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Carcinogenesis vol.31 no.3 pp.350–358, 2010
doi:10.1093/carcin/bgp181
Advance Access publication November 19, 2009
Oncofetal H19-derived miR-675 regulates tumor suppressor RB in human colorectal
cancer
Wing Pui Tsang, Enders K.O.Ng
1
, Simon S.M.Ng
2
,
Hongchuan Jin
1
, Jun Yu
1
, Joseph J.Y.Sung
1
and Tim Tak
Kwok
Department of Biochemistry,
1
Institute of Digestive Disease, Li Ka Shing
Institute of Health Sciences, State Key Laboratory in Oncology in South China
and
2
Department of Surgery, Chinese University of Hong Kong, Shatin, N.T.,
Hong Kong Special Administrative Region, China
To whom correspondence should be addressed. Science Centre, Department
of Biochemistry, Chinese University of Hong Kong, Hong Kong Special
Administrative Region, China. Tel: þ852 26036311; Fax: þ852 26037246;
Email: kwok2020@cuhk.edu.hk
H19 is an imprinted oncofetal non-coding RNA recently shown to
be the precursor of miR-675. The pathophysiological roles of H19
and its mature product miR-675 to carcinogenesis have, however,
not been defined. By quantitative reverse transcription–polymerase
chain reaction, both H19 and miR-675 were found to be upregu-
lated in human colon cancer cell lines and primary human
colorectal cancer (CRC) tissues compared with adjacent non-
cancerous tissues. Subsequently, the tumor suppressor retinoblas-
toma (RB) was confirmed to be a direct target of miR-675 as the
microRNA suppressed the activity of the luciferase reporter car-
rying the 3#-untranslated region of RB messenger RNA that con-
tains the miR-675-binding site. Suppression of miR-675 by
transfection with anti-miR-675 increased RB expression and at
the same time, decreased cell growth and soft agar colony forma-
tion in human colon cancer cells. Reciprocally, enhanced miR-675
expression by transfection with miR-675 precursor decreased RB
expression, increased tumor cell growth and soft agar colony for-
mation. Moreover, the inverse relationship between the expres-
sions of RB and H19/miR-675 was also revealed in human CRC
tissues and colon cancer cell lines. Our findings demonstrate that
H19-derived miR-675, through downregulation of its target RB,
regulates the CRC development and thus may serve as a potential
target for CRC therapy.
Introduction
Colorectal cancer (CRC) is the third most common cancer worldwide
with an estimated 1 million new cases and a half million deaths each
year (1). Screening for CRC from curable early stages has the poten-
tial to reduce both the incidence and mortality of the disease (2).
Although 5 year mortality rates of CRC have slightly declined over
the last three decades, there is still a pressing need to identify new
prognostic biomarkers and therapeutic targets for this disease. Fur-
thermore, the underlying pathophysiological mechanisms of CRC de-
velopment remain elusive (1–4).
MicroRNAs (miRNAs) are 19- to 25-nucleotide regulatory non-
coding RNAs that areinitially expressed as hairpin transcriptsof primary
miRNA. These primary miRNA hairpins are cleaved by two RNAase III
enzymes, Drosha and Dicer, to generate mature miRNAs. MiRNAs
regulate the expressions of a wide variety of genes by translation re-
pression or promoting RNA degradation and are important in the regu-
lation of various cellular processes, such as cellular proliferation,
differentiation and apoptosis (5–7). To date, .723 human miRNAs
are annotatedin the miRBase registry (miRBase version 11.0), but most
of the genes regulated by human miRNAs are not well defined.
Dysregulation of a specific spectrum of miRNAs in human malig-
nancies is frequently observed. Emerging evidence suggests miRNAs
function as both tumor suppressors and oncogenes. About 50% of
annotated human miRNAs located at chromosomal regions involved
in loss of heterozygosity, amplification or breakpoints that are asso-
ciated with cancers (5,7,8). The miRNAs downregulated in human
cancers indicate that they may function as tumor suppressors. Let-7,
which targets the oncogene RAS, has shown to be downregulated in
lung cancers (9). MiR-15 and 16, which target the antiapoptotic factor
BCL2, are downregulated in chronic lymphocytic leukemias (10).
Expression levels of miR-143 that targets ERK5 and miR-145 were
found to be decreased in colon cancer (11). In contrast, the miRNAs
upregulated in cancers may function as oncogenes. MiR-155 and its
host gene BIC are highly expressed in several types of B-cell lym-
phoma (12). The miR-17-92 cluster, which is located on chromosome
13q31, is activated by the oncogene c-Myc and is highly expressed in
B-cell lymphoma and lung cancer (13). Therefore, the importance of
miRNAs acting as a new layer of gene regulation in tumorigenesis is
emerging.
H19 is a paternally imprinted (maternally expressed) oncofetal
gene and is located on chromosome 11p15.5, close to the IGF II gene
locus. The H19 gene does not encode for a protein but instead codes
for a capped, spliced and polyadenylated 2.7 kb RNA (14–16). H19 is
highly expressed from the early stages of embryogenesis to fetal life
in many organs including the fetal adrenal, liver and placenta but is
nearly completely downregulated postnatally (17).
Emerging evidence showed that H19 expression was upregulated in
many cancers including CRC (18,19), hepatocellular carcinoma
(20), testicular cancer (21), choriocarcinoma (22), esophageal cancer
(18), ovarian cancer (23), breast cancer (24) andbladder cancer (25,26),
with or without the loss of imprinting. Patients with more H19-
positive bladder cancer cells are potentially at higher risk of recurrent
disease (27). In the tumor formed by the injection of choriocarcinoma
Jar and JEG-3 cells into the nude mice, the H19 RNA level is higher
than those cells before the injection (28). Similarly, the H19 RNA
level is greatly enhanced in tumor of human bladder carcinoma cells
formed in nude mice (25). The overexpression of H19 in cancer
tissues hints for its oncogenic function, but the exact underlying
mechanism is still not clear. Recently, H19 was reported to be the
primary miRNA precursor of miR-675 in both human and mice (29).
As both H19 and miRNAs are believed to be involved in tumorio-
genesis, this prompted us to speculate that the tumoriogenesis process
induced by H19 may be mediated through miR-675. Therefore, in this
study, we investigated the pathophysiological roles of H19 and miR-
675 in CRC carcinogenesis. Furthermore, using in silico prediction
and in vitro functional assays, we confirmed retinoblastoma (RB)
protein as a putative direct target of miR-675. This verification of
the oncogenic function of H19-miR-675-RB in CRC suggests that
this pathway may serve as the potential target for cancer therapy.
Materials and methods
Human cell lines
The human colon cancer cell lines, including 228, CaCO2, Clone A, HCT116,
HT-29, MIP101, SW480, and normal colon fibroblast cell lines, including
CCD-112CoN, CCD-18Co, were maintained routinely in Dulbecco’s modified
Eagle’s medium supplemented with 10% fetal bovine serum and 2 mM
L-glutamine (Invitrogen, Carlsbad, CA) and were grown at 37°C in a 10%
CO
2
atmosphere.
Patient samples
Primary CRC and their adjacent non-cancerous tissues were collected from
30 patients who underwent either endoscopy or surgical removal of tumors at
the Prince of Wales Hospital, Hong Kong. All patients provided written in-
formed consent for the use of their tissues. This project was approved by the
Abbreviations: CRC, colorectal cancer; mRNA, messenger RNA; miRNA,
microRNA; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide; PCR, polymerase chain reaction; RB, retinoblastoma; siRNA, small
interfering RNA; UTR, untranslated region.
ÓThe Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org 350
by guest on February 19, 2013http://carcin.oxfordjournals.org/Downloaded from
Joint The Chinese University of Hong Kong-New Territories East Cluster
Clinical Research Ethics Committee, Hong Kong. The median age of the patients
is 74 (58–89 years old). All are adenocarcinoma at stage II–IV. All tissues had
been histologically confirmed. Tissue samples were collected and immediately
snap frozen in liquid nitrogen and stored at 80°C until further analysis.
RNA extraction
Total RNA containing miRNAs was extracted from the tissues or cells using
the Trizol reagent (Invitrogen) followed by miRNeasy mini column
(miRNeasy Mini Kit, QIAGEN, Hilden, Germany) enrichment of miRNA.
In brief, tissues samples were homogenized in Trizol reagent and chloroform
was then added according to the manufacturer’s recommendations. The mix-
ture was centrifuged at 12 000gfor 15 min at 4°C, and the aqueous layer was
transferred into new tubes. Then, 1.5 volume of 100% ethanol was added to the
aqueous layer. The mixture was then applied to an miRNeasy mini column
(QIAGEN) and processed according to the manufacturer’s recommendations.
Total RNA was eluted with RNase-free water and stored at 80°C. DNase
treatment was carried out to remove any contaminating DNA. RNA concen-
trations were determined by NanoDrop spectrophotometry.
Quantitative reverse transcription–polymerase chain reaction
For detection of H19 and RB messenger RNA (mRNA), 1.5 lg total RNA was
reverse transcribed by Moloney murine leukemia virus reverse transcriptase
with oligo-dT primer according to the manufacturer’s instructions (Promega
Corporation, Madison, WI). For miR-675 detection, 3 lg total RNA was used
in the reverse transcription reaction by using the QuantMir RT Kit (System
Biosciences, Mountain View, CA). Quantitative real-time polymerase chain re-
action (PCR) was performed by using SYBR-green PCR Master Mix in a Fast
Real-time PCR 7500 System (Applied Biosystems, Foster City, CA). The mature
miR-675 DNA sequence was used as the forward primer, and the 3#universal
primer provided from the QuantiMir RT Kit as the reverse primer. The human U6
RNA was amplified in parallel as an internal control. For mRNA detection, the
gene-specific primers were: H19 (forward: 5#-TACAACCACTGCACTACCTG-
3#;reverse:5#-TGGAATGCTTGAAGGCTGCT-3#); RB (forward: 5#-AAGGA-
GACAAGTTCGCATGT-3#;reverse:5#-GCCGGTAATTGTCGTAGTTT-3#)
(30). b-Actin was amplified in parallel as the internal control. PCR reactions
were pe rformed at 95°C for 10 min, followed by 40 cycles of 95°Cfor15sand
60°C for 1 min. DCt was calculated by subtracting the Ct of U6 or b-actin RNA
from the Ct of miR-675 or the mRNA of interest, respectively. DDCt was then
calculated by subtracting the DCt of the control from the DCt of the treatment
group. Fold change of miRNA or mRNA was calculated by the equation 2
DDCt
.
Ectopic expression and gene silencing of H19 in cells
For the enhanced expression study, the full-length H19 complementary DNAwas
subcloned into pcDNA3.1 expression vector (Invitrogen). For the knockdown of
H19 expression, two complementary oligonucleotides for small hairpin RNA
targeting 5#-CATCAAAGACACCATCGGA-3#sequences were chemically syn-
thesized. The annealed hairpin small interfering RNA (siRNA) was subcloned
into pSilencer 2.1-U6 neo vector (Ambion, Austin, TX). The negative control
hairpin siRNA with no sequence homology to human genes provided by the
Fig. 1. Increased miR-675 expression in human CRCs. (A) Relative expressions of H19 mRNA and miR-675 in CRC tumor tissues and the adjacent non-
cancerous tissues. Statistical difference was analyzed by Wilcoxon signed-rank test (P50.001 for H19; P50.019 for miR-675). Both H19 mRNA and miR-675
levels were measured by quantitative reverse transcription–PCR. (B) Relative expressions of H19 mRNA (left panel) and miR-675 (right panel) in human normal
colon cell lines (CCD-18Co, CCD-112CoN) and colon cancer cell lines, Mean ± SEM, n53. (C) Positive correlation of H19 and miR-675 expression in human
colon cancer cells, r50.8784, P50.00035.
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manufacturer (Ambion) was used as the negative control. A total of 1.5 10
5
cells were seeded in 35 mm tissue culture dishes for 24 h, followed by trans-
fection with 2 lg of each respective plasmid with lipofectamine (Invitrogen) for
24 h. The cells were then subjected to RNA extraction or functional assays.
Transfection with miR-675 precursor or inhibitor
Enhanced or knockdown expressions of miR-675 were performed by trans-
fection with miR-675 precursor (Ambion) or anti-miR-675 (Ambion), respec-
tively. A total of 5 10
4
or 750 cells were plated in 35 mm culture dishes or
24/96-well plates, respectively, for 24 h and then transfected with 40 nM of
miR-675 precursor or inhibitor with lipofectamine 2000 (Invitrogen) for 24 h.
Commercially available precursor/inhibitor control (Ambion) was transfected
in parallel. The cells were then subjected to RNA/protein extraction or further
functional assays.
MiRNA target predictions
Computer-based RNA22 miRNA target detection program was used to predict
the miR-675 potential binding sites of the target mRNA (http://cbcsrv.watson
.ibm.com/rna22.html). The DNA sequence of the 3#-untranslated region
(UTR) region of RB mRNA was obtained from Genbank of the National Center
for Biotechnology Information webpage (http://www.ncbi.nlm.nih.gov/).
Luciferase activity assay
The part of RB 3#-UTR containing the 5#and 3#flanking sequences as well as
miR-675-binding sequence was amplified by a pair of primers (RB F: 5#-CT-
CTACTAGTCGTCAGTATGGTCTAACAC-3#,RBR:5#-CTCTAAGCTT-
GCTAATGCAGCTGTTTTAA-3#) and subcloned into pMIR-REPORT
vector (Ambion) immediately downstream of the luciferase gene to form the
pMIR-RB-3#-UTR construct. Restriction digestion sequences were shown in
bold letters. The pMIR-RB-3#-UTR-mut reporter construct with point muta-
tions in seed sequence was synthesized using the site-directed mutagenesis kit
(Stratagene, La Jolla, CA). A total of 5 10
4
cells were seeded in 24-well
plates for 24 h and then cotransfected with 800 ng of pMIR constructs with or
without 40 nM of miR-675 precursor/inhibitor for 24 h. Each sample was also
cotransfected with 0.05 lg of pRL-CMV plasmid-expressing Renilla luciferase
to monitor the transfection efficiency. At 24 h posttransfection, the activity of
firefly luciferase was measured by using the dual-luciferase reporter assay
system as described by the manufacturer (Promega). Relative luciferase activ-
ity was normalized with renilla luciferase activity.
MTT cell growth assay
A total of 750 cells were seeded in each well of a 96-well plate for 24 h. The
cells were then transfected with 40 nM of miR-675 precursor or inhibitor for 24
h and allowed to grow for 5 days. Thereafter, the cells were incubated in 50 ll
of 0.1 mg/ml solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) at 37°C for 3 h and then lysed in 150 ll of dimethyl sulfoxide
at room temperature for 30 min. The absorbance in each well was measured at
580 nm by a microplate reader.
Soft agar colony formation assay
Soft agar plates were prepared in six-well plates with a bottom layer of 0.6%
Noble agar in serum-free Dulbecco’s modified Eagle’s medium. The cells were
first seeded in 35 mm tissue culture dishes for 24 h and then transfected with
40 nM of miR-675 precursor or anti-miR-675. After trypsinization, 500 cells
mixed with 0.3% Noble agar in 10% fetal calf serum-supplemented Dulbecco’s
modified Eagle’s medium were seeded as the top agar layer onto the agar plates.
The cells were then incubated in a 37°C incubator for 3 weeks. The number of
colonies was counted after the colonies were stained with 0.05% crystal violet
for 1 h and washed extensively with 1phosphate-buffered saline.
Western blot analysis
Cells were lyzed in Lammeli’s lysis buffer containing 1% Triton X-100 and
scraped by a cell lifter. A 25 lg of protein was resolved in 12% sodium dodecyl
sulfate–polyacrylamide gel electrophoresis minigel and transferred onto
Immobilon-P membrane (Millipore, Billerica, MA). Membranes were probed with
primary antibody against RB (Santa Cruz Biotechnology, Santa Cruz, CA) at room
temperature for 2 h, washed extensively with 0.1% Tween-20 in phosphate-
buffered saline and incubated with secondary antibody conjugated with horse-
radish peroxidase at 1:10000 dilution. The signals were visualized with enhanced
chemiluminescence (Amersham Life Science, Buckinghamshire, UK).
Statistical analysis
The data are expressed as themean ± SEM from at least threeindependent experi-
ments. Thedifference between two groups inreal-time PCR, MTTassay,soft agar
colony formation assay and luciferase reporter assay was analyzed by two-tailed
Student’s t-test. The difference was significant for Pvalue of ,0.05. The ex-
pressions of H19 and miR-675 in CRC tissues and their matched adjacent non-
cancerous tissues were compared by Wilcoxon signed-rank test. The correlations
of miR-675 with H19 and RB mRNA expressions were examined by Pearson
correlation. The difference was considered significant for P-values of ,0.05.
Results
Confirmation of H19 as the precursor of miR-675 in human colon
cancer cells
H19 is reported to be the primary precursor of miR-675 by Cai and
Cullen, in which transfection with H19 complementary DNA contain-
ing the pri-miR-675 hairpin increased the expression of mature
miR-675 in human kidney 293T cells (29). To verify their findings,
we transfected human colon cancer cells, Clone A, HT-29, MIP101
and SW480 cells, with the H19-expressing vector and found that miR-
675 expression was greatly increased as determined by quantitative
PCR. On the other hand, deprivation of H19 expression by siRNA-
mediated knockdown remarkably reduced miR-675 expression in all
four cell lines (supplementary Figure 1 is available at Carcinogenesis
Fig. 2. The effect of miR-675 on cell proliferation in human colon cancer cells. The miR-675 expression in cells after transfection with (A) anti-miR-675 or (B)
miR-675 precursor was validated by quantitative reverse transcription–PCR. Relative miR-675 expression in cells was compared with those with the control
inhibitor or precursor, respectively. The cells were transfected with anti-miR-675 or miR-675 precursor for 24 h and allowed to grow for 5 days followed by the
MTT assay. Relative cell proliferation in cells with (A) anti-miR-675 or (B) miR-675 precursor transfection was compared with those with the control inhibitor or
precursor. Anti-control: anti-miRNA control; Pre-control: miRNA precursor control, Mean ± SEM, n53. P-values (two tailed) were calculated by Student’s
t-test, P,0.05; P,0.01.
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Fig. 3. The effect of miR-675 on the clonogenicity in soft agar of human colon cancer cells. The cells were transfected with anti-miR-675/miR-675 precursor
for 24 h. After transfection, 500 cells were plated in 0.3% soft agar for 3 weeks. Anti-control: anti-miRNA control; Pre-control: miRNA precursor control,
Mean ± SEM, n53, P,0.05; P,0.01.
H19/miR-675 regulates RB in colon cancer
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Online). Transfection with control expression vector or siRNA had no
effect on the miR-675 level in cells (data not shown). Thus, the results
were in agreement with the findings of by Cai et al. (29) that the
oncofetal H19 RNA is the primary precursor of miR-675.
Increased expression of H19 and miR-675 in primary CRC tissues and
in human colon cancer cell lines
We examined the expression of H19 and miR-675 in 20 pairs of
human CRC tissues and their adjacent normals. Both H19 and miR-
675 levels were significantly elevated in the CRC tissues than in the
adjacent non-cancerous tissues (P50.001 for H19 and P50.019 for
miR-675; Figure 1A). These results suggest that both H19 and miR-675
expressions were significantly overexpressed in human CRC. We then
examined the expressions of H19 and miR-675 in a panel of the fol-
lowing seven human colon cancer cell lines: HT-29, SW480, 228,
CaCO2, Clone A, HCT116 and MCP101. Our results showed that both
H19 and miR-675 were highly expressed in all these seven cell lines in
comparison with human colon CCD-112CoN and CCD-18Co
Fig. 4. miR-675 directly targets on RB protein. (A) Predicted binding of miR-675 with the 3#-UTR of RB mRNA. (B) The firefly luciferase activity in human
colon cancer cells after cotransfection with pMIR-Rb/pMIR-RB-mut 3#-UTR reporter construct and anti-miR-675 (Clone A, MIP101 and HT-29) or miR-675
precursor (SW480). The luciferase activity was measured by dual-luciferase reporter assay (Promega) and was normalized to Renilla luciferase activity. Mean ±
SEM, n53, P,0.01. (C) The effect of miR-675 transfection on the expression of RB protein in human colon cancer cells. The cells were transfected with
anti-miR-675 or miR-675 precursor for 24 h. Thereafter, the cells were lysed for western blot analysis of the RB protein level. Experiments were repeated at
least three times with similar results and only one of the representative results was shown. b-Actin was used as the loading control. The number beneath the protein
band is the relative expression of RB protein. The intensity of the bands was quantitated by densitometry. The expression of RB protein was first normalized with
the expression of b-actin in the sample. Relative expression of RB protein in cells with miR-675 precursor or anti-miR-675 transfection was then calculated in
relation to that of the respective control, of which the expression is designated as 1.0. Anti-control: anti-miRNA control; Pre-control: miRNA precursor control.
(D) The RB protein level in human colon cancer cells as assessed by western blotting. Experiments were repeated three times with results similarto the one shown.
b-Actin was used as a loading control, Mean ± SEM.
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fibroblast cells (Figure 1B) or human normal colon tissues (data not
shown). Furthermore, the expressions H19 and miR-675 in the cells
were correlated with each other (r50.8784; Figure 1C).
Functional effect of H19/miR-675 on cell proliferation and cellular
transformation in vitro
Overexpression of H19/miR-675 in human colon cancer cell lines and
primary CRC implied that H19/miR-675 might play a role in colo-
rectal carcinogenesis. To test this, we first investigated the functional
effect of miR-675 on cell proliferation and soft agar colony formation
of the following four selected human colon cancer cell lines: Clone A,
HT-29, MIP101 and SW480 cells. As shown in Figure 2, both knock-
down and enforced expression of miR-675 were effective. Knock-
down of miR-675 by transfection with anti-miR-675 suppressed the
tumor cell growth of all four cell lines. Approximately 20–25% re-
duction in cell growth was observed at 5 days after transfection (all
P-values ,0.05; Figure 2A). Additionally, enforced miR-675 ex-
pression by transfection with miR-675 precursor also significantly
increased 25–50% of cell growth in all four cell lines (all P-values
,0.05; Figure 2B).
Interestingly, miR-675 expression affected not only cell growth but
also malignant transformation as featured by the anchorage-indepen-
dent growth of cells in soft agar. We showed that miR-675 knockdown
by anti-miR-675 significantly reduced the clonogenicity of Clone A
(40%), HT-29 (50%), MIP101 (67%) and SW480 (50%) cells
in soft agar (all P-values ,0.05; Figure 3). On the contrary, increase
in clonogenicity was observed in Clone A (40%), HT-29 (40%),
MIP101 (70%) and SW480 (270%) cells upon transfection with
miR-675 precursor (all P-values ,0.05; Figure 3). These results pro-
vide strong evidence that miR-675 plays a role in promoting malig-
nant transformation in cells.
To elucidate whether miR-675 was the mediator for the oncogenic
function of H19, Clone A and SW480 cells were cotransfected with
H19 complementary DNA and anti-miR-675. A 40–50% increase in
the clonogenicity in soft agar was observed for cells only with H19
transfection, but such increase was, however, significantly abrogated
by the cotransfection with anti-miR-675 (supplementary Figure 2 is
available at Carcinogenesis Online). Results suggested that H19 ex-
hibited the effect on cellular transformation in human colon cancer
cells via miR-675.
MiR-675 targets RB expression
MiRNAs mainly exert their functions by targeting the 3#-UTR of the
protein-coding genes to induce mRNA degradation and/or transla-
tional repression. Using RNA22 miRNA target detection program
and setting the maximum number of the seed nucleotides as six in-
stead of seven (31), the 3#-UTR of RB mRNA was aligned with the
sequence of mature miR-675 and RB was identified to be one of the
potential targets of miR-675. The sequence alignments for miR-675
and the 3#-UTR of RB mRNA are shown in Figure 4A; miR-675
targets the nt4111–4134 of RB mRNA (GenBank accession no.
M15400.1).
To confirm whether the predicted miR-675 target site in the 3#-
UTR of RB mRNA was responsible for its regulation, the 3#-UTR of
RB mRNA flanking the entire putative target sequence or 3#-UTR
with mutated target sequence was subcloned into the firefly luciferase
reporter vector (pMIR-REPORT). The construct was then cotrans-
fected with anti-miR-675/anti-miRNA control in Clone A, HT-29,
MIP101 cells or miR-675 precursor/miRNA precursor control in
SW480 cells (the cells with the least miR-675 expression). Our results
showed that the relative luciferase activity of the pMIR-RB-3#-UTR
construct with anti-miR-675 was significantly increased in Clone A,
HT-29 and MIP101 cells (all P-values ,0.05). On the other hand,
enhanced miR-675 expression significantly decreased the relative lu-
ciferase activity in SW480 cells (P-values ,0.05). The changes in
the luciferase activity of pMIR-RB-3#-UTR upon the transfection
with miR-675 inhibitor or precursor were, however, not observed if
the miR-675-binding sequence in the reporter was mutated (Figure 4B).
By examining the RB protein level following enforced or inhibition of
miR-675 expression, our results indicated that miR-675 expression
inhibition increased the RB protein level, whereas ectopic miR-675
expression consistently decreased the RB protein level in all four cell
lines (Figure 4C). Similar results were found for RB mRNA (data not
shown). Taken together, the data from the luciferase activity assay and
western blot analysis strongly support that RB protein is a direct target
of miR-675.
The interrelationship for the expressions of H19, miR-675 and RB
was further verified in human colon cancer cells. As shown in Figure
1C, the expression of H19 is positively correlated with the level of
miR-675 in the human colon cancer cell lines. As the target of miR-
675, the level of RB protein also appears to be negatively correlated
with the levels of both H19 and miR-675 in the human colon cancer
cells (Figure 4D). Similar correction was also observed for RB mRNA
(data not shown).
The H19/miR-675 regulates RB to promote cellular transformation
As RB is a well-known tumor suppressor and hereby confirmed to be
one of the target genes of miR-675, we further investigated whether
the effect of H19/miR-675 on cellular transformation is through RB.
As expected, knockdown of RB by specific siRNA suppressed the RB
Fig. 5. The effect of miR-675/RB on soft agar clonogenicity in human colon cancer cells. The cells were transfected with RB siRNA (RBi) with or without
the anti-miR-675 for 24 h and then plated in 0.3% soft agar for 3 weeks. The sequence of RB siRNA duplex was adapted from Semizarov et al. (32). Mean ± SEM,
n53, P,0.05, P,0.01.
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protein level (40% for Clone A and 55% for SW480 cells, data not
shown) and also increased the clonogenicity of cells in soft agar
(20% for Clone A and 200% for SW480; all P-values ,0.05).
Intriguingly, the decrease in clonogenicity by the anti-miR-675,
which is known to upregulate RB, was counteracted by the cotrans-
fection with RB siRNA (Figure 5). However, such changes were not
observed in the experiment using the control siRNA. This further
confirmed that the oncogenic role of H19/miR-675 is associated with
the downregulation of RB in the cancer cells.
Expression relationship of H19, miR-675 and RB in human primary
CRC tissues
The interrelationship for the expressions of H19, miR-675 and RB has
been defined in vitro. To verify their expression relationship in human
Fig. 6. The expressions of (A) H19 mRNA, (B) miR-675 and (C) RB mRNA in human CRC tissues. Relative expressions of H19 mRNA, miR-675 and RB
mRNA in the CRC tumor tissues (T) and the matched adjacent non-cancerous tissue (N) as detected by quantitative reverse transcription–PCR, Mean ± SEM,
n53. Asterisks represent CRC samples in which both H19 and miR-675 are downregulated. (D) Correlations of miR-675 with H19 (left panel) and Rb
mRNA (right panel) in CRC tissues.
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CRC tissue samples, the expressions of H19, miR-675 and RB were
examined in an independent group of 10 pairs of human CRC tissues
with their matched adjacent non-cancerous tissues. Among this in-
dependent group analyzed, seven cases showed that the expressions of
both H19 and miR-675 were increased in the tumor tissues as com-
pared with their matched adjacent non-cancerous tissues, whereas the
RB mRNA level was also significantly reduced in the tumor tissues
(Figure 6). Such correlation is in agreement with the findings from the
present study showing that H19 is the precursor of miR-675 and the
miRNA downregulates RB expression in human colon cancer cells.
The results strongly support the probable implications of H19, miR-
675 and RB in CRC development even though other genetic factors
may also play significant role as not all CRC samples demonstrated
altered expressions of H19, miR-675 and RB.
Discussion
The present study is the first to establish the possible link between
H19/miR-675 and RB in CRC development. The overexpression of
H19 in CRC has previously been reported and it was suggested to be
due to the loss of imprinting of the gene (19). However, in this study,
we showed that both miR-675 and its precursor H19 were overex-
pressed in all seven colon cancer cell lines and the majority of primary
CRC tissues, but either was not or only very minimally expressed in
their adjacent non-cancerous tissues, suggesting the oncogenic role of
H19/miR675 in CRC development.
The oncogenic function of H19/miR-675 is featured by targeting
the well-known tumor suppressor RB. Molecular carcinogenesis of
colon cancer involves stepwise accumulation of epigenetic and ge-
netic alterations, including activating mutations of the K-Ras and
B-Raf oncogenes as well as inactivating mutations of APC and p53
tumor suppressor genes (3,33). The genetic pathways in colon carci-
nogenesis are complicated and some of the pathways may occur ran-
domly, concurrently with or even preferentially over the other
pathways (3,33,34). Recently, dysregulation of miRNAs was reported
in human CRC (11,34). The miRNAs may function as cross talk
between different colorectal carcinogenesis pathways and may there-
fore be the potential therapeutic targets. Such cross talk can be illus-
trated by the H19/miR-675/RB pathway identified in the present
study. The H19/miR-675/RB pathway was detected in human colon
cancer cell lines as well as in human CRC tissues. The role of this
pathway in CRC development is confirmed as: (i) ectopic expression
of H19-derived miR-675 promoted cell growth and induced malignant
transformation in human colon cancer cells and vice versa and (ii)
H19 and miR-675 were overexpressed in CRC tissue, whereas its
target gene, the tumor suppressor RB, was downregulated. Like
miR-675, miR-106a was also found to be overexpressed in colon
carcinoma that did not express RB (8). RB is functionally inactivated
in the majority of human cancers, subsequently leading to dysregula-
tion of cell cycles and aggressive tumor proliferation (35–38). The
role of RB in cell cycle regulation is mainly through its interaction
with transcription factor E2F. As E2F was shown to activate H19 (39),
it is therefore of interest in the future to see if there may be feedback
loop in the regulation of H19/miR-675/RB pathway.
The oncogenic role of H19 is associated with its function as the
precursor of miR-675. Even though H19 is known to have functions
related to cancer development, the underlying mechanism is still un-
clear. Although some of the H19 target genes have been identified, the
way that H19 may interact with these target genes is also still unclear.
It is believed that H19 RNA may interact directly with its target genes
or indirectly through some of the H19 interaction proteins (14–16,40).
In any case, a clear link between H19 and its target gene has yet to be
reported. The identification of H19 as the precursor of miR-675 and
the proof that the miRNA mediates the oncogenic function of H19
may provide a new perspective for the future investigation of the
action mechanism of H19. In fact, long-transcript non-coding RNA
to act as the precursor of miRNAs has been demonstrated previously,
e.g. lin 4 from lin-14 mRNA in Caenorhabditis elegans (41) and in
miR-155 from the transcript of proto-oncogene BIC (12). Neverthe-
less, the present study is the first to confirm the importance of the
miR-675 pathway in the biological function of H19.
In summary, the H19-derived miR-675 miRNA, by targeting tumor
suppressor RB, is proved to be oncogenic by promoting cell growth
and malignant transformation in human colon cancer cells. The upre-
gulation of H19 and miR-675 in CRC suggests that both H19 and
miR-675 are important factors in the tumorigenesis of CRC and that
they may also serve as potential prognosis markers as well as potential
targets for cancer therapy.
Supplementary material
Supplementary Figures 1 and 2 can be found at http://carcin
.oxfordjournals.org/
Funding
Hong Kong Research Grants Council (Earmarked Grant CUHK4270/
04M); Institute of Digestive Disease, Chinese University of Hong
Kong.
Acknowledgements
Conflict of Interest Statement: None declared.
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