ABBS 2009,41(07): BMPRII is a direct target of miR-21

*Correspondence address. Tel: +86-21-54921222;
Fax: +86-21-54921011; E-mail: [email protected]

MicroRNAs (miRNAs) are a type of small non-coding RNAs
that regulate cognate mRNA expressions at the post-transcriptional stage.
Although several miRNAs are known to be involved in various biological
processes, including developmental timing, patterning, embryogenesis,
differentiation and organogenesis, growth control, and apoptosis, many target
genes and the functions of most miRNAs are still unclear. Since there is only a
partial complementarity between miRNAs and their targets in animal cells, it is
difficult to identify the specific target genes for a given miRNA and elucidate
its function. In this study, we confirmed that bone morphogenetic protein
receptor II (BMPRII) is a direct target of miR-21, and also showed that the
protein level of BMPRII correlates inversely with the amount of miR

Keywords     microRNA; miR-21;
BMPRII; target; prostate cancer

Received: March 20, 2009 Accepted: April 7, 2009

Introduction

MicroRNAs (miRNAs) are a recently discovered class of approximately 21
nucleotide long RNA molecules that negatively regulate the expression of target
mRNAs at the post-transcriptional level [1–3]. Their importance in a variety of biological processes has become
apparent during the last decade [4–7]. Recent advances in
miRNA research have provided evidence of aberrant gene expressions in different
kinds of diseases such as cancer, cardiovascular diseases, psychological
disorders, and others [8–11].

MiR-21 has been identified as the most expressed miRNA in a number of
profiling experiments designed for the detection of miRNAs dysregulated in
cancer. MiR-21 is abundantly expressed in most cancer cell lines [12]. It has
been found that miR-21 plays a role in various fields such as development,
oncology, stem cell biology, and aging. It is one of the most studied miRNAs
[13]. Since there is only a partial complementarity between miRNAs and their
targets in animal cells, it is difficult to identify the specific target genes
for a given miRNA and elucidate its function. Although miR-21expression is
generally associated with cancer, it has several other direct targets such as
programmed cell death 4, tropomyosin 1, and phosphatase and tensin homolog
deleted on chromosome 10 [14–16].

Bone morphogenetic proteins (BMPs) are the members of the tumor growth
factor (TGF)-b super family. BMP-Smad signaling regulates
stem cell renewal, cell proliferation, differentiation, migration, and
apoptosis, and controls embryo development and postnatal tissue homoeostasis
[17–19]. BMPs exert their effects via a heteromeric receptor complex, which
consists of two types of serine-threonine kinase transmembrane receptors. BMPRII is the type 2 receptor
of BMPs; mutations in BMPRII lead to the development of hereditary pulmonary hypertension, and its
knockout results in early embryonic lethality [20,21].
BMPRII initiates intracellular signaling in response to the following specific
ligands: BMP-2, BMP-4, BMP-6, BMP-7, growth and differentiation factor (GDF)-5,
and GDF-6 [22]. BMPRII plays a critical role in the maintenance of normal pulmonary vascular
physiology, cell proliferation, cell death, and stem cell differentiation and
self-renewal [23,24]. Loss of BMPRII expression in
both prostate cancer tissues and cell lines has been shown to be associated
with the progression of prostate cancer [25]. In this study, we investigated
whether BMPRII is the direct target of miR-21 and found that the protein level of BMPRII
correlated inversely with miR-21 levels in PC3 and Lncap cells, two different
kinds of prostate cancer cells with different malignancy and metastasis status.
These findings suggest that miR-21 might be involved in the progression of
prostate cancer and self-renewal of stem cells.

Materials and Methods

Cell culture

293T, PC3, and Lncap cells were obtained from the Cell

Construction of 3‘-untranslated region-luciferase plasmid

The two upstream and the two downstream predicted binding sites on the 3‘-untranslated region (UTR) of BMPRII along with _1 kb of the contiguous sequences were cloned into the EcoRI–NdeI and NdeI–PstI sites of pGL3 (Promega, Madison, USA) and designated as Luc-BS12-BMPRII and Luc-BS34-BMPRII, respectively. In
addition, the two downstream predicted binding sites, which also included 1 kb
of the contiguous sequences, were cloned into the NdeI and PstI sites of Luc-BS12-BMPRII; this plasmid was
designated as Luc-B1234-BMPRII. The following primers were used for plasmid construction. BS12-sense
primer: 5‘-TCAG
AATTCAATCAGTTTGGACCAGTTTC-3‘; BS12-antisense
primer: 5‘-TCACATATGGTTTGATAGCAGC
CCTTC-3‘; BS34-sense primer: 5‘-TCA CATATGAATCC TCGTAAGTATGTAAAGGA-3‘; BS34-antisense primer: 5‘-TCACTGCAGAAACGGAATAACGCACCA-3‘.

Luciferase assay

For reporter assays, the cells were transiently cotransfected into 12-well
plates with the luciferase vector Luc-BS12-BMPRII, Luc-BS34-BMPRII, Luc-B1234-BMPRII, or pGL3 reporter plasmid and either
miR-21-mimcs (Ambion,

RNA isolation

Total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, USA)
according to the manufacturer’s instructions. Contaminating genomic DNA was removed from the isolated
RNA by treatment with amplification-grade DNase I (Invitrogen,

Protein extraction and western blot

Protein was extracted with protein extraction reagent (Sigma,

Results

BMPRII is a predicted target of miR-21

Using the TargetScan software (http://www.targetscan. org/), it was
predicted that there are 4 binding sites in the 3‘ UTR region of the BMPRII gene [Fig. 1(A)]. These four
predicted binding sites were located at the 527, 945, 7296, and 7480 nt in the 3‘ UTR region of the BMPRII gene, respectively. The free energy values for the 4 BSs were
approximately –19.3, –19.2, –21.4, and –26.8 Kcal/mol [Fig. 1(B)]. Generally, all the miR-21 seed region (2–8 nt position) and corresponding pairing sites
make near perfect complementarity, which is a common accepted principle in
miRNA target prediction.

Two predicted binding sites on BMPRII are broadly conserved among
vertebrates By comparing the human sequence of the BSs for interspecies homology, we
found that two of the predicted miR-21 binding sequences, BS3 and BS4, are
highly conserved among several vertebrate species, indicating this kind of
regulation via miR-21 is a conserved mechanism for fine tuning its expression
level in evolution (Fig. 2).

Luciferase assay confirmed that BMPRII could be regulated by miR-21

Since the distance between the two upstream and the two downstream
predicted binding sites was very long, we first cloned these two upstream and
downstream sites separately along with _1 kb of the contiguous sequences and designated them B12 and B34,
respectively. To determine the predicted binding sites that could be regulated
by miR-21, the fragment containing the sequences for BS12 and BS34 was cloned
separately into the 3‘ UTR region of the luciferase gene in a reporter vector pGL3. Subsequently,
the two luciferase reporter vectors with the miR-21 response elements were
transfected into 293T cells. We used the seeding region-mutated miR-21-mimics
as the negative control (NC-mimics). NC-mimics control or miR-21-mimics were
also cotransfected into these cells. Then, the activity of luciferase and
Renilla in these cells was measured. It was found that miR-21 decreased the
luciferase activity of the reporter vector containing either the miR-21
response elements B12 or B34 [Fig. 3(A,B)]. These data suggest
that miR-21 could regulate the expression of BMPRII by binding to not only the two downstream
conserved binding sites but also the two upstream predicted binding sites. To
further investigate whether all the four predicted binding sites are required
for the regulation of miR-21, all the four binding sites regions were cloned
into the 3‘ UTR of the luciferase gene in the pGL3 plasmid. The results of the
luciferase assay revealed that the expression of luciferase gene containing the
predicted binding sites was downregulated by _50% in the presence of the miR-21 mimics. The downregulation of the
luciferase gene was _30% in the presence of
ASO-miR-21 [Fig. 3(C)]. Taken together, these results confirmed that BMPRII is regulated by miR-21. The protein level
of BMPRII correlates
inversely with the amount of miR

Discussion

BMPs are one of the largest groups of cytokines within the TGF-b super family. BMPs
were originally identified as molecules that regulate the growth and
differentiation of bones and cartilage [26,27]. BMPRII
is a type II serine/threonine kinase receptor of BMPs that is required to
initiate the intracellular signaling pathway. Mutations in BMPRII lead to hereditary
pulmonary hypertension, and knock out of BMPRII results in early embryonic lethality [28,].
miR-21 is well-known as an oncogene in a variety of
tumor types. A previous study has determined several targets of miR-21, most of
which are associated with cell growth [29]. In this study, we provided evidence
that BMPRII is a direct target of
miR-21 and has four predicted target sites in its 3‘ UTR region. Among the
four predicted binding sites, two downstream sites are conserved among several
vertebrate species. Our data showed that miR-21 could regulate the expression
of BMPRII by binding to not only
the two downstream conserved binding sites but also the two upstream predicted
binding sites. We speculate that these four binding sites could function
together to enhance the regulation ability of miR-21. We confirmed that BMPRII, which is associated
with self-renewal and differentiation of stem cells, is a target of miR-21.
These data suggest that miR-21 not only regulates cell growth and stem cell
differentiation but is also involved in many other biological processes.

In this study, it was found that the protein level of BMPRII correlated
inversely with miR-21 levels in PC3 and Lncap cells. LncaP cells are
androgen-sensitive human prostate adenocarcinoma cells derived from the left
supraclavicular lymph node metastasis. PC3 cell lines were originally derived
from the advanced androgen-independent bone metastasis. The metastatic and
malignancy potential of PC3 cells is higher than that of Lncap cells, which
have moderate metastatic potential [30]. Our data showed that the expression
level of miR-21 was higher in PC3 than in Lncap cells, suggesting a possibility
that miR-21 could have potential therapeutic applications in cancer. BMPRII has
been reported to be expressed at low levels in prostate cancers, and loss of BMPRII in both prostate
cancer tissues and cell lines have been shown to have an association with the
progression of prostate cancers [31]. Our study suggested that miR-21 might be
associated with the malignance and metastatic abilities of cancer cells by
regulating BMPRII.

In conclusion, we have confirmed that BMPRII is a direct target of miR-21. In addition,
we found that the protein level of BMPRII correlates inversely with miR-21
levels in PC3 and Lncap cells. The results of this study provided further
evidence of the extensive role of miR

Funding

This work was supported by grants from the National Key Basic Research and
Development Program (2005CB724602) and the Programs from

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