger E-box-binding protein Zeb1. Mechanisms regulating Zeb1 expression have been partly uncovered, showing a critical role for themiR-200 family members. In the present study, we show that Zeb1 is regulated by the Arf GTPase-activating protein (GAP) Git2.Following the loss of Git2, we found that miR-146a maturation is enhanced, which in turn promotes the expression of Zeb1 andinduction of EMT. Furthermore, we found that Cnot6L, a validated target of miR-146a, affects the stability of Zeb1 mRNA through its
deadenylase activity. Our results present evidence for a new role for loss of Git2 in promoting EMT through a novel regulatory pathway.
Key words: Git2, miR-146a, EMT, Zeb1
IntroductionEpithelial–mesenchymal transition (EMT) (Thiery and Sleeman,
2006; Thiery et al., 2009; Sleeman and Thiery, 2011) is an
evolutionarily conserved process during morphogenesis that has
more recently been implicated as a driving force promoting the
local invasion and distant dissemination of carcinoma (Thiery,
2002; Kalluri and Weinberg, 2009). Git2 is a member of the GIT
protein family characterized by an N-terminal GTPase-activating
protein domain (Sabe et al., 2006). Git2 show GTPase-activating
activity toward Arf1 and Arf6, which influences membrane
traffic and actin remodelling (Randazzo et al., 2007). Git2
interacts with many molecules, such as G protein-coupled
receptor kinases, PIX [p21-activated kinase (PAK)-interacting
exchange factor] (Bagrodia et al., 1999) and paxillin (Turner
et al., 1999), and serve a pivotal role in focal adhesion
disassembly (Hoefen and Berk, 2006), cell polarity and the
directional motility of migrating adherent cells (Frank et al.,
2006) and neutrophils (Mazaki et al., 2006). Although Git2 is
crucial in the regulation of the migration, adhesion and polarity
of nonhematopoietic cells, part of the function of Git2 may stem
from their ability to repress Rac activation. For example, Git2
represses Rac-dependent lamellipodia extension and cell
spreading in HeLa human cervical cancer cells (Frank et al.,
2006) and in migrating adherent cells (Nishiya et al., 2005).
Because migration, adhesion and polarity are fundamental for
EMT, it is possible that the Git2 has a role in governing EMT.
Here we report loss of Git2 induces EMT by promoting the
expression of Zeb1. We found that miR-146a maturation was
regulated by Git2. Moreover, Cnot6L, a validated target of miR-
146a, affects the stability of Zeb1 mRNA through its deadenylase
activity.
ResultsGit2 is required to maintain the epithelial state inepithelial cells
After screening of a small library of siRNA that could potentially
interfere with EMT of the bladder carcinoma cell line NBT-II,
Git2 was interestingly identified because epithelial morphology
of NBT-II was lost in Git2 siRNA cells (data not shown). To
validate this observation, NBT-II cells were infected with a
lentivirus coding a short hairpin RNA (shRNA) targeting Git2 to
obtain stable clones. Knockdown efficiency tests, as determined
by western blotting, identified clone #2 as the most effective, and
it was subsequently used in this study (Fig. 1A). To test whether
Git2 shRNA was specific to knock down Git2, exogenous Git2
expression was enforced into Git2 knockdown (Git2 KD) cells.
The western blotting results demonstrated that Git2 expression
was complemented by exogenous Git2 (Fig. 1B). NBT-II cells
form epithelial-like compact colonies, with well-defined cell
contacts. However, individual cells in small colonies exhibit
lamellipodial activity at the free edge, inducing rotation of the
colony (Fig. 1C; supplementary material Movie 1). Transduced
NBT-II cells, with lentiviral constructs expressing shRNA
targeting Git2, exhibited an elongated, mesenchymal-like
morphology with modest motility and transient contact with
neighbouring cells (Fig. 1C; supplementary material Movie 2).
Wound healing assay (Fig. 1D) and transwell (Boyden chamber)
cell migration assay (supplementary material Fig. S2B) showed
that silencing of Git2 promoted the migration ability of NBT-II in
vitro. Moreover, the epithelial morphology was rescued when
Git2 was re-expressed into Git2 KD cells (Fig. 1C). Git2 is thus
essential for the maintenance of the epithelial state of NBT-II
cells. We next sought to test whether Git2 played a similar role in
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other epithelial cell lines. Two carcinoma epithelial cell lines,HepG2 and T47D, and one normal epithelial cell line, EpH4 were
selected to test the role of Git2. As anticipated, Git2 knockdownresulted in the loss of compaction of the epithelial colonies withthe most pronounced change in morphology in EpH4 cells
(Fig. 1E).
The miR-146a is augmented in Git2 KD cells
We next performed expression profiling of control and Git2 KDNBT-II cells to identify potential key regulators induced by Git2KD. Overall, 33 genes were downregulated and 15 genes were
upregulated in Git2 KD cells (supplementary material Table S1).Some of the identified genes, such as Cdh1 (E-cadherin), Vim
(vimentin), MMPs and Zeb1 are landmark genes in EMT (Thieryand Sleeman, 2006; Gregory et al., 2008). To confirm themicroarray results, the mRNA and protein levels of Cdh1, Vimand Zeb1 were further assessed by real-time quantitative PCR
and western blotting. The transcriptional and protein levels ofCdh1 were significantly reduced in Git2 KD cells, whereasVim and Zeb1 were, remarkably, increased (Fig. 2A).
Immunostaining of E-cadherin showed that the expression andlocation of E-cadherin were affected by depletion of Git2.Exogenous expression of Git2 in Git2 KD cells reversed the Git2
expression and location to control status (supplementary materialFig. S3A). Together, these results indicated that the loss of Git2repressed epithelial gene expression and promoted EMT.
Zeb1 is a well-established inducer of EMT (Gregory et al.,
2008), and here we show that Git2 inhibition increased Zeb1expression. Meanwhile, the expression of other EMT-relatedtranscriptional factors Snail2 and Twist1 was tested in control,
ShRNA control and Git2 KD cells. Silencing of Git2 didn’tchange the protein level of Snail2 or Twist1 (supplementarymaterial Fig. S2A). As such, we hypothesized that Git2 lies
upstream of Zeb1 in EMT. We speculated that the regulation ofZeb1 by Git2 might be controlled by microRNAs, since somemiRNAs have been identified as regulators of Zeb1 expression
during EMT (Gregory et al., 2008; Brabletz and Brabletz, 2010;Reshmi et al., 2011). Expression profiling of microRNAsrevealed the dramatic augmentation of miR-146a in Git2 KDcells (supplementary material Table S2; Fig. 2B), which was
further confirmed with real-time quantitative PCR (Fig. 2C). InGit2 KD cells, we noticed an increase in only the mature form ofmiR-146a, while pre-miRNA-146a transcripts remained stable
(supplementary material Table S2 microRNA microarray). Wethus suspected that Git2 affects the maturation of miR-146a. Thisnotion was confirmed via real-time PCR quantification of
precursor (pre-mir-146a) and mature miR-146a (miR-146a)transcripts in Git2 KD cells (Fig. 2D, ‘G’, green bars).Moreover, this increase in mature miR-146a transcripts wasreduced when Git2 KD cells were forced to express exogenous
Git2 (Fig. 2D, ‘G+Exo’, black bars). In NBT-II control cells, themature miR-146a was downregulated by almost 50% whenoverexpressing Git2 (Fig. 2E, ‘OE’). However, neither the
enforced Git2 expression in Git2 KD cells nor theoverexpression Git2 in control cells influenced the RNA levelof pre-miR-146a (Fig. 2D,E). Thus, only the maturation of miR-
146a was regulated by Git2.
Arf-GAP activity of Git2 is indispensable for the maturationof miR-146a
Git2 is an Arf GTPase-activating protein (GAP) belonging to theRas superfamily of small GTPases. Arf proteins cycle betweentheir active-GTP-bound and inactive-GDP-bound conformations.
Hydrolysis of bound GTP is mediated by GAP (D’Souza-Schoreyand Chavrier, 2006). The GAP activity of Arf-GAP ensure Arf-GAP bind to activated Arf (Arf-GTP) and stimulate their GTPase
activity. To evaluate the Arf-GAP activity of Git2 on theinfluence of miR-146a maturation, we firstly compared thedifference of Arf-GTP-bound form between Git2 KD and control
cells. GTP-bound Arf were captured by immobilized specific Arfeffector GGA3 (Golgi-associated, gamma adaptin ear containing,Arf binding protein 3) (Hafner et al., 2006). Enhanced levels of
Fig. 1. Git2 is required to maintain the epithelial state in NBT-II cells.
(A) Lentiviral particles expressing Git2 shRNA (constructs #1–5) were used
to infect NBT-II cells. A stable knockdown Git2 cell line was selected with
2.5 mg/ml puromycin. Git2 protein levels were measured by western blotting.
Beta-actin was used as a control. (B) The exogenous Git2 expression was
enforced into Git2 knockdown (Git2 KD) cells. The protein level of Git2 was
measured by western blotting (C, control; SC, shRNA control; G, Git2
shRNA; G+Exo, Git2 shRNA+exogenous Git2). (C) The morphology was
changed in Git2 knockdown (Git2 KD) cells (3), which could be rescued with
forced Git2 expression in Git2 KD cells (4). Scale bars: 15 mm. (D) ShRNA
control cells and Git2 shRNA-transfected NBT-II cells were seeded into a 24-
well tissue culture plate and used for a scratch wound healing assay. Images
were taken immediately after scratching (T0) and 24 hours after scratching
(T24). The silencing of Git2 promoted the migration ability of NBT-II in
vitro. (E) Three other cell lines were infected with lentiviral shRNA particles.
Loss of Git2 resulted in the acquisition of a mesenchymal-like phenotype.
Scale bars: 15 mm.
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GTP-bound Arf proteins were precipitated in NBT-II Git2 KD
cells as compared with control and shRNA control cells (Fig. 3A,
middle panel), suggesting that depletion of Git2 failed tostimulate GTPase activity of Arf and then favours the Arf-
GTP-bound form. Next, we employed wild-type (WT) Git2 and
GAP-inactive C11A mutant Git2 (Cukierman et al., 1995) into
Git2 KD NBT-II control cells. Enforced expression of the C11A
mutant, in which the critical cysteine residue for the GAP
activity, cysteine 11, was mutated to alanine to diminish the GAP
activity, unsuccessfully inhibited the mature miR-146a (Fig. 3B).
In addition, silencing of Arf6 rescued the effect of Git2 KD in
NBT-II cells. The proceeding of EMT and the synthesis of miR-146a were reduced when introducing Arf6 siRNA in Git2 KD
cells (supplementary material Fig. S1A,B). These indicate that
Arf-GAP activity of Git2 is indispensable for the maturation of
miR-146a. To further investigate the role of miR-146a, we
blocked miR-146a with a miR-146a antagomir in Git2 KD NBT-
II cells. The inhibitory efficiency was determined by real-time
PCR (supplementary material Fig. S4A). The miR-146a
antagomir successfully reverted Git2 KD NBT-II cells to an
epithelial morphology (Fig. 3C), re-suppressed the protein levelof Zeb1 and Vim and rescued the expression of Cdh1 (Fig. 3D).
This suggests miR-146a may act as a significant promoter of
EMT, in contrast to the suppressive role of miR200 family and
miR205.
Cnot6L controls the stability of Zeb1
MicroRNAs are small, non-coding RNAs that modulate gene
expression by targeting specific mRNA (Gregory et al., 2008). Toidentify the direct targets of miR-146a, we searched for the
predicted targets using the Targetscan database (http://www.
targetscan.org). We found that putative target genes, Robo1,Cds1 and Cnot6L, were also present in the downregulated gene
list (supplementary material Table S1). Following forcedexpression of Robo1, Cnot6L and Cds1 separately in Git2 KD
NBT-II cells (supplementary material Fig. S3B), we revealed that
only overexpressed Cnot6L could reduce Zeb1 protein levels inGit2 KD cells (Fig. 4A). To determine whether Cnot6L was the
direct target of miR-146a, the PCR products containing mutationof miR-146a seed recognition sequences in the 39-UTR Cnot6L
(Fig. 4C) and WT 39-UTR Cnot6L were inserted into the
luciferase reporter vector. The relative luciferase activity of 39-UTR Cnot6L was reduced in Git2 KD cells. However, the effect
was abolished by mutating the putative miR-146a binding siteswithin the 39-UTR of Cnot6L (Fig. 4B). Moreover, the inhibitory
effect was removed when introducing the inhibitor (antagomir for
miR-146a) to block miR-146a (Fig. 4B). The miR-146a antag-omir failed to change Git2 protein levels, but rescued Cnot6L
expression and repressed Zeb1 expression (Fig. 4D). In addition,NBT-II cells with enforced miR-146a expression inhibited the
expression of Cnot6L, Cdh1 and induced the expression of Vim
Fig. 2. miR-146a is augmented in Git2 KD cells. (A) The
mRNA and protein levels of Zeb1, Vim and Cdh1 were
measured in Git2-depleted and control NBT-II cells. All
experiments were repeated three times. (B) The miRNA
Affymetrix microarray revealed that miR-146a was dramatically
augmented in Git2 KD cells. (C) Quantitative real-time PCR
showed that miR-146a, rather than miR-200 family members,
were significantly increased in Git2 KD cells. All experiments
were repeated three times. (D) Exogenous Git2 was forced to
express in Git2 KD cells (G+Exo). The relative RNAs level of
precursor miR-146a and maturation miR-146a were measured.
All experiments were repeated three times. (E) The maturation
miR-146a was downregulated when overexpressing Git2 in
NBT-II control cells. All experiments were repeated three times.
SC, shRNA control; G, Git2 shRNA; G+Exo, Git2
shRNA+exogenous Git2.
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and Zeb1 (Fig. 4E). These data demonstrated that Cnot6L was a
direct target of miR-146a, and that Git2 regulates Zeb1 through
Cnot6L.
Cnot6L, the catalytic subunit of the Ccr4b–Not complex, is a
key enzyme ensuring deadenylation-dependent degradation of
specific mRNAs (Morita et al., 2007). Northern blot analysis was
carried out to test whether the deadenylase activity of Cnot6L
influenced the stability of Zeb1 mRNA. In Git2-depleted cells,
Zeb1 mRNA was prominently increased, as before. In agreement
with the protein levels in Fig. 4A, forced expression of Cnot6L
decreased Zeb1 mRNA to control levels (Fig. 5A). The
deadenylation and decay of Zeb1 mRNA were monitored by
Northern blotting analysis after transiently transfected with a
gene encoding Cnot6L in Git2 KD NBT-II cells. The amounts of
Zeb1 mRNA isolated at time intervals after transient transfection
showed a gradient reduction (Fig. 5B). To confirm that Cnot6L
affected the stability of Zeb1 mRNA, we next examined the half-
life of Zeb1 mRNA. NBT-II cells were treated with actinomycin
D to inhibit de novo transcription to determine the Zeb1 mRNA
levels at various time points after treatment (supplementary
material Fig. S4B). We found that the rate of decline in Zeb1
transcript levels was lower in Git2-depleted cells than in control
cells, and that re-expression of Cnot6L accelerated the rate of
decline (Fig. 5C). All together, the loss of Git2 seems to induce
the maturation of miR-146a. Increased amounts of the mature
form of miR-146a reduce the levels of Cnot6L, thus preventing
Zeb1 mRNA destabilization. This newly uncovered pathway
results in the execution of the EMT program (Fig. 5E).
DiscussionIn this report, we are unexpected to find that Arf-GAP protein
Git2 is involved into EMT through miR-146a mediated Zeb1
pathway. In contrast to the suppressive role of miR200 family
and miR205, mature miR-146a seems to induce EMT. Our data
demonstrate that Arf-GAP activity of Git2 is necessary for the
maturation of miR-146a. Many proteins have been shown to
regulate the maturation of miRNAs, such as SMAD (Davis et al.,
2008) and small GTPase, Ran (Lund et al., 2004). The classic
Fig. 3. Arf-GAP activity of Git2 is indispensable for the maturation of
miR-146a. (A) The immobilized GST-GGA3-PBD beads were used to
capture GTP-bound ARF. Much more ARF-GTP proteins were pulled down
in NBT-II Git2 KD cells than in control cells. (B) Git2 WT and GAP-inactive
C11A mutant were transfected into Git2 KD cells. Relative miR-146a levels
were detected. C11A mutant was incapable of reversing the RNA level of
miR-146a. (C) The miR-146a antagomir could rescue the altered cell
phenotype (2) to an epithelial cell phenotype (3) in cells treated with
Git2 shRNA. Scale bars: 15 mm. (D) The inhibitory effect of miR-146a on
Cnot6L was abrogated when applying an antagomir to miR-146a. Expression
of Vim, Zeb1 and Cnot6L reverted to control levels. (C, control; SC, shRNA
control; G, Git2 shRNA; G+antagomir, Git2 shRNA+miR-146a antagomir;
WT, wild type).
Fig. 4. Cnot6L controls the stability of Zeb1. (A) Robo1, Cds1 and
Cnot6L cDNAs were transfected into Git2 KD cells. The overexpression
(OE) of Cnot6L inhibited the expression of Zeb1. V, vector. (B) PCR
products containing mutation of miR-146a seed recognition sequences in
the 39-UTR Cnot6L (white bars) and wild type 39-UTR Cnot6L (black
bars) were inserted into the luciferase reporter vector. The relative
luciferase activity of wild type 39-UTR Cnot6L was reduced in Git2 KD
cells. The inhibitory effect was abolished by mutating the putative miR-
146a binding sites within the 39-UTR of Cnot6L. An inhibitor (antagomir
for miR-146a) was introduced into Git2 KD cells to block miR-146a
(G+146a). The miR-146a antagomir reversed relative luciferase activity
of wild type 39-UTR Cnot6L. All experiments were repeated three times.
(C) A scheme to show the construct of wild type and mutant 39-UTR
Cnot6L luciferase reporter system. (D) An antagomir to miR-146a was
applied to block miR-146a (G+146a). The inhibitory effect of miR-146a
on Cnot6L was abrogated. Expression of Zeb1 and Cnot6L were reverted
to control levels. (E) NBT-II cells were transiently transfected with
pcDNA3 vector (V) or expression plasmids for miR-146a (miR-146a).
The expression of Git2, Cnot6L and EMT markers were detected by
western blotting.
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mechanism to regulate the maturation of miRNA includes
nuclear Drosha (Lee et al., 2003) and cytoplasmic Dicer (Lee
et al., 2002). Our evidence points to the Git2-regulated
maturation of miR-146a. However, how Git2 regulate the
maturation of miR-146a? Is it mediated by Drosha or Dicer or
other proteins? These questions were required to be further
investigated.
Epithelial cells are converted into mesenchymal cells by EMT,
a mechanism hypothesized to play a key role in cancer invasion
and metastasis (Thiery and Sleeman, 2006). Traditionally, EMT
can be induced by various growth factors and their downstream
molecules. However, the three different growth factors tested
here successfully increased the expression of Zeb1, but failed to
decrease Git2 expression (Fig. 5D). Moreover, Loss of Git2
didn’t change the RNA level of miR-200 family and miR-205
(Fig. 2C; supplementary material Fig. S3C). This suggests that
there are additional transduction pathways for the fine-tuned
control of Zeb1-mediated EMT. One argument against the role of
EMT in cancer progression is that metastases exhibit phenotypes
similar to their corresponding primary tumours (Tarin et al., 2005;
Christiansen and Rajasekaran, 2006). However, there is increasingevidence to indicate that EMT is not irreversible (Guo et al., 2012;Ocana et al., 2012). The reverse process – mesenchymal to
epithelial transition (MET) – is vital for the completion ofmorphogenesis and during the final stages of differentiation for thetissue anlage (Lim and Thiery, 2011; Sleeman and Thiery, 2011).However, how EMT cells switch back to an epithelial phenotype
through MET is unclear. It is noteworthy that most carcinomaexhibit intermediate phenotypes that exhibit epithelial cellplasticity that is more likely to be responsible for the transiting
between EMT and MET. The newly identified miR-146a mediatedZeb1 pathway, compensating for miR-200 mediated Zeb1pathway, provides the possibility of flexible transition between
EMT and MET. Besides, given that Arf-GAP protein Git2 isproved to be involved in EMT, we propose a model that Arfproteins, cycling between their active-GTP-bound and inactive-
GDP-bound conformations, connect the balance of GAP and GEFto the switch between EMT and MET (Fig. 5F).
Materials and MethodsCell lines and antibodies
NBT-II, HepG2, T47D and EpH4 cells (ATCC, Manassas, VA, USA) were maintainedin complete DME (10% FBS) with antibiotics at 37 C and with 5% CO2. Antibodies andreagents were obtained from the following sources: anti-Git2 (#SAB4503703) and anti-Cnot6L (#HPA042688) antibodies (Sigma–Aldrich, St. Louis, MO, USA); anti-Zeb1antibody (#SC-10572; Santa Cruz Biotechnology Inc., Santa Cruz, CA); anti-Vimantibody (550513; BD PharmingenTM, San Diego, CA, USA); anti-Cdh1 antibody(#610182; BD Biosciences, Franklin Lakes, NJ, USA); anti-beta actin antibody(#ab8227; Abcam, Cambridge, MA, USA); anti-Snail2 antibody (#WH0006591M5;Sigma–Aldrich, St. Louis, MO, USA); anti-Twist1 antibody (#LS-C30601-100;LifeSpan BioSciences Company). As secondary antibodies, HRP-conjugated donkey-anti goat (Sigma, Poole, UK), goat-anti rabbit or goat-anti mouse antibodies (GEHealthcare, Chalfont St. Giles, UK) were used. Detection was performed usingWestpico Chemiluminescence substrate (Thermo Scientific).
Lentiviral shRNA
Git2 shRNA (#SC-40637-V) and shRNA control lentiviral particles (#SC-108080;Santa Cruz Biotechnology Inc.) (10 ml) were mixed with 1 ml 10 mg/ml polybrene(#H9268; Sigma–Aldrich) and diluted into 1 ml Dulbecco’s Modified EagleMedium (DMEM) without foetal bovine serum (FBS). The diluted medium withlentiviral particles were added to cells seeded into 24-well plates. 24–48 hourslater, the medium was replaced by DMEM with 10% FBS and 1% penicillin–streptomycin followed by positive selection using 2.5 mg/ml puromycin(#A11138-02; Invitrogen, Carlsbad, CA, USA).
Wound healing assay
NBT-II cells and Git2 KD NBT-II cells were seeded into 24-well tissue cultureplate in a density ,70–80% confluence as a monolayer. Gently and slowly scratchthe monolayer with a new 1 ml pipette tip across the center of the well. Afterscratching (T0), gently wash the well twice with medium to remove the detachedcells. Replenish the well with fresh medium. Grow cells for additional 24 hours(T24). Wash the cells twice with 16PBS. Take photos on a microscope. Eachexperimental group were repeated three times.
Transwell cell migration assay
Control, shRNA control and Git2 shRNA cells were incubated in upper layer ofBoyden chamber coated with matrigel for 24 hours. After incubation, cells onmembrane were washed with PBS three times. Images were taken undermicroscope for 20 times magnification. All experiments were performed understerile conditions.
Live cell imaging
Git2 KD and control NBT-II cells were seeded in 12-well plates. Live cell imagingwas acquired by Zeiss Axiovert 200M Cell Imaging System (Zeiss Microimaging,Thornwood, NY), with a 206objective, and exposure times of 40 ms and 5-minuteintervals.
cDNA and plasmid transfection
cDNAs were obtained from Origene (Rockville, MD, USA): Git2 cDNA(#RG214925), Cnot6l cDNA (#RG219766), Robo1 cDNA (#SC109740), and
Fig. 5. (A) Cnot6L cDNAs were forced to express in Git2 KD cells
(G+Cnot6L). Zeb1 RNA levels were analysed by northern blotting. 28S was
used as a control. Zeb1 mRNA was increased in Git2 KD cells. Forced
expression of Cnot6L decreased Zeb1 mRNA to control levels. (B) Git2 KD
cells were transiently transfected with a gene encoding Cnot6L. The amounts
of Zeb1 mRNA were monitored by northern blotting analysis at time intervals
after transient transfection as indicated. (C) Cells were treated with
actinomycin D to inhibit de novo transcription. Zeb1 mRNA levels were
determined by real time PCR at various time points after treatment. The rate
of decline in Zeb1 transcript levels was lower in Git2-depleted cells than in
control cells. Re-expression of Cnot6L (G+Cnot6L) accelerated the rate of
decline. (D) Three different growth factors (HGF, hepatocyte growth factor;
PDGF, platelet-derived growth factor; TGFb, transforming growth factor-b)
were used to induce EMT. Git2 and Zeb1 protein expression were measured
after 24 hours of stimulation. Ctr, control. (E,F) Schemes to describe the
mechanism of Git2 regulating EMT (E) and a possible connection between
the EMT/MET switch and the Arf-GDP/GTP switch (F).
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Cds1 cDNA (#SC119345). cDNAs (2 mg) were mixed with 3 ml FuGENER 6Transfection Reagent (#E2691; Promega, Madison, WI, USA) and incubated for15 minutes at room temperature. The mixture was added to NBT-II or Git2 KDNBT-II as indicated. The transfected cells were selected in the presence of 600 mg/ml G418 (Invitrogen, #10131-035), and resistant clones were further confirmed bywestern blotting.
The miR-146a antagomir construct (#RmiR-AN0196 am02; GeneCopoeia,Rockville, MD, USA) was transfected into Git2 KD NBT-II cells as describedabove. The transfected cells were harvested and washed in PBS containing 0.2%BSA. Cells (16105 cells per sample) were sorted on a FACSAria II (BDBiosciences) cell sorter equipped with a 594-nm laser.
NBT-II cells were transiently transfected with pcDNA3 vector or expressionplasmids for miR-146a (Addgene plasmid 15902). 48 hours later, cells were lysedby RIPA lysis buffer. Whole proteins were collected and applied for westernblotting.
Git2 KD cells were transfected with 2 Arf6 siRNA (#SI00268100 #SI03054947;Qiagen). 48 hours later, cells were lysed by RIPA lysis buffer. Whole proteinswere collected and applied for western blotting.
Quantitative real-time PCR
For Cdh1, Vim and Zeb1Total RNAs were extracted by Trizol (#15596-026; Invitrogen) andcomplementary DNA (cDNA) was synthesized by reverse transcription kit(#205310; Qiagen). Real-time PCR was subsequently performed in triplicatewith a 1:4 dilution of cDNA using the SYBRH Green RT-PCR Reagents Kit. Thecomparative Ct method was used to compute relative expression values. AllmRNA quantification data were normalized to GAPDH.
For mature miR-146a and pre-mir-146aTotal miRNA was extracted using miRNA Iso Kit (#AM1560; AppliedBiosystems, Foster City, CA, USA), according to the manufacturer’sinstructions. MiRNAs were reversed transcribed with TaqmanH microRNA RTKit (#4366596; Applied Biosystems). Real-time PCR was subsequently performedin triplicate using Taqman kits for mature miR-146a (#4427975), pre-mir-146a(#4427012) and miR-200a (#4378069; Applied Biosystems). Data were collectedand analysed using the Rotor-gene software. Fold changes of miR-146a and miR-200a were normalized to control cells. The miR-146a and pre-mir-146aquantification data were normalized to miR-200a.
Microarray analysis
For Git2 microarray analysisTotal RNA samples from Git2 KD and control NBT-II cells were isolated using theRNA isolation kit (#74104; Qiagen) according to the manufacturer’s protocol.Total RNA (100 ng) was reverse transcribed to produce cDNA/mRNA hybridmolecule, which was subsequently used as a template to create double strandedcDNA with a unique DNA/RNA heteroduplex at one end. The cDNA was thenamplified via SPIA (Single Primer Isothermal Amplification), which producessingle stranded anti-sense DNA. Post-SPIA modification generates sense targetcDNA that was fragmented, biotin labeled and hybridized to Affymetrix Rat Gene1.0 ST arrays for 18 hours at 45 C rotated at 60 rpm (Affymetrix, Santa Clara, CA,USA). Arrays were then washed and stained using the FS450_0007 fluidicsprotocol and scanned using an Affymetrix 3000 7G scanner.
For miRNA microarray analysisTotal RNA samples from Git2 KD and control NBT-II cells were isolated by usingthe miRNeasy Mini Kit (#217004; Qiagen) according to the manufacturer’sprotocol. Genisphere FlashTag Biotin HSR labeling techniques was utilized tohybridize samples to Affymetrix miRNA 2.0 Arrays. The miRNA 2.0 Arrayprovides probe sets for 131 species including rat, and comprises more than 15,000unique probe sets for maturation miRNA based on Sanger miRBase (v.15), over2,000 pre-miRNA sequences (Affymetrix). Mature miRNAs are assigned the‘rno_’ prefix and pre-miRNAs ‘hp_ rno_’ as displayed in supplementary materialTable S2.
39-UTR luciferase reporter analysis
The Cnot6l luciferase reporters were purchased from Applied Biological Materials,Inc. (Richmond, BC, USA). The mutant construct with a mutation of the miR-146aseed sequence was generated with the mutagenic oligonucleotide primers (F: 59-AUACUUUCUAGAACUCAGAAUAA R: 59-AUGAGAGAAAAUUUGGGCA-UU), according to the manual of GeneTailor Site-Directed Mutagenesis System(Invitrogen). Cells were co-transfected with each reporter construct and the Renillaluciferase vector pRL-TK (#E2241; Promega), and incubated with passive lysisbuffer, according to the dual-luciferase assay manual. The luciferase activity wasmeasured with a luminometer (Lumat LB9507, Berthold Tech., Bad Wildbad,Germany). The firefly luciferase signal was normalized to the Renilla luciferasesignal for each individual analysis. All experiments were performed in triplicatewith data pooled from at least two independent experiments.
Northern blot analyses
Total RNAs were isolated with ISOGEN according to the manufacturer’s protocol(Nippon Gene, Tokyo, Japan). Northern blot analyses were carried out as describedpreviously (Yoshida et al., 2000). To prepare the probes, DNA fragments of Zeb1 and28S were amplified by PCR. The probes were labelled with [a-32P] dCTP by a random-prime labelling system and were hybridized at 65 C for 2 hours in ExpressHybHybridization Solution (Clontech Laboratories, Inc. Mountain View, CA, USA).
Assays of mRNA stability
Git2 KD cells were transiently transfected with Cnot6L cDNA plasmids, and totalRNA was extracted at the indicated time points and analysed by Northern blotting. Thelevel of Zeb1mRNA was monitored by Northern blotting. 28S was used as control.
Half-life assay
ShRNA control, Git2 KD and Cnot6L overexpressed Git2 KD cells were treatedwith actinomycin D (2.5 mg/ml). Total RNAs were extracted by Trizol (#15596-026; Invitrogen) and complementary DNA (cDNA) was synthesized by reversetranscription Kit (#205310; Qiagen). Real-time PCR was subsequently performedat various time points after actinomycin D treatment in triplicate with a 1:4 dilutionof cDNA using the SYBRH Green RT-PCR Reagents Kit.
Fluorescent staining of E-cadherin
ShRNA control, Git2 KD and Git2 exogenous Git2 KD cells were 100% methanolfixed (5 minutes) and then incubated in 1%BSA/10% normal goat serum/0.3 Mglycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein–protein interactions. The cells were then incubated with the E-cadherin antibody (#ab11512) at 5 mg/ml overnight at +4 C. The secondary antibody(green) was Alexa FluorH 488 goat anti-rat IgG (H+L) used at a 1/1000 dilution for1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 mM.
Pull down assay
Arf6-GTP pull down assay was carried out according to the manual (#16122;Pierce Biotechnology, Rockford, IL, USA). Briefly, Git2 KD cells, shRNA controlcells and control NBT-II cells were washed with PBS at 4 C, harvested into 500 mllysis buffer (200 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1% TritonX-100, 0.1% SDS, 0.5% deoxycholate, 5% glycerol, 1 mM phenylmethylsulfonylfluoride, 9 nM pepstatin, 9 nM antipain, 10 nM leupeptin, and 10 nMchymostatin). Cell extracts were incubated with 30 mg of GST-GGA3 or GSTimmobilized on glutathione-Sepharose for 1 hour at 4 C. The pellets were washedthree times with lysis buffer. Bound proteins were eluted by 30 ml elution buffer.The reactions were analysed by immunoblotting with an ARF6-specificmonoclonal antibody.
Western blotting
Total cell lysates (40 mg of protein) were separated by sodium dodecyl sulfate-PAGE (SDS-PAGE) in 8% gels and electrotransferred on to nitrocellulosemembranes. After blocking with 5% skim milk, the membranes were probed withprimary antibodies, respectively, at 4 C overnight (dilution, 1:1000). Themembrane was incubated with appropriate peroxidase-labelled secondaryantibodies and developed by Super Signal chemiluminescence substrate(#34077; Pierce Biotechnology). Beta-actin protein levels were used as a controlfor adequacy of equal protein loading.
Statistics
All data were expressed as mean6s.e.m. Statistical analysis was performed usingthe Student’s t-test as indicated.
AcknowledgementsWe thank Dr Chu Yeh-shiu for support with microscopy; Dr JormayLim for help with Targetscan; Dr Jing Ma and Sim Wen Jing for helpwith cell culture. We thank Prof. Hisataka Sabe for the C11A mutantplasmid. The authors confirm that they have no competing financialinterests.
Author contributionsZ.W. designed and performed the experiments. J.P.T. wrote thearticle. Both authors read and approved the final manuscript.
FundingThis work was supported by A*STAR core funding.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.126367/-/DC1
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ReferencesBagrodia, S., Bailey, D., Lenard, Z., Hart, M., Guan, J. L., Premont, R. T., Taylor,
S. J. and Cerione, R. A. (1999). A tyrosine-phosphorylated protein that binds to animportant regulatory region on the cool family of p21-activated kinase-bindingproteins. J. Biol. Chem. 274, 22393-22400.
Brabletz, S. and Brabletz, T. (2010). The ZEB/miR-200 feedback loop – a motor ofcellular plasticity in development and cancer? EMBO Rep. 11, 670-677.
Christiansen, J. J. and Rajasekaran, A. K. (2006). Reassessing epithelial tomesenchymal transition as a prerequisite for carcinoma invasion and metastasis.Cancer Res. 66, 8319-8326.
Cukierman, E., Huber, I., Rotman, M. and Cassel, D. (1995). The ARF1 GTPase-activating protein: zinc finger motif and Golgi complex localization. Science 270,1999-2002.
D’Souza-Schorey, C. and Chavrier, P. (2006). ARF proteins: roles in membrane trafficand beyond. Nat. Rev. Mol. Cell Biol. 7, 347-358.
Davis, B. N., Hilyard, A. C., Lagna, G. and Hata, A. (2008). SMAD proteins controlDROSHA-mediated microRNA maturation. Nature 454, 56-61.
Frank, S. R., Adelstein, M. R. and Hansen, S. H. (2006). GIT2 represses Crk- andRac1-regulated cell spreading and Cdc42-mediated focal adhesion turnover. EMBO J.
25, 1848-1859.Gregory, P. A., Bert, A. G., Paterson, E. L., Barry, S. C., Tsykin, A., Farshid, G.,
Vadas, M. A., Khew-Goodall, Y. and Goodall, G. J. (2008). The miR-200 familyand miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 andSIP1. Nat. Cell Biol. 10, 593-601.
Guo, W., Keckesova, Z., Donaher, J. L., Shibue, T., Tischler, V., Reinhardt, F.,Itzkovitz, S., Noske, A., Zurrer-Hardi, U., Bell, G. et al. (2012). Slug and Sox9cooperatively determine the mammary stem cell state. Cell 148, 1015-1028.
Hafner, M., Schmitz, A., Grune, I., Srivatsan, S. G., Paul, B., Kolanus, W., Quast,T., Kremmer, E., Bauer, I. and Famulok, M. (2006). Inhibition of cytohesins bySecinH3 leads to hepatic insulin resistance. Nature 444, 941-944.
Hoefen, R. J. and Berk, B. C. (2006). The multifunctional GIT family of proteins.J. Cell Sci. 119, 1469-1475.
Kalluri, R. and Weinberg, R. A. (2009). The basics of epithelial-mesenchymaltransition. J. Clin. Invest. 119, 1420-1428.
Lee, Y., Jeon, K., Lee, J. T., Kim, S. and Kim, V. N. (2002). MicroRNA maturation:stepwise processing and subcellular localization. EMBO J. 21, 4663-4670.
Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark,O., Kim, S. et al. (2003). The nuclear RNase III Drosha initiates microRNAprocessing. Nature 425, 415-419.
Lim, J. and Thiery, J. P. (2011). Alternative path to EMT: regulation of apicobasalpolarity in Drosophila. Dev. Cell 21, 983-984.
Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E. and Kutay, U. (2004). Nuclearexport of microRNA precursors. Science 303, 95-98.
Mazaki, Y., Hashimoto, S., Tsujimura, T., Morishige, M., Hashimoto, A., Aritake,
K., Yamada, A., Nam, J. M., Kiyonari, H., Nakao, K. et al. (2006). Neutrophildirection sensing and superoxide production linked by the GTPase-activating proteinGIT2. Nat. Immunol. 7, 724-731.
Morita, M., Suzuki, T., Nakamura, T., Yokoyama, K., Miyasaka, T. and
Yamamoto, T. (2007). Depletion of mammalian CCR4b deadenylase triggerselevation of the p27Kip1 mRNA level and impairs cell growth. Mol. Cell. Biol. 27,4980-4990.
Nishiya, N., Kiosses, W. B., Han, J. and Ginsberg, M. H. (2005). An alpha4 integrin-paxillin-Arf-GAP complex restricts Rac activation to the leading edge of migratingcells. Nat. Cell Biol. 7, 343-352.
Ocana, O. H., Corcoles, R., Fabra, A., Moreno-Bueno, G., Acloque, H., Vega, S.,
Barrallo-Gimeno, A., Cano, A. and Nieto, M. A. (2012). Metastatic colonizationrequires the repression of the epithelial-mesenchymal transition inducer Prrx1.Cancer Cell 22, 709-724.
Randazzo, P. A., Inoue, H. and Bharti, S. (2007). Arf GAPs as regulators of the actincytoskeleton. Biol. Cell 99, 583-600.
Reshmi, G., Sona, C. and Pillai, M. R. (2011). Comprehensive patterns in microRNAregulation of transcription factors during tumor metastasis. J. Cell. Biochem. 112,2210-2217.
Sabe, H., Onodera, Y., Mazaki, Y. and Hashimoto, S. (2006). ArfGAP family proteinsin cell adhesion, migration and tumor invasion. Curr. Opin. Cell Biol. 18, 558-564.
Sleeman, J. P. and Thiery, J. P. (2011). SnapShot: The epithelial-mesenchymaltransition. Cell 145, 162 e161.
Tarin, D., Thompson, E. W. and Newgreen, D. F. (2005). The fallacy of epithelialmesenchymal transition in neoplasia. Cancer Res. 65, 5996-6000.
Thiery, J. P. (2002). Epithelial-mesenchymal transitions in tumour progression. Nat.
Rev. Cancer 2, 442-454.
Thiery, J. P. and Sleeman, J. P. (2006). Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev. Mol. Cell Biol. 7, 131-142.
Thiery, J. P., Acloque, H., Huang, R. Y. and Nieto, M. A. (2009). Epithelial-mesenchymal transitions in development and disease. Cell 139, 871-890.
Turner, C. E., Brown, M. C., Perrotta, J. A., Riedy, M. C., Nikolopoulos, S. N.,
McDonald, A. R., Bagrodia, S., Thomas, S. and Leventhal, P. S. (1999). PaxillinLD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAPprotein: A role in cytoskeletal remodeling. J. Cell Biol. 145, 851-863.
Yoshida, Y., Tanaka, S., Umemori, H., Minowa, O., Usui, M., Ikematsu, N., Hosoda,
E., Imamura, T., Kuno, J., Yamashita, T. et al. (2000). Negative regulation ofBMP/Smad signaling by Tob in osteoblasts. Cell 103, 1085-1097.
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