Cancer Letters 342 (2014) 43–51
Contents lists available at ScienceDirect
Cancer Letters
journal homepage: www.elsevier.com/locate/canlet
MicroRNA let-7c inhibits migration and invasion of human non-small
cell lung cancer by targeting ITGB3 and MAP4K3
Bingtian Zhao a,1, Haibo Han b,1, Jinfeng Chen a, Zhiqian Zhang b, Shaolei Li a, Fang Fang a, Qingfeng Zheng a,
Yuanyuan Ma a, Jianzhi Zhang a, Nan Wu a, Yue Yang a,⇑
a
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Thoracic Surgery II, Peking University Cancer Hospital and Institute,
Beijing 100142, People’s Republic of China
b
Department of Cell Biology, Peking University Cancer Hospital and Institute, Beijing 100142, People’s Republic of China
a r t i c l e
i n f o
Article history:
Received 17 February 2013
Received in revised form 9 July 2013
Accepted 18 August 2013
Keywords:
MicroRNA
Let-7c
NSCLC
Migration and invasion
a b s t r a c t
MicroRNAs play an important regulatory role in carcinogenesis and cancer metastasis. Different members
of let-7 family have been reported to be decreased in human lung tumors. However, the effect of specific
let-7 member on metastasis of NSCLC remains undefined. Our current study detected the expression of
let-7 members in 94 cases of NSCLC and a significant association was noticed between low levels of
let-7c expression and metastasis, venous invasion, advanced TNM stages and poor survival of NSCLC
patients. Consistently, ectopic expression of let-7c in relatively highly metastatic cells remarkably suppressed their migration and invasion. Inhibition of let-7c in cells with relatively low metastatic potential
promoted their motility and invasion. We then analyzed the potential targets of let-7c and found that
ITGB3 and MAP4K3 were directly repressed by let-7c. Upon restoring the expression of ITGB3 and
MAP4K3, the effects of let-7c on tumor metastasis were partially reversed, and more importantly, the
expression levels of ITGB3 and MAP4K3 were inversely correlated with let-7c in 64 NSCLC tissues. Collectively, our results suggest that let-7c, by degrading ITGB3 and MAP4K3, prevents NSCLC metastasis.
Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Lung cancer is the leading cause of cancer mortality worldwide,
with nearly 1,400,000 deaths each year [1]. Non-small cell lung
cancer (NSCLC) accounts for almost 85% of lung cancer. About
40% stage I and 60% stage II NSCLC patients are dying of distant
metastases within 5 years after curative tumor resection [2]. Thus,
identification of new molecules involved in tumor metastasis is a
critical step toward the development of novel therapeutics. These
new agents are expected to prevent distal metastasis, thereby significantly increasing the survival rate.
MicroRNAs (miRNAs) are recognized as important post-transcriptional regulators of gene expression [3]. Mounting evidence
suggests that the aberration or alteration of miRNAs is involved
in carcinogenesis, progression and metastasis in many human cancers including NSCLC [4]. Yanaihara and colleagues, by comparing
104 NSCLC samples to corresponding normal lung tissues, have
demonstrated that 43 miRNAs are differentially expressed in cancer samples [5]. High levels of mir-328 and mir-378 are found to
⇑ Corresponding author. Address: Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, People’s Republic of China. Tel./fax: +86
1088196568.
E-mail address: [email protected] (Y. Yang).
1
These authors contribute to this work equally.
0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.canlet.2013.08.030
be associated with brain metastasis from NSCLC [6,7]. Low level
of mir-451 expression is reportedly associated with lymph node
involvement of NSCLC [8]. Recently, meta-analysis of prospective
trials reveals that mir-21 and mir-155 are reliable predictors for
recurrence, metastasis and poor survival of NSCLC patients [9,10].
Thus, the potential role of miRNAs on NSCLC metastasis warrants
further investigation.
Let-7 family has been shown to act as tumor suppressors by
inhibiting oncogenes and key regulators of mitogenic pathways
including RAS, MYC, HMGA2, etc. [11]. As for lung cancer, some
let-7 members are reported to be correlated with tumor growth
and pathologic classification. Let-7a and let-7f are firstly reported
to be down-regulated in human lung cancers and are proved to
be correlated with poor prognosis [12]. Ectopic overexpression levels of let-7b and let-7g are able to effectively suppress cancer
growth in mouse models of lung cancer [13–15]. Expression profiles of let-7 members are also a promising method for classification of adenocarcinoma (AD) and squamous cell carcinoma (SCC)
[16]. These studies suggest that specific member of let-7 family
plays a crucial role in the growth and development of lung cancer.
However, the influence of let-7 members on metastasis in lung
cancer remains largely undefined. Our previous findings suggest
that let-7c is a suppressor in migration and invasion of colorectal
cancer (CRC) [17]. Our current study aimed to generate more results consolidating the correlation between expression of let-7
44
B. Zhao et al. / Cancer Letters 342 (2014) 43–51
members (let-7a, 7b, 7c) and metastasis of human lung cancer
cells. We focused on let-7c because its expression was significantly
down-regulated both in the lung cancer cell lines with relatively
highly metastatic potential and in tumor tissues with metastasis.
The effect of let-7c on migration and invasion of lung cancer cells
was directly verified by cell functional assays after overexpression
and inhibition of let-7c in cells. To explore the potential mechanism underlying let-7c mediated metastatic suppression in lung
cancer cells, a list of candidate genes were predicted by bioinformatics’ software and further confirmed by dual-luciferase reporter
system. As a result, both ITGB3 and MAP4K3 are identified as target
genes responsible for the functions of let-7c. ITGB3 (integrin b3,
also known as CD61), a member of integrin family, has been shown
to be involved in cell adhesion and ‘‘outside-in’’ signaling transduction [18]. MAP4K3, a member of the MAP4K family, is an important
mediator in different signaling pathways [19]. Our study first demonstrates that let-7c, in addition to its suppressive role in tumor
growth, also inhibits tumor metastasis by directly destabilizing
the mRNAs of ITGB3 and MAP4K3 in NSCLC.
2. Materials and methods
2.1. Cell lines and clinical samples
Human NSCLC cell lines (SKMES-1, H520, H157, A549, GLC82,
H1299) and human lung epithelium cell (HLEC) were cultured in
RPMI 1640 medium supplemented with 10% fetal bovine serum
(FBS), 100 U/ml penicillin, and 100 lg/ml streptomycin (Invitrogen, Grand Island, NY) in a humidified atmosphere of 5% CO2 at
37 °C incubator.
The NSCLC specimens (n = 94) without any preoperative radiotherapy or chemotherapy and matched adjacent normal tissues obtained from the patients undergoing surgery at Beijing Cancer
Hospital during 2006–2008 with informed consent following the
protocols approved by the Ethics Committee of Peking University
Hospital.
was then added to SKMES-1 cells and screened by Blasticidin
(5 lg/ml) and G418 (500 lg/ml) to generate TET-ON let-7c expression cells.
To rescue expression of MAP4K3, the open reading frame (ORF)
of MAP4K3 was amplified from cDNA library by PCR and cloned
into the lentiviral shuttle vector plenti6 (Invitrogen). The lentiviral
plasmid containing ITGB3 was obtained from GeneCopoeia (EXE2219-LV105).
2.4. Cell proliferation assay
To observe the effect of let-7c on cell proliferation, SKEMS-1
cells with tetracycline-inducible let-7c expression were seeded
into 96-well plates and induced by doxycycline (Dox, 2 lg/ml). Cell
Counting Kit-8 (CCK-8) was used to monitor cell growth at 0, 1, 2,
3, 4 day and the number of viable cells was assessed by measurement of absorbance at 450 nm by FlUOstar OPTIMA (BMG LABTECH, Offenburg, Germany).
2.5. Wound healing assay
About 3 105 cells were seeded in 6-well dishes and an incision
was made in the central area of the confluent culture to create an
artificial wound. Images of the wound area were captured by
microscope (Leica, Wetzlar, Germany) 24 h after injury.
2.6. Transwell cell migration and invasion assay
To evaluate the effect of let-7c on cell migration and invasion,
2.5 104 cells in 100 ll RPMI1640 with 1% FBS were plated to
the upper chamber of the transwells (8 lm pore size, Corning,
NY, USA) with and without matrigel. The lower chamber was
loaded with 450 ll of RPMI1640 with 10% FBS as chemoattractants
for cells. After 24 h, migrated or invaded cells were counted and
imaged by microscope after fixing with 2% methanol and stained
with 1% crystal violet solution.
2.2. RNA extraction and quantitative RT-PCR
2.7. Bioinformatic prediction and dual-luciferase reporter assay
Total RNA was extracted from tissues and cells according to the
protocol of miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). For mature miRNAs quantification, 100 ng total RNA was added with a
polyA tail by polyA polymerase (New England Biolabs, Beverly,
MA, USA), following by reverse transcription with an oligo-dT
adapter primers and Moloney murine leukaemia virus (MMLV,
Invitrogen). For mRNA detection, cDNAs were synthesized from
2 lg total RNA using oligo-d(T)15 primers and MMLV. Sequences
of all primers were listed in Table S1. qRT-PCR was performed by
using SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) on LightCyclerÒ 480 Real-Time PCR System (Roche, USA). The relative
amount of miRNAs or genes was normalized to U6 or GAPDH. Data
was calculated based on 2DCt where DCt = Ct (Target) – Ct (Reference). Fold change was calculated by the 2DDCt method.
miRGen was used to analyze the putative target genes of let-7c.
The 30 -UTRs of ITGB3 and MAP4K3 containing predicted let-7c
binding sites were amplified from cDNA library. The corresponding
mutant 30 -UTRs were obtained by overlap-extension PCR. The sequences of all primers were listed in Table S1. All PCR products
were cloned into downstream of firefly luciferase gene of the
pGL3-control plasmid (Promega, Madison, WI). SKMES-1 cells were
cotransfected with 200 ng of pGL3 constructions, 600 ng of
pcDNA3.0 or pcDNA3.0-let-7c and 26 ng of pRL-TK in 24-well
plates by Lipofectamine 2000. Luciferase activity (Firefly and Renilla) was measured with a dual-luciferase reporter assay system
(Promega) at 24 h after transfection.
2.8. Protein extraction and western blot analysis
2.3. Plasmid construction and cell transfection
pcDNA3.0-pre-Let-7c was constructed in our previous study
[17] and transfected into SKMES-1 cells by lipofectamine 2000
(Invitrogen) following the manufacturer’s protocol. Stable single
let-7c overexpressing cell clones were screened by G418 (500 lg/
ml) and then expanded culture. Pre-Let-7c was also subcloned into
plenti6-TREpitt vectors to construct tetracycline-inducible let-7c
expression system. Shuttle vector constructs and the ViraPower
Packaging Mix (Invitrogen) were cotransfected 293FT cells according to the manufacturer’s protocol to obtain lentivirus. Lentivirus
Protein was extracted from cells using RIPA buffer containing
complete protease inhibitor cocktail (Roche, Mannheim, Germany).
Protein (20 lg) was separated by electrophoresis on SDS–PAGE
gels and blotted onto the PVDF membrane (Millipore). Rabbit
anti-ITGB3 (1:1000 dilution, Epitomics, USA), rabbit anti-MAP4K3
(1:1000 dilution, Cell Signaling Technology, USA), mouse anti-b-actin (1:50,000 dilution, Roche) were used as primary antibodies.
HRP-conjugated goat anti-rabbit or anti-mouse IgG (cwBiotech,
China) was used as secondary antibody respectively. Signals were
visualized by using chemiluminescence (Millipore).
45
0.8
0.4
0.0
B
n=38
100
1.96
1
0.57
0.1
M ann-Whitney test p=0.0002
0.01
LE
C
LC
82
H
52
0
H
15
7
H
1
SK 29
M 9
ES
-1
A5
49
Normal
tissues
G
H
D
1.0
Probability of Overall Survival
Probability of Tumor-Free
Survival
C
let-7c high expression n=47
0.8
0.6
let-7c low expression n=47
0.4
0.2
Log-rank test
p=0.0342
0.0
0
20
40
Months
60
n=38
10
2 -ΔΔCt
1.2
2 -ΔΔCt
Relative let-7c expression
A
Relative let-7c expression
B. Zhao et al. / Cancer Letters 342 (2014) 43–51
80
Carcinoma
tissues
1.0
let-7c high expression n=47
0.8
0.6
let-7c low expression n=47
0.4
0.2
Log-rank test
p=0.0386
0.0
0
20
40
Months
60
80
Fig. 1. Analysis of let-7c expression in NSCLC cell lines and specimens. (A) The levels of let-7c in H1299, SKMES-1 and A549 cell lines with relatively highly invasive properties
were greatly lower than others. Columns, mean of three replicate calculation; bars, SD. (B) The expression of let-7c in cancer tissues was significantly lower than that in
normal tissues (n = 38). (C and D) Kaplan–Meier survival curves demonstrated that low level of let-7c was associated with poor tumor-free survival and overall survival. Each
sample was analyzed in triplicate and normalized to reference U6.
2.9. Overexpression or inhibition of let-7c by miRNA mimics or
inhibitors
The synthetic let-7c mimics (50 -UGAGGUAGUAGGUUGUAUGG
UU-30 ) and negative control miRNA (50 -CAGUACUUUUGUGUAGUACAA-30 ) were synthesized by GenePharma (Shanghai, China).
A chemically-modified let-7c-inhibitor (50 -AACCAUACAACCUAC
UACCUCA-30 ) and negative control (50 -UUUGUACUACACAAAAGUACUG-30 ) were purchased from RiboBio (Guangzhou, China). Cells
were transiently transfected with oligonucleotides using lipofectamine 2000 and were used to evaluate migratory and invasive
behavior. At the same time, cells were also collected to examine
the expression of let-7c, ITGB3 and MAP4K3.
2.10. Statistical analysis
Continuous variables with normal distribution were presented
as mean ± SD, otherwise were presented as median. Dichotomous
variables were presented as number and percentage values. Differences between each group were assessed by two-tailed Students’ttest or Mann–Whitney test by SPSS13.0 unless specified. p values
of less than 0.05 was denoted as statistically significant.
3. Results
3.1. Let-7c down-regulated in human lung cancer cells with highly
metastatic ability
The invasive potential of lung cancer cell lines used in this study
was firstly tested by transwell assay as described in Supplementary
Fig. 1. The expression levels of let-7c in these cell lines were detected to determine the potential effect of let-7c expression on
metastasis. Fig. 1A showed that the relative expression level of
let-7c was lower in cells with relatively highly metastatic ability
(H1299, SKMES-1 and A549) than those with relatively low or no
metastatic potential (H157, H520, GLC82, HLEC). These results suggested that low level of let-7c might be associated with NSCLC
metastasis.
3.2. Decreased let-7c expression associated with metastasis in NSCLC
patients
To determine the relationship between let-7c and metastasis in
clinical samples, we firstly examined let-7c expression in 38 human
NSCLC tissues and matched normal tissues. As shown in Fig. 1B, let7c expression was significantly down-regulated in NSCLC tissues
compared to normal tissues. Because we focused on the role of let7c expression in tumor metastasis, we further examined let-7c
expression in 94 NSCLC samples. As a result, low level expression
of let-7c was associated with venous invasion, advanced TNM stage
and lymph node or distant metastasis. As listed in Table 1, the median value of relative let-7c expression in tissues with venous invasion was about 50% lower than in tissues without venous invasion
(p = 0.0219), 38% lower in tissues with advanced TNM stage than
with TNM I-II stage (p = 0.0357) and 45% lower in tissues with
metastasis than those without metastasis (p = 0.0098). A statistical
significance was found among different pathologic types, for example, let-7c expression was higher in adenocarcinoma (AD) than in
squamous cell carcinoma (SCC). Although there seemed an inverse
correlation between let-7c expression and advanced node stage, statistical analysis did not reveal a significant association. In contrast,
there were no significant differences in let-7c expression with regards to the genders, ages and T grades.
3.3. Decreased let-7c expression associated with poor survival of
NSCLC patients
Kaplan–Meier curves were used to analyze survival of patients
with NSCLC categorized by a cut-off value according to median
46
B. Zhao et al. / Cancer Letters 342 (2014) 43–51
Table 1
Relationship between let-7c expression and pathological features in NSCLC.
1
Variable
Case number (%)
Let-7c expression (2DDCt)
Median
Sex
Male
Female
65 (69.1)
29 (30.9)
0.6832
0.8129
Age
<62 yr
>62 yr
50 (53.2)
44 (46.8)
0.6529
0.8343
Differentiation
Well
Moderate
Poor
Undifferentiated
11 (11.7)
41 (43.6)
39 (41.5)
3 (3.2)
1.501
0.5819
0.6977
0.4607
Histological type
Adenocarcinoma
Squamous carcinoma
Adenosquamous carcinoma
Large-cell carcinoma
58 (61.7)
30 (31.9)
3 (3.2)
3 (3.2)
0.9498
0.5074
Venous invasion
Yes
No
15 (16.5)
79 (83.5)
0.4402
0.8617
Tumor stage
T1
T2
T3
T4
21 (22.3)
53 (56.4)
16 (17.0)
4 (4.3)
0.8648
0.6811
0.7541
0.3883
Node stage
No
N1
N2
40 (42.6)
28 (29.8)
26 (27.7)
0.9794
0.774
0.5422
TNM stage
I-II
III
55 (58.5)
39 (41.5)
0.973
0.5353
Metastasis
Yes
No
32 (34)
62 (66)
0.5422
0.8848
p value1
0.7997
0.9486
0.1628
0.0139
0.0219
0.0918
0.1947
0.0357
0.0098
Mann-Whitney test was used to evaluate the significant associations for two groups and Kruskal–Wallis test for more groups.
expression level of let-7c (0.725). As shown in Fig. 1C and D, tumor
free survival (TFS) and overall survival (OS) time with low let-7c
expression were significantly shorter than those with high let-7c
expression. The 75 percentile of TFS for low versus high let-7c
expression group were 11.43 versus 42.88 months (p = 0.0342),
and the 75 percentile of OS for the low versus high let-7c expression group were 25 versus 41 months (p = 0.0386). Taken together,
low level of let-7c expression may predict a short survival time of
NSCLC. However, COX regression failed to demonstrate the expression of let-7c as an independent prognostic factor (data not
shown).
74% and 77% in transfected H1299 and A549 respectively, compared with control cells. Moreover, invasive cells were also decreased by 69% and 88% in transfected H1299 and A549
respectively, compared with control cells. Consistent with data
from SKMES-1 cells, overexpression of let-7c in both cells also
showed a reduced cell migration and invasion by transwell assay.
Collectively, overexpression of let-7c may impede cellular proliferation, cell migration and invasion in vitro.
3.4. Let-7c inhibited growth, migration and invasion of SKMES-1 cells
in vitro
To investigate the mechanism through which let-7c overexpression suppresses tumor growth and metastasis, we firstly predicted
the target genes of let-7c by miRGen and screened out 28 candidate genes associated with cell growth and invasion. To identify
the genuine targets, the changes of these genes were detected in
SKMES-1 cells with induced let-7c expression. Among these genes,
the expression levels of MAP4K3 and ITGB3 were significantly repressed after let-7c overexpression both in mRNA and protein levels compared to control cells (Fig. 3A and B).
The binding sites in 30 -UTRs of ITGB3 and MAP4K3 for let-7c
and the corresponding mutated sequences were depicted in
Fig. 3C. To determine whether the regulation is direct, the 30 -UTRs
of ITGB3 and MAP4K3 containing let-7c binding-sites and corresponding mutations were cloned into the pGL3-control vector. As
shown in Fig. 3D, the relative luciferase activity was significantly
decreased in the wild-type 30 -UTRs of ITGB3 and MAP4K3
The relationship of reduced expression of let-7c and metastasis
of NSCLC drove us to explore the possible biological functions of
let-7c in lung cancer cells. We first induced let-7c expression in
the highly metastatic SKMES-1 cells. As shown in Fig. 2A, the relative expression of let-7c peaked 48 h after adding 2 lg/ml DOX.
Overexpression of let-7c in SKMES-1 cells showed distinct patterns
of growth, spreading and invasion inhibition compared with control cells (Fig. 2B–F). To improve the result, we further transiently
transfected the other two highly metastatic cell lines H1299 and
A549 with let-7c mimics. After trasfected with mimics, let-7c
expression in H1299 and A549 cells were strongly increased compared with control cells (Supplementary Fig. 2). As shown in
Fig. 2G and H, cells migrated through transwell were reduced by
3.5. Let-7c directly down-regulated ITGB3 and MAP4K3 expression by
binding to their 30 -UTRs
47
B. Zhao et al. / Cancer Letters 342 (2014) 43–51
B 120
DOX-
+
0
1
3
4
F
p=0.0002
4000
3000
2000
1000
0
x-
Do
DOX+
DOX-
2
Days
E
DOX+
DOX+
p<0.0001
1500
1000
500
0
x+
Do
+
Migration
Two-way ANOVA p<0.001
0
0
24h
48h
SKMES-1 Tet-on-let-7c
SKMES-1
Invasion
40
D
ox
+
DOX-
-
D
-
80
Migrated cells/view
0
Dox
Time
Cell Line
24 h
ox
2
0h
SKMES-1 let-7c Dox+
D
4
C
SKMES-1 let-7c Dox-
Invaded cells/view
Absorbance 450nm (%)
6
H
G
A549
Migration
H1299
mimics
control
mimics
Invasion
control
control
mimics
control
mimics
Cell number per view
2 -ΔΔCt
Relative let-7c expression
A
3000
H1299
A549
2000
1000
0
Matrigel
-
miR-let7c
control
mimics
+
+
control
mimics
Fig. 2. Overexpression of let-7c inhibited proliferation, migration and invasion in SKMES-1 cell. (A) The relative level of let-7c was highly improved in SKMES-1 cell after
inducing by 2 lg/ml DOX at 48 h. Inducing let-7c expression inhibited proliferation of SKMES-1 cells by CCK-8 assay (B), impeded cell spread by wound-healing assay (C),
inhibited migration and invasion using transwell assay without and with matrigel. Bar: 50 lm. (D). (E and F) Data represented the mean ± SD of three independent
experiments with four random fields counted for each chamber. (G and H) Overexpression of let-7c in H1299 and A549 cells also showed a reduced ability of migration and
invasion by transwell assay. Bar: 50 lm.
compared to corresponding mutant constructs and the control.
These data revealed that let-7c directly decreased the expression
of these genes through binding to their 30 -UTRs.
To confirm the correlation of let-7c and target genes in lung
cancer tissues, we examined the expression of let-7c and the
mRNA levels of ITGB3 and MAP4K3 in the same set of 64 NSCLC tissues. As expected in Fig. 4, let-7c expression was inversely correlated with the mRNA expression levels of two target genes. The
results supported that let-7c targeted ITGB3 and MAP4K3 in vivo.
3.6. Inhibition of let-7c enhanced cell migration and invasion in GLC82
cells by increasing the expression of ITGB3 and MAP4K3
To further confirm the results, we inhibited the expression of
let-7c by miRNA inhibitors in relatively low metastatic cell lines,
including GLC82, H520 and H157 cells. The levels of let-7c expression were decreased about 50% after transfected with let-7c inhibitor compared with control cells (Fig. 5A). Consistent with the
results of overexpressing let-7c in SKMES-1 cells, the inhibition
of let-7c expression in GLC82, H157 and H520 cells enhanced the
cell motility and invasion (Fig. 5B and C). At the same time, the protein levels of ITGB3 and MAP4K3 were up-regulated after inhibition of let-7c in GLC82 cells compared with negative control
(Fig. 5D). These data supported that inhibition of let-7c expression
increased cell motility and invasion through terminating the degradation of its target genes ITGB3 and MAP4K3.
3.7. Rescue of ITGB3 and MAP4K3 reversed the effect of let-7c on
metastasis
To investigate whether ITGB3 and MAP4K3 have an important
role in let-7c-mediated inhibition of metastasis, we rescued the
expression of ITGB3 and MAP4K3 in let-7c overexpressing
SKMES-1 cells (Fig. 6A and B). The inhibitory effect of let-7c on cell
migration and invasion was overcome after rescue of ITGB3 and
MAP4K3 (Fig. 6C–E). Collectively, these results suggested that let7c acted as a metastatic suppressor in lung cancer cells by repressing the expression of ITGB3, MAP4K3, and possibly other
molecules.
4. Discussion
In this study we firstly determine the expression level of let-7
members in lung cancer cell lines and clinical samples. The decreased expression of let-7c is found to be associated with highly
invasive ability in lung cancer cells and metastasis in NSCLC. Then
we investigate the mechanism underlying the let-7c-mediated
suppression of metastasis. As a result, ITGB3 and MAP4K3 are identified as direct target genes of let-7c. After rescuing expression of
ITGB3 and MAP4K3, the suppressive effects of let-7c on migration
and invasion of NSCLC cells are partially reversed. Taken together,
our work suggests that let-7c exerts its suppressive effect on
metastasis of NSCLC by targeting ITGB3 and MAP4K3.
48
B. Zhao et al. / Cancer Letters 342 (2014) 43–51
A
B
Relative gene expression
5
4
MAP4K3
Dox-
3
Dox+
Dox-
Dox+
2
1
β-actin
0
+
+
MAP4K3
ITGB3
SKMES-1 Tet-on-let-7c
Dox
Gene
Cell line
C
ITGB3
MAP4K3-3’UTR-Mut
AAAUAAUUUAGUGCAGCAAGAC
MAP4K3-3’UTR
5’... AAAUAAUUUAGUUACUACCUCA...
hsa-let-7c
3’...UUGGUAUGUUGGAUGAUGGAGU
D
ITGB3-3’UTR-Mut
ITGB3-3’UTR
hsa-let-7c
GCAGGUGUUCUUCAUGCUUGUAG
5’...GCAGGUGUUCUUCAUUACCUCAG...
3’...UUGGUAUGUUGGAUGAUGGAGU
ITGB3-3’UTR-Mut
ITGB3-3’UTR
hsa-let-7c
GAUUGUAUUUCUUGGUGCUUGAU
5’... GAUUGUAUUUACCUUCUACCUCU...
3’... UUGGUAGGUUGGAUGAUGGAGU
Relative luciferase activity
1.2
** p<0.01
Control
3’UTR-mut
3’UTR
0.8
**
**
0.4
0.0
MAP4K3
ITGB3
Fig. 3. Let-7c targeted ITGB3 and MAP4K3 directly in SKEMS-1 cells. (A and B) The relative expression levels of MAP4K3 and ITGB3 both in mRNA and protein levels were
suppressed in SKEMS-1 cells after inducing expression of let-7c. (C) Putative let-7c binding sites for let-7c and corresponding mutations in 30 -UTRs of ITGB3 and MAP4K3. (D)
Luciferase reporter assay demonstrated that let-7c inhibited the wild-type, but not the mutant of 30 -UTRs of ITGB3 and MAP4K3 reporter activities compared with the vector
control (p < 0.05). Data represented the mean ± SD of three independent experiments with quadruplicates of each sample.
B
Spearman correlation p=0.012
0.0
r= - 0.3124
-0.5
-1.0
-1.5
-2.0
-2.5
Relative let-7c expression, log (2 -Δct )
Relative let-7c expression, log (2 -Δct )
A
Spearman correlation p=0.0167
0.0
r= - 0.2982
-0.5
-1.0
-1.5
-2.0
-2.5
-5
-4
-3
-2
-1
0
Relative ITGB3 expression, log (2-Δct )
-3
-2
-1
0
1
Relative MAP4K3 expression, log (2 -Δct )
Fig. 4. The correlation analysis suggested significantly negative correlation between let-7c expression and MAP4K3 and ITGB3 mRNA levels in 64 NSCLC tissues. p values
were obtained by two-tailed Spearman’s test.
Our previous study has demonstrated that let-7c is a metastatic
suppressor in colorectal cancer [17]. It is also reported that let-7c
expression is significantly decreased in metastatic prostate cancer
compared to high grade but localized carcinoma [20]. Here, we
demonstrate that let-7c also plays a suppressive role in migration
and invasion of NSCLC cells. In addition, we confirm that reduced
let-7c expression is associated with venous invasion, advanced
TNM stage, and poor survival of NSCLC. Furthermore, Fassina and
49
B. Zhao et al. / Cancer Letters 342 (2014) 43–51
3
B
control
inhibitor
inhibitor
control
inhibitor
control
inhibitor
Migration
control
2
2-ΔΔCt
1
Invasion
Relative let-7c expression
A
0
GLC82
Cell number per view
C
H520
GLC82
H157
H520
H157
D
3000
GLC82
H520
H157
MAP4K3
2000
ITGB3
control 7c-inhibitor
control 7c-inhibitor
1000
0
β-actin
Matrigel
-
-
+
+
miR-let7c
control
inhibitor
control
inhibitor
B
5
2
Rescue
1
β-actin
Cell line
C
None
ITGB3
None
MAP4K3
SKMES-1 pcDNA3.0-let-7c
7c
7c+ITGB3
7c+MAP4K3
AP
N
M
on
G
IT
Migrated cells/view
D
0
Rescue
e
B3
e
on
3
4K
3
4
N
A
Relative gene expression
Fig. 5. Inhibition of let-7c up-regulated the ITGB3 and MAP4K3 expression and elevated the migration and invasion in GLC82 cell line. (A) The expression level of let-7c was
decreased about 50% by let-7c inhibitor. (B and C) Cell migration and invasion were increased after inhibition of let-7c in GLC82, H520 and H157 cells. Each bar represented
the mean ± SD of the counts from three respectively experiments. Bar: 50 lm. (D) The protein levels of ITGB3 and MAP4K3 were increased after inhibition of let-7c compared
to the control cells.
4000
** p<0.001
**
3000
**
2000
1000
4K
3
B3
AP
7c
+M
7c
2000
7c
+I
TG
E
Invaded cells/view
Invasion
Migration
0
** p<0.001
**
1500
1000
**
500
0
7c
7
G
IT
c+
B
3
+M
7c
A
P
4K
3
Fig. 6. Rescue of ITGB3 and MAP4K3 alone overcame the effects of let-7c on migration and invasion in let-7c overexpressing SKMES-1 cells. (A and B) Expression levels of
ITGB3 and MAP4K3 were increased both in mRNA and protein levels after introducing two genes in let-7c overexpressing SKMES-1 cells. (C) Cell migration (upper) and
invasion (lower) abilities were partially reversed after introducing two genes in let-7c overexpressing SKMES-1 cells. Bar: 50 lm. (D and E) Data represented the mean ± SD of
three independent experiments with four random fields counted for each chamber. p < 0.001.
50
B. Zhao et al. / Cancer Letters 342 (2014) 43–51
colleagues have shown that let-7c expression is higher in AD than
that in SCC [16], which has been also identified in our study. Collectively, the observations made by other groups and our own suggest that let-7c, in addition to its role in proliferation [21],
immunocyte differentiation [22], drug resistance [23] and carcinogenesis [24], is closely involved in tumor metastasis.
Bcl-xL, HMGA2 and MYC have been identified as the let-7c targets in different human cancers [25–27]. In the present study, we
also investigate the mechanism responsible for the let-7c mediated
inhibition of NSCLC metastasis and first demonstrate that ITGB3 and
MAP4K3 are two target genes of let-7c as mediators for NSCLC
migration and invasion. ITGB3 (integrin b3), a molecule involved in
several diseases including cancer [28,29], can promote ROS-induced
migration and invasion in colorectal cancer cells [30]. It works as a
downstream molecule of let-7a to incite development and metastasis of malignant melanoma [31]. ITGB3, as a target gene of mir-30, is
up-regulated in breast tumor-initiating cells, leading to an antiapoptotic effect [32]. Our results indicate that ITGB3 is a direct target
of let-7c. Accordantly, rescuing ITGB3 expression can reverse the
inhibitory effects of let-7c on migration and invasion in NSCLC. Furthermore, we reveal an inverse correlation between let-7c expression and ITGB3 expression in 64 NSCLC tissues. Together with the
observations that let-7a inhibits metastasis of malignant melanoma
by increasing ITGB3 expression [31], it is plausible for us to postulate
that different let-7 members may target same gene for regulating
similar biological function in different cancer cells. MAP4K3 (GLK)
is a member of MAP4K family containing a conserved N-terminal kinase domain, C-terminal citron homology domain and proline-rich
motifs in center [19]. Previous data have demonstrated that MAP4K3
is connected with JNK [19], apoptosis [33], EGFR [34] and mTOR
[35,36] signaling pathways. Our own findings suggest that MAP4K3
is a target gene of let-7c and restoration of MAP4K3 expression abolishes the inhibitory effect of let-7c on cell migration and invasion. In
addition, let-7c expression is inversely correlated with expression
levels of MAP4K3 in NSCLC tissues. Thus, our results suggest that
ITGB3 and MAP4K3 are let-7c target genes involved in NSCLC progression, which may help us to identify new therapeutic targets
for lung cancer.
Interestingly, our previous study has shown that the let-7c target genes also include MMP11 and PBX3, both of which are involved in the let-7c-mediated suppression of migration and
invasion in CRC [17]. To determine whether there is a signaling
pathway through ITGB3 or MAP4K3 to induce expression of
MMP-11, we find links between ITGB3 or MAP4K3 and MMP-11
by the BioGraph (http://biograph.be/) as shown in Attachment 1.
Results from BioGraph prediction suggest that the potential pathways for increasing expression of MMP11 through ITGB3 and/or
MAP4K3 are so complicated. As for the emerging data suggests female sex hormones play a key role in stimulating NSCLC progression [37], whether ITGB3 induces MMP11 expression through
estradiol and progesterone pathway is still worthy of further study.
Given that the inhibitory effect of tumor metastasis is mediated by
different target genes, further studies are warranted to establish
either a linear signal cascade or interactive crosstalk among ITGB3,
MAP4K3, MMP11 and/or PBX3.
To summarize, our study indicates that let-7c plays a pivotal
role in progression and metastasis of NSCLC by down-regulating
its target genes ITGB3 and MAP4K3. These findings lend novel insight to the mechanism underlying NSCLC metastasis and might
facilitate the development of prognostic biomarkers as well as
effective treatment regimens.
Conflict of Interest statement
None declared.
Acknowledgements
This work was supported partially by Beijing Natural Science
Foundation (Grant Nos. 11G2633 and 5122012), National Natural
Science Foundation (Grant Nos. 81201964 and 81071733), the National High Technology Research and Development Program of China (863 Program, No. 2012AA02A502), and the National Basic
Research Program of China (973 Program, No. 2010CB529402).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.canlet.2013.
08.030.
References
[1] A. Jemal, M.M. Center, C. DeSantis, E.M. Ward, Global patterns of cancer
incidence and mortality rates and trends, Cancer Epidemiology, Biomarkers
and Prevention: A Publication of the American Association for Cancer Research,
Cosponsored by the American Society of Preventive Oncology 19 (2010) 1893–
1907.
[2] G.M. Strauss, Adjuvant chemotherapy of lung cancer: methodologic issues and
therapeutic advances, Hematology/Oncology Clinics of North America 19
(2005) 263–281. vi.
[3] W. Filipowicz, S.N. Bhattacharyya, N. Sonenberg, Mechanisms of posttranscriptional regulation by microRNAs: are the answers in sight?, Nature
Reviews Genetics 9 (2008) 102–114
[4] G. Di Leva, C.M. Croce, Roles of small RNAs in tumor formation, Trends in
Molecular Medicine 16 (2010) 257–267.
[5] N. Yanaihara, N. Caplen, E. Bowman, M. Seike, K. Kumamoto, M. Yi, R.M.
Stephens, A. Okamoto, J. Yokota, T. Tanaka, G.A. Calin, C.G. Liu, C.M. Croce, C.C.
Harris, Unique microRNA molecular profiles in lung cancer diagnosis and
prognosis, Cancer Cell 9 (2006) 189–198.
[6] S. Arora, A.R. Ranade, N.L. Tran, S. Nasser, S. Sridhar, R.L. Korn, J.T. Ross, H.
Dhruv, K.M. Foss, Z. Sibenaller, T. Ryken, M.B. Gotway, S. Kim, G.J. Weiss,
MicroRNA-328 is associated with (non-small) cell lung cancer (NSCLC) brain
metastasis and mediates NSCLC migration, International Journal of Cancer.
Journal International Du Cancer 129 (2011) 2621–2631.
[7] L.T. Chen, S.D. Xu, H. Xu, J.F. Zhang, J.F. Ning, S.F. Wang, MicroRNA-378 is
associated with non-small cell lung cancer brain metastasis by promoting cell
migration, invasion and tumor angiogenesis, Medical Oncology 29 (2012)
1673–1680.
[8] X.C. Wang, L.L. Tian, X.Y. Jiang, Y.Y. Wang, D.G. Li, Y. She, J.H. Chang, A.M. Meng,
The expression and function of miRNA-451 in non-small cell lung cancer,
Cancer Letters 311 (2011) 203–209.
[9] M. Yang, H. Shen, C. Qiu, Y. Ni, L. Wang, W. Dong, Y. Liao, J. Du, High expression
of miR-21 and miR-155 predicts recurrence and unfavourable survival in nonsmall cell lung cancer, European Journal Cancer (2012).
[10] Z.L. Liu, H. Wang, J. Liu, Z.X. Wang, MicroRNA-21 (miR-21) expression
promotes growth, metastasis, and chemo- or radioresistance in non-small
cell lung cancer cells by targeting PTEN, Molecular and Cellular Biochemistry
372 (2013) 35–45.
[11] I. Bussing, F.J. Slack, H. Grosshans, Let-7 microRNAs in development, stem cells
and cancer, Trends in Molecular Medicine 14 (2008) 400–409.
[12] J. Takamizawa, H. Konishi, K. Yanagisawa, S. Tomida, H. Osada, H. Endoh, T.
Harano, Y. Yatabe, M. Nagino, Y. Nimura, T. Mitsudomi, T. Takahashi, Reduced
expression of the let-7 microRNAs in human lung cancers in association with
shortened postoperative survival, Cancer Research 64 (2004) 3753–3756.
[13] A. Esquela-Kerscher, P. Trang, J.F. Wiggins, L. Patrawala, A. Cheng, L. Ford, J.B.
Weidhaas, D. Brown, A.G. Bader, F.J. Slack, The let-7 microRNA reduces tumor
growth in mouse models of lung cancer, Cell Cycle 7 (2008) 759–764.
[14] P. Trang, J.F. Wiggins, C.L. Daige, C. Cho, M. Omotola, D. Brown, J.B. Weidhaas,
A.G. Bader, F.J. Slack, Systemic delivery of tumor suppressor microRNA mimics
using a neutral lipid emulsion inhibits lung tumors in mice, Molecular Therapy
: The Journal of the American Society of Gene Therapy 19 (2011) 1116–1122.
[15] M.S. Kumar, S.J. Erkeland, R.E. Pester, C.Y. Chen, M.S. Ebert, P.A. Sharp, T. Jacks,
Suppression of non-small cell lung tumor development by the let-7 microRNA
family, Proceedings of the National Academy of Sciences of the United States of
America 105 (2008) 3903–3908.
[16] A. Fassina, R. Cappellesso, M. Fassan, Classification of non-small cell lung
carcinoma in transthoracic needle specimens using microRNA expression
profiling, Chest 140 (2011) 1305–1311.
[17] H.B. Han, J. Gu, H.J. Zuo, Z.G. Chen, W. Zhao, M. Li, D.B. Ji, Y.Y. Lu, Z.Q. Zhang,
Let-7c functions as a metastasis suppressor by targeting MMP11 and PBX3 in
colorectal cancer, Journal Pathology 226 (2012) 544–555.
[18] J.S. Bennett, F. Catella-Lawson, A.R. Rut, G. Vilaire, W. Qi, S.C. Kapoor, S.
Murphy, G.A. FitzGerald, Effect of the Pl(A2) alloantigen on the function of
beta(3)-integrins in platelets, Blood 97 (2001) 3093–3099.
[19] K. Diener, X.S. Wang, C. Chen, C.F. Meyer, G. Keesler, M. Zukowski, T.H. Tan, Z.
Yao, Activation of the c-Jun N-terminal kinase pathway by a novel protein
B. Zhao et al. / Cancer Letters 342 (2014) 43–51
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
kinase related to human germinal center kinase, Proceedings of the National
Academy of Sciences of the United States of America 94 (1997) 9687–9692.
K.R. Leite, J.M. Sousa-Canavez, S.T. Reis, A.H. Tomiyama, L.H. Camara-Lopes, A.
Sanudo, A.A. Antunes, M. Srougi, Change in expression of miR-let7c, miR-100,
and miR-218 from high grade localized prostate cancer to metastasis, Urologic
Oncology 29 (2011) 265–269.
N. Nadiminty, R. Tummala, W. Lou, Y. Zhu, X.B. Shi, J.X. Zou, H. Chen, J. Zhang,
X. Chen, J. Luo, R.W. deVere White, H.J. Kung, C.P. Evans, A.C. Gao, MicroRNA
let-7c is downregulated in prostate cancer and suppresses prostate cancer
growth, PloS One 7 (2012) e32832.
A. Pelosi, S. Careccia, V. Lulli, P. Romania, G. Marziali, U. Testa, S. Lavorgna, F.
Lo-Coco, M.C. Petti, B. Calabretta, M. Levrero, G. Piaggio, M.G. Rizzo, miRNA let7c promotes granulocytic differentiation in acute myeloid leukemia, Oncogene
(2012).
K. Sugimura, H. Miyata, K. Tanaka, R. Hamano, T. Takahashi, Y. Kurokawa, M.
Yamasaki, K. Nakajima, S. Takiguchi, M. Mori, Y. Doki, Let-7 expression is a
significant determinant of response to chemotherapy through the regulation
of IL-6/STAT3 pathway in esophageal squamous cell carcinoma, Clinical Cancer
Research: An Official Journal of the American Association for Cancer Research
18 (2012) 5144–5153.
S. Willimott, S.D. Wagner, Stromal cells and CD40 ligand (CD154) alter the
miRNome and induce miRNA clusters including, miR-125b/miR-99a/let-7c
and miR-17-92 in chronic lymphocytic leukaemia, Leukemia: Official Journal
of the Leukemia Society of America, Leukemia Research Fund, U.K 26 (2012)
1113–1116.
S. Shimizu, T. Takehara, H. Hikita, T. Kodama, T. Miyagi, A. Hosui, T. Tatsumi, H.
Ishida, T. Noda, H. Nagano, Y. Doki, M. Mori, N. Hayashi, The let-7 family of
microRNAs inhibits Bcl-xL expression and potentiates sorafenib-induced
apoptosis in human hepatocellular carcinoma, Journal of Hepatology 52
(2010) 698–704.
K. Motoyama, H. Inoue, Y. Nakamura, H. Uetake, K. Sugihara, M. Mori, Clinical
significance of high mobility group A2 in human gastric cancer and its
relationship to let-7 microRNA family, Clinical Cancer Research: An Official
Journal of the American Association for Cancer Research 14 (2008) 2334–2340.
N. Nadiminty, R. Tummala, W. Lou, Y. Zhu, J. Zhang, X. Chen, R.W. eVere White,
H.J. Kung, C.P. Evans, A.C. Gao, MicroRNA let-7c suppresses androgen receptor
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
51
expression and activity via regulation of Myc expression in prostate cancer
cells, The Journal of Biological Chemistry 287 (2012) 1527–1537.
Y. Huo, K. Ley, Adhesion molecules and atherogenesis, Acta Physiologica
Scandinavica 173 (2001) 35–43.
N.J. Yoo, Y.H. Soung, S.H. Lee, E.G. Jeong, Mutational analysis of proapoptotic
integrin beta 3 cytoplasmic domain in common human cancers, Tumori 93
(2007) 281–283.
Y. Lei, K. Huang, C. Gao, Q.C. Lau, H. Pan, K. Xie, J. Li, R. Liu, T. Zhang, N. Xie, H.S.
Nai, H. Wu, Q. Dong, X. Zhao, E.C. Nice, C. Huang, Y. Wei, Proteomics
identification of ITGB3 as a key regulator in reactive oxygen species-induced
migration and invasion of colorectal cancer cells, Molecular and Cellular
Proteomics: MCP 10 (2011) (M110 005397).
D.W. Muller, A.K. Bosserhoff, Integrin beta 3 expression is regulated by let-7a
miRNA in malignant melanoma, Oncogene 27 (2008) 6698–6706.
F. Yu, H. Deng, H. Yao, Q. Liu, F. Su, E. Song, Mir-30 reduction maintains selfrenewal and inhibits apoptosis in breast tumor-initiating cells, Oncogene 29
(2010) 4194–4204.
D. Lam, D. Dickens, E.B. Reid, S.H. Loh, N. Moisoi, L.M. Martins, MAP4K3
modulates cell death via the post-transcriptional regulation of BH3-only
proteins, Proceedings of the National Academy of Sciences of the United States
of America 106 (2009) 11978–11983.
D.E. Hammond, R. Hyde, I. Kratchmarova, R.J. Beynon, B. Blagoev, M.J. Clague,
Quantitative analysis of HGF and EGF-dependent phosphotyrosine signaling
networks, Journal of Proteome Research 9 (2010) 2734–2742.
G.M. Findlay, L. Yan, J. Procter, V. Mieulet, R.F. Lamb, A MAP4 kinase related to
Ste20 is a nutrient-sensitive regulator of mTOR signalling, The Biochemical
Journal 403 (2007) 13–20.
L. Yan, V. Mieulet, D. Burgess, G.M. Findlay, K. Sully, J. Procter, J. Goris, V.
Janssens, N.A. Morrice, R.F. Lamb, PP2A T61 epsilon is an inhibitor of MAP4K3
in nutrient signaling to mTOR, Molecular Cell 37 (2010) 633–642.
D.C. Marquez-Garban, V. Mah, M. Alavi, E.L. Maresh, H.W. Chen, L. Bagryanova,
S. Horvath, D. Chia, E. Garon, L. Goodglick, R.J. Pietras, Progesterone and
estrogen receptor expression and activity in human non-small cell lung
cancer, Steroids 76 (2011) 910–920.