MiR-126 inhibits proliferation of small cell lung cancer cells by targeting SLC7A5

Abstract

Despite intensive efforts to improve therapies, small cell lung cancer (SCLC) still has a dismal median survival of 18 months. Since miR-126 is under-expressed in the majority of SCLC tumors, we investigated the effect of miR-126 overexpression on the proliferation and cell cycle distribution of H69 cells. Our results demonstrate that miR-126 inhibits proliferation of H69 cells, by delaying the cells in the G1 phase. Short interfering RNA (siRNA) mediated suppression of SLC7A5, a predicted target of mir-126, has the same effect on H69 cells. We also show for the first time that SLC7A5 is a direct target of miR-126.

Full text

miR-126 inhibits proliferation of small cell lung cancer cells by targeting SLC7A5
Edit Miko
a
, Zoltán Margitai
b
, Zsolt Czimmerer
a
, Ildikó Várkonyi
c
, Balázs Dezs}
o
c
, Árpád Lányi
d
,
Zsolt Bacsó
b
, Beáta Scholtz
a,
⇑
a
Dept. of Biochemistry and Molecular Biology, Clinical Genomics Center, University of Debrecen Medical and Health Science Center (UDMHSC), Hungary
b
Dept. of Biophysics and Cell Biology, UDMHSC, Hungary
c
Dept. of Pathology, UDMHSC, Hungary
d
Institute of Immunology, UDMHSC, Hungary
article info
Article history:
Received 30 December 2010
Revised 16 March 2011
Accepted 16 March 2011
Available online 23 March 2011
Edited by Tamas Dalmay
Keywords:
Small cell lung cancer
miR-126
SLC7A5
G1 delay
abstract
Despite intensive efforts to improve therapies, small cell lung cancer (SCLC) still has a dismal median
survival of 18 months. Since miR-126 is under-expressed in the majority of SCLC tumors, we inves-
tigated the effect of miR-126 overexpression on the proliferation and cell cycle distribution of H69
cells. Our results demonstrate that miR-126 inhibits proliferation of H69 cells, by delaying the cells
in the G1 phase. Short interfering RNA (siRNA) mediated suppression of SLC7A5, a predicted target of
mir-126, has the same effect on H69 cells. We also show for the first time that SLC7A5 is a direct tar-
get of miR-126.
Ó
2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
Small cell lung cancer (SCLC) is an aggressive form of lung can-
cer, and it appears that current therapeutical regimens have
reached their maximum potential for this type of cancer. Therefore,
development of targeted therapies for SCLC seems to be inevitable
to improve treatment. SCLC is a fast-growing tumor, whose prolif-
eration is being fuelled in part by several aberrantly activated pro-
proliferative signalling pathways. The role of microRNAs (miRNAs)
in regulating cell proliferation in normal and tumor cells is well
established. In addition, due to the nature of miRNA–mRNA inter-
actions, the same miRNA may regulate a given pro-proliferative
pathway through multiple proteins, or may affect parallel path-
ways through different targets. miR-126 appears to have a complex
role in regulating cellular proliferation. It has an anti-proliferative
effect in several tumor types including non-small cell lung cancer
(NSCLC) cells and colon cancer cells, through targeting different
members of the PI3K/Akt pathway [1–3], or in breast cancer cells,
by targeting IRS1 [4]. In addition to its functions regulating the cell
cycle, miR-126 is a key regulator of vessel development, by target-
ing SPRED1 and PIK3R2 in endothelial cells [5,6]. Lastly, miR-126
may be involved in the metastatic process, as evidenced by its
effects on mammary or gastric carcinoma cell migration [7–9].
Since our previous work has shown that miR-126 is uniformly
under-expressed in primary SCLC tumors [10], in the present study
we investigated its role in regulating the proliferation of SCLC cells.
2. Materials and methods
2.1. Cell culture and transfection
Human H69 and HTB-172 SCLC cell lines were purchased from
the American Type Culture Collection. The cells were grown in
RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and
penicillin/streptomycin at 37 °C with 5% CO
2
. miRNA Precursor
Molecule hsa-miR-126 (PM12841) and Pre-miR Negative
Control –1 (AM17110) were obtained from Ambion
(Austin,Texas). Short interfering RNAs (siRNAs) targeting SLC7A5
(s15653), PLK2 (s64) and Silencer Select Negative Control –1 siR-
NA (4390843) were obtained from Applied Biosystems (Foster Ci-
ty,CA). Cells were transfected with pre-miR-126, SLC7A5, PLK2
siRNAs and controls at a final concentration of 50 nM in all exper-
iments, using Lipofectamine 2000 (Invitrogen). Cells were incu-
bated with the transfection complexes for 6 h before replacing
the medium. Cells were refed daily with fresh growth medium.
0014-5793/$36.00 Ó2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2011.03.039
Abbreviations: SCLC, small cell lung cancer; ECM, extracellular matrix; miRNA,
microRNA; NSCLC, non-small cell lung cancer; AML, acute myeloid leukemia;
siRNA, short interfering RNA; qPCR, real-time quantitative polymerase chain
reaction; UTR, untranslated region; RNAi, RNA interference
⇑
Corresponding author. Address: Dept. of Biochemistry and Molecular Biology,
H-4010 Debrecen, Egyetem tér 1, Hungary. Fax: +36 52 314 989.
E-mail address: scholtz@dote.hu (B. Scholtz).
FEBS Letters 585 (2011) 1191–1196
journal homepage: www.FEBSLetters.org

2.2. Establishing the growth curves
H69 cells: 1.2 10
6
cell/well were plated in 6-well plates and
transfected with miRNA precursors. Forty-eight hours post-
transfection 1/10th of the cells were replated in 24-well plates,
and grown further. Cell numbers were determined by trypan blue
exclusion at different time points (48, 72, 96 h) post-transfection.
Results represent the mean of three independent experiments.
Transfection and proliferation analysis of HTB-172 cells is
described in Supplementary figure legends,Fig. 1.
2.3. Cell cycle analysis
1.2 10
6
cell/well were plated in 6-well plates and transfected
with pre-miRs and siRNAs. Forty-eight hours post-transfection 1/
5th of the cells from each transfection were replated and grown fur-
ther. Cells were harvested at 72 and 96 h post-transfection, washed
in PBS and fixed in ice-cold 95% methanol at 20 °C. Fixed cells were
washed twice in PBS, then resuspended in 0.5 ml PBS containing
propidium iodide (50
l
g/ml) and RNase A (200
l
g/ml) and were
stained overnight at 4 °C. Measurements were performed on a FACS-
Array 96-well plate flow cytometric bioanalyzer (Becton Dickinson).
The DNA dye was excited with the 532 nm laser line and emission
was collected in the yellow channel in linear mode with a 585/
42 nm bandpass filter. Cell clusters were gated out using FSC-A/
SSC-A (Area) and FSC-W/SSC-W (Width) 2D-histograms. Fluores-
cence intensity data were fit with the automatic one cycle diploid
model of the Modfit LT 3.0 software (Verity Software House) with
the AutoDebris compensation, AutoAggregate Compensation and
Apoptosis Model turned on. In the measurements the G1–G2 linear-
ity ratio was around 1.92 and the R.C.S. of the fit (reduced
v
2
,a
measure of goodness of fit) was less than 5. All samples were
prepared in triplicates and generally 50 000 cells were collected
from each well.
2.4. Total RNA extraction and qRT-PCR
Total RNA was isolated using Trizol reagent (Invitrogen), fol-
lowed by DNase I treatment (Ambion). For quantification of
mRNAs, mature miR-126 and RNU38B, reverse transcription was
performed by using High-Capacity cDNA Reverse Transcription
Kit (Applied Biosystems). RT primers for mature miR-126 and
RNU38B were supplied by Applied Biosystems. Real-time quantita-
tive polymerase chain reaction (qPCR) was performed with Fast-
Start SYBR Green Master Mix (Roche) with 0.3
l
M of forward
and reverse primers on an ABI 7900 HT sequence detection system.
The PCR program used for amplification was: 95 °C for 10 min, fol-
lowed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, 72 °C for 45 s.
HPRT1 was used for normalization. PCR primer sequences are in
Supplementary Table 1. For semi-quantitative PCR of mature
miR-126 and RNU38B, specific primers were supplied by Applied
Biosystems. The PCR program used for semi-quantitative amplifi-
cation was: 95 °C for 10 min, followed by 15 cycles of 15 s at
95 °C and 60 s at 60 °C. PCR products were separated by agarose
gel electrophoresis and visualized by EtBr staining.
2.5. Western blot
Cells were washed with PBS and lysed in 2X SDS loading buffer.
Proteins were separated on 10% SDS polyacrylamide gels and
transferred onto nitrocellulose membrane by electroblotting.
Membranes were blocked with 2.5% nonfat milk, and probed
with primary antibody against human SLC7A5 (3157-1, 1:1000,
Epitomics), PLK2 (Snk/H90, sc-25421, 1:100, Santa Cruz
Fig. 1. Effects of miR-126 overexpression on the proliferation of H69 cells. H69 cells were transfected with pre-miR-126 or NegmiR (negative control). Asterisks indicate
significant t-test results (P< 0.05). (A) MiR-126 overexpression inhibits proliferation of H69 cells. Following transfection, viable cells were counted by trypan blue exclusionat
the indicated time points. (B) Representative cell cycle analysis of H69 cells at 96 h post-transfection, analyzed on a FACSArray bioanalyzer. (C) MiR-126 overexpression
delays H69 cells in G1 phase of the cell cycle. Cells were analyzed on a FACSArray bioanalyzer at 72 or 96 h post-transfection.
1192 E. Miko et al. / FEBS Letters 585 (2011) 1191–1196

Biotechnology) and GAPDH (6C5, sc-32233, 1:1000, Santa Cruz
Biotechnology). The membranes were further probed with
horseradish peroxidase-conjugated secondary antibodies
(1:10,000; anti-mouse or anti-rabbit; Amersham) and proteins
were visualized by SuperSignal West Pico chemiluminescent
substrate (Pierce).
2.6. SLC7A5 3
0
untranslated region (UTR) cloning and luciferase
reporter assay
For luciferase reporter assays, 331 bp from 3
0
UTR of SLC7A5
gene, including the miR-126 target site, was amplified by PCR
using F1 and R1 primers with XhoI and NotI sites. PCR was per-
formed on H69 cDNA created by High-Capacity cDNA Reverse
Transcription Kit (Applied Biosystems). The XhoI/NotI-digested
PCR product was cloned into the XhoI/NotI-digested psiCHECK2
dual luciferase vector (Promega). F1/R2 and F2/R1 primers were
used to delete the miR-126 target site from the 3
0
UTR. After mixing
the two PCR products, and digestion with XhoI and NotI, the 3
0
UTR
fragment with a deleted miR-126 binding site was cloned into
XhoI/NotI-digested psiCHECK2 vector. Primer sequences are given
in Supplementary Table 2. H69 cells were cotransfected with
500 ng psiCHECK2 constructs (WT-UTR or DEL-UTR) and 50 nM
pre-miR-126 or pre-miR-negative control in 6-well plates. Forty-
eight hours post-transfection, firefly and renilla luciferase activities
were measured using Dual-Glo Luciferase assay system (Promega)
in a Perkin Elmer Victor3V Multilabel Plate Reader. The ratio of the
luminescent signals from renilla versus firefly was used to deter-
mine the target specificity of miR-126. All experiments were done
in triplicate.
2.7. Statistical analysis
Statistical analysis was done using GraphPad Prism IV software.
Pvalues were calculated by paired t-test. Pvalues <0.05 were
considered significant.
2.8. Immunohistochemistry
Tissue microarrays of formalin-fixed paraffin-embedded surgical
specimens representing primary SCLC tumors were constructed.
These tumor specimens were characterized in our previous study
[8] as chromogranin-A/synaptophysin and thyroid-transcription fac-
tor-1 (TTF1) positive tumors (>70%). Following hematoxylin-eosin
staining, serial sections and antigen retrieving were made for immu-
nohistochemistry (IHC) labeling using rabbit monoclonal antibody to
SLC7A5 (1:300 dilution; Epitomics, Burlingame, CA, USA) for 1 h at
room temperature. Envision (biotin-free) peroxidase-based detection
kit (Dako, Glostrup, Denmark) for mouse/rabbit antibodies was then
used with the red AEC or brown DAB substrate-chromogen (Vector
Labs, Burlingame, CA) followedby hematoxylin nuclear counterstain-
ing as previ ously described [25,26]. Alternatively, we performeddou-
ble immunofluorescent (IF) staining[25] where SLC7A5 antibody was
visualized with the use of horse-radish peroxidase (HRP)-coupled
anti-rabbit IgG(Fab)
2
and tyramide-FITC for green fluorescence
followed by mAB to TTF1 and biotinylated secondary antibody treat-
ments (all from Dako), developed with streptavidin-texas red for red
fluorescence (Vector). Specificity of the IHC-staining was determined
by negativecontrol staining, using non-immune control rabbit serum
(Dako) in place of the primary antibody (data not shown). Finally,
tissue-reactivities for the antibodies (percentage of positive cells)
were evaluated for each case, as described earlier [26],byusinga
3D-Histotech-Zeiss slide-scanner and Mirax-viewer software
program (3D-Histotech, Budapest, Hungary).
3. Results
3.1. Overexpression of miR-126 inhibits proliferation of SCLC cells by
causing delay in the G1 phase
To understand the role of miR-126 in the proliferation of SCLC
cells, H69 cells were transiently transfected with miR-126
Fig. 2. miR-126 suppresses SLC7A5 protein production. H69 cells were transfected with pre-miR-126 or NegmiR (negative control). Cell lysates and total RNA were prepared
at 96 h post-transfection. (A) Representative western blot for SLC7A5 and PLK2 in transfected H69 cells. GAPDH protein levels were used for normalization. The numbers
above the blot indicate normalized protein amounts relative to the negative control, as determined by densitometry. (B) Overexpression of mature miR-126 in transfected
cells determined by semi-quantitative RT-PCR. PCR products were visualized after electrophoresis in an EtBr-stained agarose gel. (C) SCL7A5, but not PLK2 protein production
is suppressed by miR-126, as determined by western analyses. (D) Effect of miR-126 overexpression on SLC7A5 and PLK2 mRNA levels, as determined by qRT-PCR.
E. Miko et al. / FEBS Letters 585 (2011) 1191–1196 1193

precursor, or the negative control miRNA, and cell numbers were
monitored for 96 h. As expected, transfection of pre-miR-126 into
H69 cells resulted in increased miR-126 expression compared to
non-transfected or NegMiR control-transfected cells (Fig. 2B). Over-
expression of miR-126 resulted in a significantly decreased prolifer-
ation of H69 cells, evident from 72 h post-transfection (Fig. 1A).
Similar observations were made for another SCLC cell line, HTB-
172 (Supplementary Fig. 1) On the other hand, overexpression of
miR-199a, which is also down-regulated in H69 cells, had no effect
on H69 cell proliferation (data not shown). Flow cytometric cell
cycle analysis at two time points (72 and 96 h post-transfection)
revealed an increasing percentage of miR-126-transfected cells in
the G1 phase over time, and a concomitant decrease in the percent-
age of cells in the G2/M phase. (Fig. 1B and C).
3.2. Overexpression of miR-126 suppresses SLC7A5 expression at both
the RNA and the protein level
To identify potential targets for miR-126 that might play a role
in regulating proliferation of SCLC cells, we first performed an in
silico analysis using the miRNA target prediction databases Target-
Scan and PicTar (Table 1). However, with the exception of SLC7A5,
none of the validated or doubly-predicted target genes are known
to be overexpressed in SCLC cell lines or tumors [11]. SLC7A5 pro-
tein overexpression in SCLC is in accordance with the previously
described downregulation of miR-126 expression. Therefore, we
selected SLC7A5 for further studies to analyze the role of
miR-126 in the cell cycle regulation of SCLC.
Table 1
Predicted and validated targets for miR-126.
Gene symbol Enscmbl ID Target
scan
PicTar Experimentally validated
CRK NM 016823 + + +
PLK2 NM 006622 + + +
SLC7A5 NM 003486 + +
PTPN9 NM 002833 + +
FBX033 NM 203301 + +
RGS3 NM 021106 + +
SPRED1 NM 152594 + +
TOM1 NM 005488 + +
IRS1 NM 005544 + +
HOXA9 NM 002142 + +
VCAM1 NM 001078 +
PIK3R2 NM 005027 +
SOX2 NM 003106 +
Fig. 3. Suppression of SLC7A5 production by RNAi delays H69 cells in the G1 phase. H69 cells were transfected with siRNAs specific to SLC7A5 (siSLC7A5) or PLK2 (siPLK2), or
with the negative control siRNA. (A) Representative western blot for SLC7A5 and PLK2 in siRNA-transfected H69 cells. GAPDH protein levels were used for normalization. The
numbers above the blot indicate normalized protein amounts relative to the negative control, as determined by densitometry. (B) Representative cell cycle analysis of H69
cells at 96 h post-transfection, analyzed on a FACSArray bioanalyzer. (C) Suppression of SLC7A5 production by RNAi delays H69 cells in the G1 phase. Cells were analyzed on a
FACSArray bioanalyzer at 72 or 96 h post-transfection. Asterisks indicate significant t-test results (P< 0.05).
1194 E. Miko et al. / FEBS Letters 585 (2011) 1191–1196

We next investigated the effect of miR-126 overexpression on
SLC7A5 and PLK2 expression. miR-126 overexpression in H69 cells
caused more than a 50% reduction in SLC7A5 mRNA levels, and also
a slight suppression of PLK2 mRNA expression, as determined by
qRT-PCR (Fig. 2C). Subsequent western blot analysis of SLC7A5 and
PLK2 demonstrated that while miR-126 overexpression resulted in
decreased SLC7A5 protein levels, PLK2 protein levels did not change
significantly (Fig. 2A and B). SLC7A5 expression was also suppressed
in pre-miR-126 transfected HTB-172 cells (Supplementary Fig. 2).
3.3. Suppression of SLC7A5 by RNA interference (RNAi) delays SCLC
cells in the G1 phase
To better understand the effect of SLC7A5 in SCLC cell cycle con-
trol, we utilized RNAi to specifically suppress SLC7A5 production in
H69 cells, and performed cell cycle analysis by flow cytometry at
72 and 96 h post-transfection. Transfection of specific siRNA into
H69 cells resulted in significantly lower SLC7A5 expression when
compared to the negative control siRNA (Fig. 3A). Similarly to the
effect of miR-126 overexpression, suppression of SLC7A5 resulted
in an increasing percentage of transfected cells in the G1 phase
over time, and a concomitant decrease in the percentage of cells
in the G2/M phase, when compared to the negative control siRNA
(Fig. 3B and C). In contrast, specific suppression of PLK2 expression
by RNAi had no such effect on the cell cycle distribution of trans-
fected H69 cells.
3.4. SLC7A5 is a direct target of miR-126
To confirm that SLC7A5 is a molecular target of miR-126, as sug-
gested by the previous experiments, we constructed a luciferase
reporter vector containing 331 bp of the SLC7A5 3
0
UTR, including
the predicted miR-126 binding site (WT-UTR). We also constructed
a control luciferase vector with the miR-126 binding site deleted
from the SLC7A5 3
0
UTR (DEL-UTR) (Fig. 4A). The sequenced
plasmids showed 100% identity with the SLC7A5 3
0
UTR, and the
intended deletion in the control vector (data not shown). H69 cells
were transiently transfected with the WT-UTR-luciferase or the
DEL-UTR-luciferase vector and with pre-miR-126. Co-transfection
of WT-UTR with pre-miR-126 resulted in a significant decrease in
luciferase protein levels; however, deletion of the miR-126 binding
site from the SLC7A5 3
0
UTR abolished this effect of miR-126
(Fig. 4B).
The correlation of miR-126 and SLC7A5 expression was also
investigated in 12 primary SCLC tumor samples, using immunohis-
tochemistry (IHC) with SLC7A5-specific antibody. SLC7A5 expres-
sion was not detectable in normal lung tissue, which is in
accordance with previous observations (Supplementary Fig. 3A, in-
set, and [12]). In contrast, the SCLC tumors tested positive for
SLC7A5 protein expression – in fact, 8 tumors contained more than
70% SLC7A5-positive cells. As demonstrated by the double IF
stained specimens, the majority of tumor cells exhibited nuclear
staining for TTF1 (typical feature for SCLC) with SLC7A5 co-expres-
sion (Supplementary Fig. 3B). The tumor samples analyzed with
IHC were the same samples analyzed before for aberrant miR-
126 expression [8]. Since all 12 SCLC tumors overexpressed SLC7A5
and under-expressed miR-126, the inverse correlation between the
expression levels of miR-126 and its target could be corroborated
in primary tumors (Fig. 5).
4. Discussion
In the present work we demonstrate for the first time that
miR-126 overexpression has a negative effect on SCLC cell prolifer-
ation, by delaying cells in the G1 phase of the cell cycle. However,
miR-126 is not just an anti-proliferative miRNA; rather, it appears
to have multiple functions depending on the cell type and the
actual cellular environment. This is underscored by the observa-
tions, that not all validated miR-126 target mRNAs are affected
by miR-126 in every cell type. Interestingly, in SCLC cells
miR-126 overexpression does not suppress PLK2 expression, even
though PLK2 was shown to be a bona fide target for miR-126 in
CBF acute myeloid leukemia (AML) cells. A similar observation
can be made for TOM1, which is targeted by miR-126 in CF airway
epithelium cells, but not in MCF7 cells [4,12]; or for SPRED1, which
is targeted in HUVEC cells, but not in AML cell lines [5,13].Itis
presently unclear how certain target mRNAs are presented to,
and others are protected from miR-126 in these experimental
setups, but the resulting target selectivity could contribute to the
varied functions of miR-126.
Fig. 4. SLC7A5 is a direct target of miR-126. (A) The predicted miR-126 binding site
in the wild type SLC7A5 3
0
UTR (WT-UTR), and in the deleted construct (DEL-UTR).
(B) Relative luciferase activity of the SLC7A5 WT-UTR and the DEL-UTR luciferase
constructs in H69 cells transfected with miR-126 or the negative control (NegmiR).
Fig. 5. SLC7A5 and miR-126 expression levels are inversely correlated in primary
SCLC tumors. Relative expression levels of mature miR-126 (left Yaxis) were
determined in primary SCLC tumor specimens by qRT-PCR [8]. Overexpression of
SLC7A5 in the same panel of primary SCLC tumors was determined by immuno-
histochemistry, using an SLC7A5-specific monoclonal antibody. Percentage of
SLC7A5-positive neoplastic cells was determined for each tumor specimen (right
Yaxis). Normal lung tissue exhibited no SLC7A5-specific staining (Supplementary
Fig. 3).
E. Miko et al. / FEBS Letters 585 (2011) 1191–1196 1195

Importantly, we identified a novel target of miR-126 in SCLC
cells, SLC7A5, which is the light chain of the heterodimeric 4F2
amino acid transporter. SLC7A5 is overexpressed in many cancer
types, including SCLC, and its expression levels are usually corre-
lated to cancer progression and aggressiveness [14–16].We
demonstrated that in SCLC cells, similarly to other tumor types
[17–19], suppression of SLC7A5 expression has an anti-proliferative
effect. SCL7A5 suppression or miR-126 overexpression both delay
SCLC cells in the G1 phase, suggesting that the effect of miR-126
on the cell cycle is at least in part mediated through SLC7A5. Con-
sequently, decreased miR-126 expression contributes to high
SLC7A5 expression in SCLC cells, and, thus, may ensure efficient
transport of essential amino acids in the rapidly proliferating
tumor cells.
On the other hand, miR-126 may also be involved in a more
direct regulation of the cell cycle in SCLC. Glutamine–leucine
exchange by SLC7A5 was shown to activate the nutrient and
growth factor integrating kinase mTOR, which in turn phosphory-
lates p70S6 kinase 1 and 4EBP1, leading to the production of
growth promoting proteins [20]. Removal of miR-126 from the
regulatory network may enhance the existing positive feedback
between SLC7A5 and mTOR [21,22], and can contribute signifi-
cantly to the proliferative potential of the tumor cells. In addition,
miR-126 is capable of targeting the PI3K/Akt pathway as well,
either through PIK3R2 or some other mechanisms [1,2,5]. It should
be noted that both the PI3K/Akt and the mTOR pathways are
indeed active in a large percentage of SCLC tumors [23–26].
In summary, our work has identified miR-126 as an important
negative regulator of the growth and proliferation of SCLC cells,
which probably fine-tunes the activity of the PI3K/Akt/mTOR
network through multiple targets, including SLC7A5. However,
miR-126 may have additional functions in the tumor stroma: in
normal endothelial cells it is a positive regulator of angiogenesis,
and it may have interesting functions in regulating the immune
response. Therefore, more research is needed to understand the
complex role of miR-126 in the growth, survival and progression
of SCLC tumors in vivo, and to determine how miR-126 may poten-
tially be exploited as an anti-tumor agent.
Acknowledgments
This work was supported by grants from the National Office for
Research and Technology (B.S.: NKFP 2004 OM-00427 and GVOP-
3.1.1.-2004-05-0263/3.0; Z.B.: GVOP-3.2.1-2004-04-0351/3.0;
B.D.: NKTH-TECH-08-A1-2008-0228; Á.L.: TÁMOP 4.2.1./B-09/1/
KONV-2010-0007), by grants from the Hungarian Research Foun-
dation (Z.B.: OTKA T046945; Á.L.: OTKA 81676), and by grants from
the Hungarian Scientific Research Fund (Z.B.: OMFB-01626/2006).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.febslet.2011.03.039.
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