MicroRNA-34a Modulates c-Myc Transcriptional
Complexes to Suppress Malignancy in Human Prostate
Soichiro Yamamura, Sharanjot Saini, Shahana Majid, Hiroshi Hirata, Koji Ueno, Guoren Deng, Rajvir
Department of Urology, San Francisco Veterans Affairs Medical Center and University of California San Francisco, San Francisco, California, United States of America
MicroRNA-34a (miR-34a), a potent mediator of tumor suppressor p53, has been reported to function as a tumor suppressor
and miR-34a was found to be downregulated in prostate cancer tissues. We studied the functional effects of miR-34a on c-
Myc transcriptional complexes in PC-3 prostate cancer cells. Transfection of miR-34a into PC-3 cells strongly inhibited in vitro
cell proliferation, cell invasion and promoted apoptosis. Transfection of miR-34a into PC-3 cells also significantly inhibited in
vivo xenograft tumor growth in nude mice. miR-34a downregulated expression of c-Myc oncogene by targeting its 39 UTR
as shown by luciferase reporter assays. miR-34a was found to repress RhoA, a regulator of cell migration and invasion, by
suppressing c-Myc–Skp2–Miz1 transcriptional complex that activates RhoA. Overexpression of c-Myc reversed miR-34a
suppression of RhoA expression, suggesting that miR-34a inhibits invasion by suppressing RhoA through c-Myc. miR-34a
was also found to repress c-Myc-pTEFB transcription elongation complex, indicating one of the mechanisms by which miR-
34a has profound effects on cellular function. This is the first report to document that miR-34a suppresses assembly and
function of the c-Myc–Skp2–Miz1 complex that activates RhoA and the c-Myc-pTEFB complex that elongates transcription
of various genes, suggesting a novel role of miR-34a in the regulation of transcription by c-Myc complex.
Citation: Yamamura S, Saini S, Majid S, Hirata H, Ueno K, et al. (2012) MicroRNA-34a Modulates c-Myc Transcriptional Complexes to Suppress Malignancy in
Human Prostate Cancer Cells. PLoS ONE 7(1): e29722. doi:10.1371/journal.pone.0029722
Editor: Alfons Navarro, University of Barcelona, Spain
Received December 27, 2010; Accepted December 3, 2011; Published January 3, 2012
Copyright: ? 2012 Yamamura et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by Grants R01CA138642, T32DK007790 (NIH), VA Research Enhancement Award Program (REAP) and Merit Review grants.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
MicroRNAs (miRNAs) are highly conserved, single stranded,
non-coding RNAs of approximately 22 nucleotides that regulate
gene expression by posttranscriptional silencing of specific target
mRNAs, by repressing translation or cleaving RNA transcripts .
miRNAs regulate diverse cellular processes such as cell-cycle
progression, proliferation, apoptosis and development. miRNAs
have been shown to function as oncogenes or tumor suppressor
The p53 tumor suppressor is deleted or mutated in more than
50% of human tumors and is a key molecule which suppresses
malignancies . p53 has been found to target the miR-34 family
[4,5,6] and the ectopic expression of miR-34 genes has drastic
effects on cell proliferation and survival. Ectopic miR-34a causes
cell-cycle arrest in the G1 phase [6,7] and apoptosis [7,8]. As p53
has been found to target miR-34a and since, cell-cycle arrest and
apoptosis are also end points of p53 activation, the miR-34a gene
may be a mediator of p53 function.
The proto-oncogene c-Myc regulates cell proliferation and
transformation both transcriptionally and non-transcriptionally
and is frequently deregulated in human cancers [9,10]. c-Myc is a
basic helix–loop–helix and leucine zipper transcription factor
which binds to Enhancer Box elements (E-boxes) and activates the
transcription of genes which stimulate cell cycle progression and
cell growth. c-Myc suppresses the transcription of genes which
arrest the cell cycle, through Miz1, the c-Myc associated protein.
c-Myc also has a function to recruite histone acetyltransferases
(HATs). c-Myc non-transcriptionally interacts with components of
the replication machinery to positively regulate DNA synthesis,
leading to genomic instabilities.
c-Myc was reported to activate MiR-17-92, a polycistronic
microRNA cluster consisting of miR-17, 18a, 19a, 20a, 19b and
92a [11,12]. miR-19 was found to be the principal oncogenic
component of this cluster, targeting the tumour suppressor PTEN
. miR-34c has been shown to negatively regulate c-Myc in
response to DNA damage and to inhibit c-Myc-induced DNA
synthesis . During oncogene-induced senescence, miR-34a
was also found to target c-Myc .
Rho GTPases are small G proteins that regulate various cellular
processes, including cytoskeletal dynamics, migration, vesicle
[16,17,18]. Rho GTPases, their regulators, and their effectors
have been suggested to control tumor formation and progression.
RhoA has been shown to control cancer metastasis and
progression [19,20,21]. Recently, c-Myc complex was found to
activate the RhoA gene .
The positive transcription elongation factor b (P-TEFb)
regulates the promoter-proximal pause release of the elongation
phase of transcription by Pol II  and integrates mRNA
PLoS ONE | www.plosone.org1 January 2012 | Volume 7 | Issue 1 | e29722
synthesis with histone modification, pre-mRNA processing and
mRNA export . P-TEFb is composed of cyclin (CycT1,
CycT2a, CycT2b or CycK) and cyclin-dependent kinase 9 (Cdk9)
. c-Myc interacts with cyclin T1 (CycT1), the regulatory
component of P-TEFb, and controls the elongation phase of
transcription by Pol II [25,26,27].
The c-Met pathway is dysregulated in most human malignan-
cies and regulates tumor formation and progression [28,29]. c-Met
interacts with hepatocyte growth factor/scatter factor and has
been implicated in tumor invasion and migration including PC-3
We report here that miR-34a was downregulated in prostate
cancer tissues. miR-34a inhibited cell proliferation in vitro, in vivo
tumor growth and promoted apoptosis in prostate cancer cells. We
also found miR-34a targets c-Met and inhibits PC-3 cell invasion.
We investigated the effects of miR-34a on the two c-Myc
transcriptional complexes. By targeting c-Myc, miR-34a reduced
the c-Myc-Miz-Skp2 complex which induces RhoA transcription
and inhibited cell invasion, showing that miR-34a indirectly
regulates RhoA. miR-34a also suppressed the c-Myc-P-TEFb
complex that plays a key role in controlling the elongation phase of
transcription by RNA polymerase II (Pol II), indicating one of the
mechanisms by which miR-34a has dramatic effects on cellular
function. Our results demonstrate that in prostate cancer PC-3
cells miR-34a suppresses assembly and function of the c-Myc
complex that activates or elongates transcription, revealing a novel
role of miR-34a in the regulation of transcription by c-Myc.
miR-34a expression is downregulated in prostate cancer
We examined the expression levels of miR-34a in laser capture
microdissected (LCM) prostate cancer tissues (n=10) and matched
adjacent normal regions by real-time PCR. All cancer tissues
showed lower miR-34a levels compared with matched adjacent
normal regions, demonstrating that miR-34a is downregulated in
cancer (Fig. 1A). Histological data shows that these tissues are
adenocarcinoma with Gleason scores of 6 or 7. Gleason patterns
and other clinical data are shown in Table S1.
We also assayed miR-34a expression levels in malignant (PC-3,
LNCaP and DU145 cells) and non-malignant prostate RWPE-1
cells. Real-time RT-PCR revealed that the expression level of
miR-34a was markedly lower in PC-3 cells compared with non-
malignant epithelial prostate cell RWPE-1 cells (data not shown).
This result was consistent with the previously reported data using
PrEC normal human prostate epithelial cells . We also
compared the expression levels of miR-34a in malignant prostate
cells and the laser capture microdissected (LCM) prostate normal
tissues (n=10). Real-time RT-PCR showed that the expression
level of miR-34a was lower in malignant prostate cells compared
with the average of the expression level of miR-34a in the normal
tissues (data not shown). These results were also consistent with the
previously reported data using normal human tissues .
miR-34a inhibits cell proliferation of PC-3 cells
To study the effect of miR-34a on the growth of prostate cancer
cells, we transiently transfected several cell lines with pre-miR
negative control (NC) or pre-miR-34a. The transient transfection
of pre-miR-34a increased miR-34a levels in prostate cancer cells
(Fig. S1A). MTS assay showed that miR-34a inhibited PC-3 cell
proliferation by about 40% on day 4 but had no significant effect
on LNCaP and DU145 cells (Fig. S2A). A recent report indicates
that PC-3 cells have characteristics of prostatic small cell
carcinoma and not of adenocarcinoma , however we used
PC-3 cells for further study because the proliferation suppression
effect of miR-34a was significant, which was consistent with the
previously reported data . Transient transfection of pre-miR-
34a also caused significant morphological changes in PC-3 cells
We employed a lentiviral system to express miR-34a. The HIV
lentiviral system, expressing miR-34a or vector control, was used
to infect PC-3 cells and the infected cells were selected with
puromycin. The stable transfection of pre-miR-34a increased
miR-34a levels in PC-3 cells (Fig. S1B). We performed soft agar
colony formation assay using the infected PC-3 cells expressing
miR-34a or control. miR-34a reduced colony numbers to about
20% of that of control, showing that miR-34a significantly
inhibited the colony forming ability of PC-3 cells (Figs. 1B and C).
To examine the effects of the ectopic expression of miR-34a on
in vivo tumor growth, we subcutaneously injected the stable miR-
34a or the control cell line into nude mice. Tumor volumes were
measured every 7 days for 5 weeks following the injection.
Xenograft tumors from PC-3 cells overexpressing miR-34a were
smaller than xenograft tumors from the control cells. At week 5,
tumor sizes of control xenografts were about 5 times larger than
those of miR-34a xenografts, indicating that the ectopic expression
of miR-34a significantly suppressed tumor growth in vivo (Figs. 1D
Since ectopic expression of miR-34a suppressed PC-3 cell
proliferation, we next studied the effects of miR-34a on apoptosis
in PC-3 cells using flow cytometry. We found that ectopic
expression of miR-34a increased PC-3 cell apoptosis to about4
times of that controls (Fig.1F and Fig. S3), demonstrating that
miR-34a has apoptotic activity in PC-3 cells.
miR-34a inhibits cell invasion and migration
We performed a transwell invasion assay using Matrigel to
investigate the effect of miR-34a on the invasive ability of PC-3
cells. We transiently transfected PC-3 cells with pre-miR negative
control (NC) or pre-miR-34a and subjected the transfected cells to
transwell invasion assay. The results clearly revealed that miR-34
reduced invasion of PC-3 cells to 20% of that of controls (Figs. 2A
and B). Wound-healing assay also showed that miR-34a markedly
reduced the migration of PC-3 cells (Figs. 2C and D).
miR-34a targets c-Myc and c-Met
Oncogenes, c-Met and c-Myc, have predicted binding sites for
miR-34a in their 39-UTRs (Fig. S4). c-Met has 2 predicted binding
sites for miR-34a in its 39-UTRs (Fig. S3). We cloned the putative
miR-34a targets in the 39UTRs into a luciferase construct.
Luciferase reporter assays with miR-34a-expressing PC-3 cells
showed that miR-34a repressed luciferase activity. Mutation of the
putative miR-34a target sites in these UTRs decreased the
response to miR-34a. These results indicate that miR-34a binds
to the 39-UTRs of c-Myc and c-Met (Fig. 3A).
We examined the effects of miR-34a transfection on the protein
levels of these genes. Transfection of miR-34a reduced the protein
levels of c-Met and c-Myc, protein in PC-3 cells (Fig. 3B). These
results confirm that miR-34a directly targets c-Met and c-Myc via
binding to their 39UTRs in PC-3 cells. miR-34a also reduced
levels of transcription factor E2F1 which regulates the cell cycle,
DNA replication and cell proliferation (Fig. 3B).
miR-34a inhibits RhoA and suppresses assembly of c-Myc
The c-Myc–Skp2–Miz1 transcriptional complex has been found
to activate RhoA gene and to be critical for cell invasion and
miR-34a Targets c-Myc Transcriptional Complexes
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cancer metastasis . Since we found that c-Myc is a target of
miR-34a, thus downregulating the expression of c-Myc, we
examined whether the downregulation of c-Myc by miR-34a
results in reduction of RhoA expression by suppressing assembly of
the c-Myc–Skp2–Miz1 complex. Real-time PCR showed miR-34a
decreased RhoA mRNA level (Fig. 4A) and Western blot revealed
that miR-34a reduced RhoA protein expression (Fig. 4B).
We performed immunoprecipitation (IP) to study assembly of
the c-Myc–Skp2–Miz1 transcriptional complex in miR-34a
transfected cells. IP (Fig. 4C) revealed that anti-c-Myc immuno-
precipitates contained Miz1, Skp2 and Max (lane 5) but not anti-
IgG (control) immunoprecipitates (lane 3), indicating that the c-
Myc–Skp2–Miz1 complex was formed in PC-3 cells. IP also
showed that miR-34a decreased the levels of these components in
the c-Myc immunoprecipitates [lanes 4 (control) and 6], indicating
that miR-34a suppressed assembly of the c-Myc–Skp2–Miz1
transcriptional complex in PC-3 cells. miR-34a did not signifi-
cantly reduce Skp2 in the immunoprecipitates compared with the
We performed chromatin immunoprecipitation (ChIP) assays to
examine whether miR-34a reduces binding of c-Myc to the RhoA
promoter region containing E-boxes 5 and 6, which is a primary
binding site of c-Myc . The ChIP assay showed that
endogenous c-Myc binds to E-boxes 5 and 6 and control
experiments demonstrated that RNA polymerase II binding to
the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pro-
Figure 1. miR-34a inhibits cell growth. (A) Relative miR-34a expression in laser capture microdissected (LCM) prostate cancer tissues (C) and
matched adjacent normal regions (N). (B) miR-34a overexpression suppresses soft agar colony formation of a stably transfected miR-34a PC-3 cell line.
After 14 days incubation, colonies with over 50 cells were counted. The values are normalized to those of control. (C) Representative images of the
colonies. (D) miR-34a inhibits in vivo tumor growth. Time course of tumor growth in nude mice after subcutaneous injection of a stably transfected
miR-34a PC-3 cell line or control cell line. *, P,0.05 compared with control. (E) Representative images of tumors in nude mice at 5 weeks after
subcutaneous injection of a stably transfected miR-34a PC-3 cell line or control cell line. (F) miR-34a induces apoptosis in PC-3 cells. PC-3 cells were
transfected with pre-miR negative control (NC) or pre-miR-34a for 3 days. PC-3 cells were stained with AnnexinV-FITC/7-AAD and apoptosis was
analyzed by flow cytometry. The data shows the percentage of early apoptotic and apoptotic cells out of the total cell population of PC-3 cells.
*, P,0.05 compared with control.
miR-34a Targets c-Myc Transcriptional Complexes
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moter was not altered (Fig. 4D). These results suggested that miR-
34a reduced the recruitment of the c-Myc–Skp2–Miz1 complex to
the RhoA promoter and reduces RhoA expression.
Overexpression of c-Myc reverses inhibition of invasion
To determine whether inhibition of invasion by miR-34a could
be reversed via restoration of c-Myc, we transfected PC-3 cells
with a c-Myc expression plasmid together with pre-miR-34a. c-
Myc overexpression partially rescued RhoA expression (Fig. 5A,
compare lane 4 with 3) and miR-34-induced suppression of
invasion (Fig. 5B), suggesting that miR-34a inhibits invasion, at
least partially, via RhoA reduction by targeting c-Myc.
miR-34a suppresses assembly of c-Myc-P-TEFb complex
The positive transcription elongation factor b (P-TEFb) plays a
key role in controlling the elongation phase of transcription by
RNA polymerase II (Pol II) . It is a cyclin-dependent kinase
comprised of Cdk9 andcyclin. c-Myc has been shown to interact
with P-TEFb through CycT1 and regulate transcription elonga-
tion [25,26,27]. Since miR-34a targets c-Myc, we performed
Figure 2. miR-34a inhibits invasion and migration of PC-3 cells. (A) miR-34a inhibits invasion of PC-3 cells. PC-3 cells were transiently
transfected with pre-miR negative control (NC) or pre–miR-34a for 48 h. The cells were harvested and subjected to transwell invasion assay. The
values are normalized to those of NC. *, P,0.05 compared with control. (B) Representative images of the invaded cells. (C) miR-34a inhibits wound-
healing of PC-3 cells. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 48 h. The cells were harvested and
subjected to wound-healing assay. The width of the remaining open wound calculated in relation to separation at time 0 h. *, P,0.05 compared with
control. (D) Representative images of the wound healing assay.
miR-34a Targets c-Myc Transcriptional Complexes
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immunoprecipitation (IP) to study assembly of the c-Myc-pTEFB
transcription elongation complex in mir-34a-transfected PC-3
cells. IP (Fig. 6A) revealed that anti-c-Myc antibody pulled down
CycT1, Cdk9 and Max (lane 5) but IgG (control) did not pull
down these proteins (lane 3), indicating that c-Myc interacted with
P-TEFb. IP also showed that miR-34a decreased the amount of p-
TEFb in the c-Myc immunoprecipitates in PC-3 cells [lanes 4
(control) and 6], indicating that miR-34a suppressed assembly of
the c-Myc–P-TEFb complex in PC-3 cells.
c-Myc was reported to activate transcription of CAD,
carbamoyl-phosphate synthase/aspartate transcarbamoylase/di-
hydroorotase, and NUC by stimulating promoter clearance and
elongation, via recruitment of P-TEFb [25,39,40]. We studied
whether miR-34a affected the expression level of CAD and NUC
which specifically suppresses expression of c-Myc-responsive
CAD and NUC by inhibiting P-TEFb . We found that
miR-34a decreased CAD and NUC mRNA expression in PC-3
cells while DRB restored the expression (Fig. 6B). These results
indicate that miR-34a repressed these genes by suppressing the c-
Overexpression of c-Met reverses inhibition of invasion
It has been suggested that miR-34a inhibits cell invasion in a c-
Met-dependent manner [41,42]. Therefore, we studied effects of
miR-34a on c-Met and invasion since miR-34a targets c-Met
Figure 3. miR-34a targets oncogenes in PC-3 cells. (A) PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-
34a or pre–miR-con for 8 h, followed by transient transfection with control reporter plasmids or 39UTR plasmids for 48 h. 39UTR reporter activity was
measured by luciferase assay and normalized to activity of Renilla luciferase. *, P,0.05 compared with control. (B) PC-3 cells were transiently
transfected with pre-miR negative control (NC) or pre–miR-34a or pre–miR-con for 72 h. Protein expression level was analyzed by Western blot.
miR-34a Targets c-Myc Transcriptional Complexes
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