ONCOLOGY LETTERS 2: 1101-1106, 2011
Abstract. MicroRNAs (miRNAs) are involved in a number
of biological processes, including tumour biology. Pre-clinical
studies have shown that miRNA-126 regulates signalling
downstream of vascular endothelial growth factor receptor 2
(VEGFR-2) and, consequently, angiogenesis. The aim of
this study was to analyse the possible relationship between
miRNA-126, VEGFR-2 and angiogenesis in tumour tissue
from patients with colorectal cancer (CRC). Tumour tissue was
obtained from 81 patients. The miRNA-126 and VEGFR-2
gene expression levels were analysed by PCR and the protein
concentrations of VEGFR-2 were analysed by ELISA.
Angiogenesis, visualised by the endothelial cell marker CD105
combined with caldesmon, was assessed by immunohisto-
chemistry and the microvessel density (MVD) technique.
In situ hybridisation was performed for miRNA-126. Tumours
were classied as low or high miRNA‑126‑expressing using
the median as the cut-off. The median gene expression levels of
VEGFR‑2 were signicantly lower in the tumours expressing
low levels of miRNA-126, 0.30 (95% CI, 0.24-0.36), compared
to those expressing high levels of miRNA-126, 0.48 (95% CI,
0.28-0.60), p=0.02. A positive association was observed
with VEGFR-2 protein concentrations, p=0.06. The median
MVD was signicantly lower in the tumours expressing low
levels of miRNA-126, 5.8 (95% CI, 5.33-6.67), compared to
those expressing high levels, 8.0 (95% CI, 6.33-9.00), p<0.01.
miRNA-126 was detected in endothelial cells by in situ
hybridisation analysis. These results suggest that high levels
of miRNA-126 in CRC are associated with high VEGFR-2
mRNA and protein levels and a higher density of newly formed
microvessels. However, further studies should be conducted to
analyse the clinical value of miRNA-126 in CRC.
Angiogenesis, the development of capillaries from pre-
existing blood vessels, is essential for the growth of malignant
tumours (1). The vascular endothelial growth factor (VEGF)
system plays a signicant role in regulating this process (2), and
the clinical benet from targeting this system in patients with
metastatic colorectal cancer (mCRC) is well documented (3).
Pre-clinical studies demonstrated a role of microRNAs
(miRNAs) in the regulation of angiogenesis and the VEGF
system (4,5). miRNA-126 has been reported to play an essen-
tial role in angiogenesis by modulating VEGFR-2-related
signal transduction through the RAS/ERK and PI3K/AKT
pathways by inhibiting regulatory units (6-9).
miRNAs are a group of small single-stranded non-coding
RNAs with a length of approximately 22 nucleotides. They
negatively regulate gene transcripts at a post-transcriptional
level by targeting mRNAs leading to mRNA degradation or
translational repression, depending on the complementarities
between miRNA and its targets (10). It has been demonstrated
that one miRNA may target several different mRNAs, and that
one mRNA may be targeted by several miRNAs; several other
principles have been suggested to explain miRNA-mediated
Approximately 1,000 unique human miRNAs have been
identied (11). Most of the miRNAs are located in introns of
mRNA transcripts, whereas others have been identied in
exons, in the 3' untranslated region (3'UTR) of a gene tran-
script or in non-coding transcripts, including miRNA clusters.
A large fraction of the identied miRNAs are located in areas
of the genome known to be involved in the development of
cancer and are consequently thought to play a prominent role
in malignant transformation (10). The expression levels of
miRNAs are often dysregulated in malignant tissue compared
to the corresponding normal tissue, which enables miRNAs
Elevated microRNA-126 is associated with high vascular
endothelial growth factor receptor 2 expression levels
and high microvessel density in colorectal cancer
TORBEN FRØSTRUP HANSEN1, CLAUS LINDBJERG ANDERSEN4, BOYE SCHNACK NIELSEN5,
KAREN-LISE GARM SPINDLER1, FLEMMING BRANDT SØRENSEN2,
JAN LINDEBJERG5, IVAN BRANDSLUND3 and ANDERS JAKOBSEN1
Departments of 1Oncology, 2Clinical Pathology, and 3Biochemistry, Vejle Hospital, Vejle;
4Department of Molecular Medicine, Aarhus University Hospital Skejby, Aarhus;
5Exiqon A/S, Diagnostic Product Development, Vedbaek, Denmark
Received February 28, 2011; Accepted July 22, 2011
Correspondence to: Dr Torben Frøstrup Hansen, Department of
Oncology, Vejle Hospital, Kabbeltoft 25, 7100 Vejle, Denmark
Key wo rds: angiogenesis, colorectal neoplasm, microRNAs,
microvessel density, vascular endothelial growth factor receptor 2
HANSEN et al: miRNA-126 IN CRC
to act as either tumour suppressors or oncogenes depending
on the function of their target networks (10). An increasing
number of reports on the clinical signicance of miRNAs in
various types of cancer, including CRC, have been published.
Differences in expression levels between CRC and normal
colorectal tissue have been described (12-14), and certain
studies have also provided evidence of prognostic (15,16) and
predictive (17) value related to miRNAs.
At present, little is known about the possible relationship
between miRNAs and the VEGF system in patients with
CRC. The aim of this study was to analyse the relationship
between miRNA-126, VEGFR-2 and neo-angiogenesis in
patients with CRC.
Materials and methods
Study population. This study included 81 consecutive patients
with CRC during the period between February 2004 and July
2005, all undergoing surgical resection for histologically veri-
ed adenocarcinomas of the colon or rectum at the Department
of Surgery, Vejle Hospital, Denmark. Patients who had
received preoperative chemoradiation for rectal cancer were
excluded. Pre-treatment examinations included a chest X-ray
and ultrasound or CT scan of the abdomen. Postoperatively,
the tumours were histologically classied and staged
according to the pTNM system. Information regarding patient
characteristics was based on patient records and registries.
The study was approved by the Regional Scientic Ethical
Committee for Southern Denmark according to Danish law,
J.nr. S-VF-20040047. Informed consent was obtained from all
patients enrolled in the study.
Tissue sampling. Immediat ely after surgery the removed bowel
segment was brought to the Department of Clinical Pathology
and tissue from the tumour was sampled by a pathologist.
Samples for mRNA and miRNA analyses were placed in
RNAlater™ (Qiagen, CA, USA) and stored at ‑20˚C. Samples
for quantitative protein analysis were frozen and stored at
‑80˚C. The samples were frozen within 30 min of surgical
removal. Samples intended for later immunohistochemistry
(IHC) and in situ hybridisation (ISH) followed routine xa-
tion and parafn embedding. Samples for quantitative protein
analysis and IHC were sampled in close proximity to each
other. Based on a microscopic examination of H&E-stained
FFPE tumour sections, it was semi-quantitatively estimated
that the tissue used for quantitative protein analyses was
dominated by carcinoma cells (>50%).
VEGFR-2 mRNA and protein analyses. The VEGFR-2
gene expression and protein analyses have previously been
described (18). Briey, tissue samples were homogenised and
total RNA was isolated according to the RNeasy® mini hand-
book of June 2001 (Qiagen, MD, USA). RNA was quantied
using spectrophotometry (Eppendorf, Hamburg, Germany)
followed by cDNA synthesis using a M-MLV RT kit
(Invitrogen Co., Carlsbad, CA, USA). The geometrical mean
of β-2-microglobulin and β-actin was used for normalisation.
Gene expression analyses were performed using uorescence‑
based real-time reverse transcription-polymerase chain
reaction (RT-PCR). The analyses were performed on the
ABI PRISM 7900 HT fast real-time PCR system, TaqMan
(Applied Biosystems, Foster City, CA, USA).
After protein extraction, VEGFR-2 was analysed
using Quantikine ELISA kits (DVR200; R&D Systems,
Minneapolis, MN, USA). The controls were also purchased
from R&D Systems. The assay employs the quantitative sand-
wich enzyme immunoassay technique. Results are presented in
pg/mg of total protein. Tissue samples, standards and controls
were assayed in duplicate and the mean was recorded. The
total coefcients of variation on three levels (low, medium and
high concentrations of VEGFR-2) were 11.3, 10.1 and 8.3%,
CD105 and caldesmon immunostaining and MVD counting.
The staining and counting procedures have previously been
described (19). Briey, MVD was measured using tissue
sections stained by antibodies against CD105 and caldesmon
in order to visualise immature microvessels. A kit from
Dako (Glostrup, Denmark; code K5361, EnVision™ G⎪2
doublestain system, rabbit/mouse, DAB+/permanent red)
was used for the detection of primary antibodies. The anti-
CD105 antibody was obtained from Novocastra (Bristol, UK;
endoglin NCL-CD105, clone 4G11) and used at a dilution of
1:25. The anti-caldesmon antibody was obtained from Dako
(code M3557, clone h-CD) and used at a dilution of 1:50.
Microvessels were counted at the invasive tumour front by
two observers unaware of the clinical parameters. The mean
MVD from three hotspots was used for statistical analysis.
Any stained endothelial cell or endothelial cell cluster clearly
separated from adjacent microvessels by tumour cells and/or
stroma elements was considered a single countable microvessel.
Vessel lumen was not necessary for a structure to be counted
as a microvessel. Consensus counts resolved any discrepancy
between the observers. Regarding the CD105 (brown) and
caldesmon (red) staining, only microvessels without associated
red staining were counted in order to preferentially estimate
the MVD of the immature vessels.
miRNA-126 gene expression analyses. miRNA quantication
was carried out using predesigned TaqMan miRNA assays
(Applied Biosystems). cDNA synthesis, pre‑amplication
and qPCR were performed according to the manufacturer's
instructions. In brief, RNA was reversely transcribed using
the TaqMan miRNA reverse transcription kit in combination
with the stem-loop Megaplex™ primer pool A v2.0, allowing
simultaneous reverse transcription of 377 unique miRNAs.
Subsequently, the Megaplex RT product was pre‑amplied
using TaqMan PreAmp master mix and Megaplex PreAmp
primer pool A v2.0. RT‑PCR amplication of miRNA‑126
(part no. 4395339) was performed in TaqMan Universal PCR
master mix. Cycling conditions were as follows: 95˚C for
10 min followed by 40 cycles of 95˚C for 15 sec and 60˚C
for 1 min. PCR reactions were performed on the ABI PRISM
7900 HT Fast RT-PCR system (Applied Biosystems). Raw
Cq values were calculated using the SDS software v2.1 using
automatic baseline and threshold settings. Expression levels
of Hsa-miR-340 (part no. 4427975) were used for normali-
sation, since this miRNA was found by the NormFinder
algorithm (20), a rigorous statistical approach for evaluating
expression variation of normalisation gene candidates, to be
ONCOLOGY LETTERS 2: 1101-1106, 2011 110 3
the most stably expressed transcript throughout the tissue
miRNA-126 in situ hybridisation. ISH was performed as
described elsewhere (21). In brief, ISH was carried out on 6-µm
tissue sections containing CRC and normal colorectal tissue
using double DIG-labelled LNA probes for human miRNA-
126 (Exiqon A/S, Ved baek, Denmark). The DIG-labelled
probe was detected with alkaline phosphatase-conjugated
sheep anti-DIG Fab fragments followed by NBT-BCIP chro-
mogenic staining and nuclear fast red staining.
Statistical analysis. The Fisher's exact test was used for
two-group comparisons. Median values were compared
using the Wilcoxon rank sum test. Disease-free survival
(DFS) was dened as the time from surgery until the rst
documented tumour recurrence or death. Progression-free
survival (PFS) was dened as the time from surgery until the
rst documented tumour recurrence, progression or death.
Overall survival (OS) was dened as the time from surgery
until death. Survival curves were determined according to
the Kaplan-Meier method, and the log-rank test was used to
test for differences between the groups. Survival data from
patients diagnosed with a new malignancy following their
surgical resection for CRC (5 patients) were censored from the
date of their new cancer diagnosis. These data were excluded
to prevent any potential bias related to the presence of a new
cancer or the chemotherapeutic treatments used. Statistical
calculations were carried out using NCSS statistical software
(NCSS Statistical Software, version 2007; Kaysville, UT,
USA). P<0.05 was considered to be statistically signicant and
all tests were two-sided.
Patient characteristics. The patient characteristics, along
with miRNA-126 expression levels in CRC obtained by PCR
analysis, are shown in Table I. Female patients presented
with a signicantly lower median miRNA‑126 expression
in their tumour tissue compared to their male counterparts.
Furthermore, non‑signicant associations were observed with
regard to age at diagnosis and tumour localisation. Younger
patients and patients with left-sided colon cancers presented
with a higher miRNA-126 expression. No differences were
observed between the patient characteristics, as listed in
Table I, and gene or protein levels of VEGFR-2 or MVD. The
5-year survival rate for the entire cohort was 60%.
miRNA-126 expression. Paired samples of CRC and normal
colorectal tissue were obtained from 28 patients. The
median gene expression of miRNA-126 in CRC, 0.85 (95%
CI, 0.71‑0.97), did not differ signicantly from the median
gene expression in normal colorectal tissue, 1.01 (95% CI,
0.80-1.11), p=0.56. The distribution of miRNA-126 expres-
sion levels in the CRC samples for the entire patient cohort
is shown in Fig. 1A. Tumours were classied as low or high
miRNA-126-expressing tumours using the median as a cut-off.
The distribution of VEGFR-2 gene expression in CRC
based on miRNA-126 levels is shown in Fig. 1B. The
median gene expression level of VEGFR‑2 was signicantly
lower in the tumours expressing low levels of miRNA-126,
0.30 (95% CI, 0.24-0.36), compared to those expressing high
levels, 0.48 (95% CI, 0.28- 0.60), p= 0.02.
Table I. Patient characteristics.
No. (%) miRNA-126
(n=81) Median p-value
Male 37 (46) 1.00 (0.87-1.28)
Female 44 (54) 0.83 (0.59-1.08)
Age (years) 0.09
Mean (SD) 71.7 (11.1)
>mean 44 (54) 0.85 (0.89-1.48)
<mean 37 (46) 1.02 (0.89-1.48)
T category 0.72
1-3 63 (78) 0.97 (0.82-1.12)
4 18 (22) 0.79 (0.59-1.48)
N category 0.81
0 46 (57) 0.99 (0.72-1.13)
1-2 35 (43) 0.96 (0.77-1.28)
M category 0.57
0 65 (80) 0.96 (0.81-1.12)
1 16 (20) 0.90 (0.70-1.69)
I-II 43 (53) 1.01 (0.68-1.13)
III-IV 38 (47) 0.95 (0.77-1.28)
Rectum 20 (25) 0.88 (0.70-1.48) 0.88
Colon 61 (75) 0.98 (0.77-1.23) 0.07
Left colon 28 (46) 1.05 (0.94-1.47)
Right colon 33 (54) 0.77 (0.56-1.13)
MSI status 0.33
MSI 15 (19) 0.72 (0.38-1.24)
MSS 66 (81) 0.96 (0.82-1.08)
Tumour grade 0.74
1 and 2 61 (75) 0.95 (0.79-1.08)
3 20 (25) 0.96 (0.59-1.58)
Vascular invasion 0.42
Yes 10 (12) 1.08 (0.70-1.73)
No 71 (88) 0.96 (0.79-1.02)
Neuronal invasion 0.18
Yes 10 (12) 1.32 (0.70-1.87)
No 71 (88) 0.94 (0.77-1.02)
Yes 17 (21) 0.77 (0.59-1.36)
No 64 (79) 0.97 (0.83-1.12)
CI, condence interval; SD, standard deviation; MSI, microsatellite
instable; MSS, microsatellite stable.
HANSEN et al: miRNA-126 IN CRC
The distribution of VEGFR-2 protein concentrations in
CRC based on miRNA-126 levels is shown in Fig. 1C. The
median protein concentration of VEGFR-2 was slightly lower,
although not signicant, in the tumours expressing low levels of
miRNA-126, 115 pg/mg (95% CI, 98-140), compared to those
expressing high levels, 134 pg/mg (95% CI, 121-180), p=0.06.
The median MVD was signicantly lower in the tumours
expressing low levels of miRNA-126, 5.8 (95% CI, 5. 33 -
6.67), compared to those expressing high levels, 8.0 (95% CI,
6.33-9.00), p<0.01 (Fig. 1D).
miRNA-126 in situ hybridisation. ISH analyses using high-
afnity LNA probes revealed intense ISH signals in endothe-
lial cells. miRNA-126-positive vessels were observed in the
tumour stroma, as well as in the lamina propria of normal
mucosa and in the deep unaffected bowel wall (Fig. 2).
miRNA-126 and prognosis. The possible prognostic value of
miRNA-126 expression in CRC was assessed by comparing
patients with a low expression of miRNA-126 to those with a
high expression, using the median as the cut-off. Neither PFS
[hazard ratio 0.85 (95% CI, 0.44-1.64)] nor OS [hazard ratio
0.99 (95% CI, 0.49‑1.98)] differed signicantly between the
two groups (p=0.64 and p=0.98, respectively).
Our patient cohort included a total of 28 patients with micro-
satellite stable (MSS) stage II CRC and, within this subgroup,
short DFS was signicantly linked to high miRNA‑126 expres-
sion levels (Fig. 3).
In the present pilot study, a direct correlation was found
between miRNA-126, VEGFR-2 and angiogenesis in samples
from patients with CRC.
Paired samples of CRC and normal colorectal tissue
were obtained from 28 patients, and the median miRNA-126
expression level in the two tissue types did not differ signi-
cantly. Guo et al (7) demonstrated that the miRNA-126 level
was signicantly lower in colon cancer compared to normal
colon tissue in a panel of 6 paired samples. In both cases, the
results are based on a rather limited number of paired samples
and should therefore be interpreted with caution. Furthermore,
the use of different normalisation methods (Guo et al used
5S rRNA) may also be signicant in this context.
The miRNA‑126 expression in CRC was signicantly
lower in females compared to males, and a possible, albeit
non‑signicant, difference was also observed regarding age
and tumour localisation. The miRNA-126 expression tended
to be lower in the older group of patients and in patients with
right-sided colon cancers.
Based on these ndings, a correlation with microsatellite
instable (MSI) status was expected. However, no such correla-
tion was found in the present study. Díaz et al (15) analysed
several miRNAs in 110 patients who underwent surgery for
CRC. miRNA-126 expression was found to be signicantly
lower in the youngest group of patients and no signicant
association with gender was documented. These discrepancies
Figure 1. (A) Distribution of miRNA-126 gene expression in CRC tissue. (B) Distribution of VEGFR-2 gene expression in CRC tissue according to m iRNA-126
levels, p=0.02. (C) Distribution of VEGFR-2 protein concentration in CRC tissue according to miRNA-126 levels, p=0.06. (D) Distribution of CD105-positive
microvessel density in CRC tissue according to m iRNA-126 levels, p<0.01. Black bars, medians, n=81.
ONCOLOGY LETTERS 2: 1101-1106, 2011 110 5
are difcult to explain since the two studies involved a
similar number of patients, included Caucasians only, used
almost the same methodology, although Díaz et al used 5S
for normalisation, and also included patients with stage I-IV
disease. The study by Díaz et al used fresh-frozen tumour
tissue compared to the present study in which miRNA was
extracted from tissue preserved in RNAlater. A possible effect
on miRNA-126 expression levels by directly comparing pres-
ervation in RNAlater and freezing of the tissue specimens
remains to be claried.
A high miRNA-126 expression was correlated to a high
gene expression of VEGFR-2 and likely also to high protein
concentrations of VEGFR-2 in CRC, although the latter
correlation did not reach statistical signicance (p=0.06).
Furthermore, a high miRNA‑126 expression was signicantly
correlated to a higher density of newly formed micro
vessels. These results, obtained in a clinical setting, suggest
a relationship between the VEGF system, neo-angiogenesis
and miRNA-126, and correlate well with results reported
in the pre-clinical literature. Two recent studies (6,9) have
demonstrated the signicant role of miRNA‑126 in media‑
ting developmental angiogenesis and identied two specic
targets for miRNA-126, i.e., the sprouty-related, EVH1
domain-containing protein 1 (SPRED-1) and the phospho-
inositol-3 kinase regulatory subunit 2 (PIK3R2), both acting
as negative regulators of the RAS/ERK and PI3K/AKT
pathways, respectively. miRNA-126 is capable of potenti-
ating the angiogenic signalling downstream of VEGFR-2 by
modulating the signals through these pathways. Other studies
have demonstrated a similar regulatory role of miRNA-126
with regard to these pathways (7,22). In 2010, Nicoli et al
(8) published results suggesting that blood ow stimulates
angiogenesis in the zebrash by a genetic pathway in which
a transcription factor (klf2a) induces the expression of
miRNA-126 and thereby enhances VEGF-A signalling. These
pre‑clinical studies, conrm that high levels of miRNA‑126
increase signalling downstream of VEGFR-2, ultimately
resulting in increased angiogenesis. To a certain extent, this
increase is supported by the present results obtained in a
Our results showed a relationship between miRNA-126,
VEGFR-2 and neo-angiogenesis estimated by CD105 MVD,
suggesting that miRNA-126 expression was correlated to
tissue vascularisation. The miRNA-126 ISH analyses revealed
an expression in endothelial cells in malignant and normal
colorectal tissue similar to results from various in vitro
studies (5,6,8,9). These observations indicate high and specic
miRNA-126 expression levels in endothelial cells. The clinical
value of the miRNA-126 ISH analyses, which may be an alter-
native to the MVD technique based on IHC, deserves further
analysis using larger patient cohorts.
The possible prognostic value of miRNA-126 expression
in CRC was analysed. However, no signicant correlation
with survival was found in the unselected patient cohort,
which corresponds to the results obtained by Díaz et al (15). A
subgroup analysis consisting of patients with stage II disease
only and MSS tumours is relevant due to the need to identify
new prognostic markers in stage II CRC, and the well-known
prognostic difference between MSI and MSS tumours (23)
justies this selection. Although this subgroup only consisted
of 28 patients in the present study, it is signicant that
Figure 2. miRNA-126 ISH. (A and B) Expression of miRNA-126 in normal
colon tissue. (C) Expression in colon cancer tissue. The miRNA-126
ISH signal is observed in endothelial cells and is not conned to tumour‑
Figure 3. Kaplan-Meier DFS curves according to miRNA-126 expression in
patients with stage II and MSS tumours, n=28 (14 patients in each group).
The solid line pertains to patients with a low miRNA-126 expression and the
dashed line to patients with a high miRNA-126 expression, p=0.03.
HANSEN et al: miRNA-126 IN CRC
our observations are consistent with the ndings reported
by Schepeler et al (16). The se authors demonstrated that
miRNA-126, among other miRNAs, was up-regulated
in the primary tumour tissue from patients with stage II
MSS colon cancers who experienced relapse signicantly
later compared to those that did not. A meaningful multi-
variate survival analysis was not conducted due to the small
sample size. Therefore, although miRNA-126 expression
in CRC does not appear to be of prognostic value in the
entire patient cohort, it may harbour a prognostic impact
for a selected group of CRC patients. The meta-analysis by
Des et al demonstrated a correlation between high MVD
and poor prognosis in CRC (24). The present relationship
between high MVD and high miRNA-126 expression in the
present study provides a plausible explanation for these nd-
ings, although this hypothesis requires validation in a larger
In conclusion, signicant associations were found between
miRNA-126 levels in CRC, VEGFR-2 gene expression and
neo-angiogenesis, although a great overlap was observed
between the compared groups. Conclusions should therefore
be drawn with caution, and additional testing in larger and
more homogeneous study populations is necessary to validate
the results. The present results correspond well with the pre-
clinical literature, indicating a pivotal role of miRNA-126 in
the modulation of angiogenesis (6-9,25). Thus, miRNA-126
may harbour predictive information with regard to anti-
angiogenetic therapy. A better understanding of how this
miRNA is regulated is of great signicance. Further studies
that analyse the clinical value of miRNA-126 in CRC, are
The authors are grateful for the technical assistance provided
by Lone Frischknecht, Lone Hartmann Hansen, Sara Egsgaard,
Birgit Roed Sørensen, Pamela Celis and Karin Larsen. This
study was supported by The Cancer Foundation, which had no
inuence on any part of the study.
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