CD24 Induces Expression of the Oncomir miR-21 via Src, and CD24 and Src Are Both Post-Transcriptionally Downregulated by the Tumor Suppressor miR-34a
and CD24 and Src Are
Both Post-Transcriptionally Downregulated by the Tumor Suppressor miR-34a. PLoS ONE 8(3): e59563. doi:10.1371/journal.pone.0059563
CD24 Induces Expression of the Oncomir miR-21 via Src, and CD24 and Src Are Both Post-Transcriptionally Downregulated by the Tumor Suppressor miR-34a
Santoshi Muppala 0
Giridhar Mudduluru 0
Jo rg H. Leupold 0
Daniel Buergy 0
Jonathan P. Sleeman 0
Heike Allgayer 0
Alfons Navarro, University of Barcelona, Spain
0 1 Department of Experimental Surgery, University of Heidelberg , Mannheim and Molecular Oncology of Solid Tumors, DKFZ, Heidelberg, Germany , 2 Department of Anesthesiology and Intensive Care Medicine, Medical Faculty Mannheim, University of Heidelberg , Mannheim, Germany , 3 Centre for Biomedicine and Medical Technology Mannheim (CBTM), Universita tsmedizin Mannheim, University of Heidelberg , Mannheim, Germany, 4 KIT Karlsruhe Campus Nord, Eggenstein-Leopoldshafen , Germany
Cancer is a complex disease process that evolves as a consequence of multiple malfunctions in key regulatory molecular networks. Understanding these networks will be essential to combat cancer. In this study, we focussed on central players in such networks. In a series of colon and breast cancer cell lines, we found that CD24 activates Src, and induces the activation of c-Jun and expression of c-Jun and c-Fos. Thereby CD24 increases the promoter activity and expression of miR-21, which in turn suppresses expression of Pdcd4 and PTEN. Co-transfection of a CD24 expression construct and an siRNA that silences Src showed that CD24-dependent upregulation of miR-21 is mediated by Src. Additionally, we found that miR-34a posttranscriptionally downregulates CD24 and Src expression, leading to the deactivation of c-Jun, reduced expression of c-Jun and c-Fos, inhibition of miR-21, and upregulation of Pdcd4 and PTEN. Furthermore, miR-34a-mediated inhibition of Src expression reduced migration and invasion of colorectal cancer cells. Resected tumor tissues from 26 colorectal patients showed significantly lower expression of Pdcd4 and miR-34a, and higher expression of CD24, Src and miR-21 compared to the corresponding normal tissues. Moreover, CD24 positively correlated with the amount of Src protein in tumor tissues, and a trend towards an inverse correlation between miR-34a and Src protein levels was also observed. Our results reveal essential players in the complex networks that regulate the progression of solid tumors such as colorectal cancer. These findings therefore identify novel therapeutic approaches for combating tumor growth and progression.
Funding: SM was supported by a doctoral stipendium awarded to JPS and HA by the Medical Faculty Mannheim, University Heidelberg, under the auspices of
the Forschungsschwerpunkt Oncologie initiative. GM was supported by the Stiftung fur krebs- und Scharlachforschung Mannheim, from the University of
Heidelberg. JPS gratefully acknowledges funding from the European Union under the auspices of the FP7 collaborative project TuMIC, contract no.
HEALTH-F22008-201662. HA was supported by the Alfried Krupp von Bohlen und Halbach Foundation (Award for Young Full Professors), Essen, Hella-Bu hler-Foundation,
Heidelberg, the Hector Foundation, Weinheim, the FRONTIER Excellence Initiative of the University of Heidelberg, the BMBF, Bonn, the Walter Schulz Foundation,
Munich, Deutsche Krebshilfe, Bonn, Germany, and the German-Israeli Cooperation (DKFZ-MOST). 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.
. These authors contributed equally to this work.
Tumorigenesis is a multistep process that is regulated by
complex molecular networks whose activity is perturbed by
sequential alterations in a variety of oncogenes, tumor-suppressor
genes and microRNA genes . These alterations are usually
somatic events, although germ-line mutations can predispose a
person to heritable or familial cancer. Subsequent tumor
progression ultimately leads to the metastatic spread of tumor
cells into distant organs , which again is driven by a network of
regulatory and effector proteins. Despite many years of basic and
clinical research aimed at curbing tumor growth, metastasis
remains the prime reason why cancer patients succumb to their
disease , largely because of the lack of understanding of the
complex molecular networks that regulate tumor progression.
Also known as heat stable antigen, CD24 is a
glycosylphosphatidylinositol (GPI)-anchored membrane protein that has been
implicated in tumorigenesis, progression, metastasis and poor
prognosis for a variety of tumor types . Thus expression of
CD24 repeatedly emerges from transcrptional profiling as being
correlated with tumorigenesis and tumor progression [5,6].
Functionally CD24 can promote invasiveness and metastasis
formation in vivo [7,8]. CD24 may act in several ways to exert
these effects. It can support rolling of tumor cells on endothelial
monolayers due to its ability to bind to P-selectin , a protein
expressed on thrombin-activated platelets [10,11] and endothelial
cells [11,12]. CD24 also regulates the activity of CXCR4 , as
well as proliferation, motility and integrin-mediated adhesion .
However, much remains to be learned about the activity of CD24
in the context of cancer.
Little is known about the molecular regulatory networks that are
addressed by CD24. Our own recent findings suggest that CD24
activates Src within lipid rafts . Src plays a central role in the
regulation of invasion and metastasis . Its activity is normally
tightly controlled in non-transformed cells, but in may types of
cancer, enhanced Src kinase activity is found that correlates with
poor prognosis [16,17]. Activated Src induces AP-1 activation
mainly through the MAPK pathway, thus inducing cell migration
and invasion . AP-1 family members in turn are key players in
multistep tumorigenesis due to their transcriptional activation
miRNAs are non-coding RNA molecules that
post-transcriptionally regulate gene expression, and can act to either promote or
inhibit tumor formation and progression. For example, miR-21 is
an oncomir that inhibits the expression of tumor suppressor and/
or metastasis suppressor genes such as Pdcd4 and PTEN [20,21],
and is transcriptionally regulated by AP-1 family members [22,23].
Conversely, miR-34a is a tumor suppressor microRNA that is
regulated by the tumor suppressor gene p53 , and
downregulates expression of tumor progression-associated genes such as
Axl and c-Met .
In this study we investigated further the molecular pathways
addressed by CD24, and thereby have uncovered a regulatory
network in which miRNAs play a central role. Specifically we
found that CD24-dependent activation of Src increases miR-21
expression, and thereby inhibits expression of Pdcd4 and PTEN.
This pathway is counter-regulated miR-34a, which
post-transcriptionally inhibits expression of CD24 and Src, resulting in
diminished miR-21 expression, and thus enhanced expression of
Pdcd4 and PTEN.
Materials and Methods
Cell Culture and Antibodies
The human colorectal cancer cell lines (HT-29, HCT-116, Rko,
SW480, Colo206f and WiDr) and the human breast cancer cell
line MDA-MB-231 were purchased from American Type Culture
Collection (ATCC, Manassas, USA), and grown according to the
recommended conditions. The human colorectal cancer cell line
Geo, a gift from Prof. Douglas Boyd (MD Anderson Cancer
Center, Houston, USA), was cultivated in DMEM/10%FCS as
published before [20,25,26]. Media and FCS were obtained from
Invitrogen (Karlsruhe, Germany) and Sigma (Taufkirchen,
Germany). Lipofectamine was purchased from Invitrogen
(Karlsruhe, Germany), transwell chambers (1 cm2/8 mm pore size) were
from Costar (Cambridge, USA), and Matrigel was obtained from
BD Biosciences (Bedford, USA). Antibodies against Pdcd4
(ab51495) were purchased from Abcam (Cambridge, UK),
pSrc-Y416 (#2101), Src-36D10 (#2109), c-Jun-60A8 (#9165) and
PTEN-138G6 (#9559)-antibodies were from Cell Signalling
(NEB-Frankfurt, Germany), and phospho-c-Jun (sc-822X), c-Fos
(sc-52X), anti-IgG control (sc-2338X) and b-actin (sc-1616R)
antibodies were from Santa Cruz Biotechnology (Heidelberg,
Germany). Mouse monoclonal CD24 antibody (SWA-11) was a
kind gift from Prof. Peter Altevogt (Department of Immunology,
DKFZ, Heidelberg, Germany) [14,27].
Control miR/Scrambled (AM17110), pre-miR-34a (PM-34a)
(ID:PM11030), and anti-miR-34a (AM-34a) (ID: AM11030), as
well as negative control siRNA (#AM4635), siRNA-CD24
(#ID:s2616) and siRNA-Src (#ID:s13414) were obtained from
Ambion (Austin, USA). Taqman primer-probes for the
quantification of miR-21 (ID: 000397), miR-34a (ID: 00426), RNU6B (ID:
001093) and Pdcd4 (ID: Hs00377253_m1) were purchased from
Applied Biosystems (Foster City, USA), and oligonucleotides for
39UTR cloning and RT-PCR for CD24 and Src were purchased
from Metabion (Martinsried, Germany).
Expression Plasmids and 39-UTR-luciferase Reporter
CD24 cDNA was amplified using the primers indicated in
Table S1 and cloned into the pCDNA3.1 vector. The identity of
the insert was confirmed by sequencing. The full-length 39-UTR
of CD24 (634 nt) and Src (1814 nt) was amplified using genomic
DNA from Geo cells and cloned into the HindIII site of pMIR
(Ambion) and the Xba I site of pGL3 (Promega, Madison, WI,
USA), respectively. The identity of the insert was confirmed by
sequencing. Sitedirected mutagenesis (Stratagene, Heidelberg,
Germany) to mutate the seed sequences of miR-34a was
performed using Luc-CD24-39-UTR and Src-39-UTR wild-type
sequences as a template. The sequences of cloning primers are
provided in Table S1. The constitutively active chicken Src
expression plasmid CA10-SrcY527F (A-Src) was used as described
Cells were co-transfected in 24 well plates with either 0.5 mg of
luciferase construct and pRL-TK (50 ng, Renilla Luciferase;
Promega), or together with 50 nM of control miR or
PM/AMmiR-34a using lipofectamine 2000. pRL-TK served as an internal
control, and its luminescence was measured to normalize
transfection efficiency. Dual reporter assays were performed
according to the manufacturers protocol using the dual-luciferase
assay system (Promega). Briefly, 48 h post transfection, cells were
washed twice with PBS and lysed with 100 ml passive lysis buffer
(Promega) for 20 min, then 20 ml of cell lysate was used for the
measurements. Assays for all samples were performed in triplicate,
and the results were averaged.
Preparation of RNA, Protein Extracts, RT-PCR and
RNA, protein isolation, RT-PCR and western blot analysis
were performed as described previously . Expression of CD24
and Src mRNA was determined by SyBr green PCR and actin as a
normalizing control (for primer sequences see Table S1). Mature
miRNA expression of miR-34a (ID:000426) and miR-21
(ID:000397) were determined by the Taqman miRNA assay
(Applied Biosystems, Foster City, CA, USA), and normalized using
the 22DDCt method relative to U6-snRNA (RNU6B; ID:001093
from applied Biosystem, USA). All PCRs were performed in
Chromatin Immunoprecipitaion Assay (ChIP)
ChIP assays were performed as described previously .
Briefly, Rko cells were transfected either individually or in
combination with the CD24 expression construct, Src siRNA,
PM-34a or the A-Src expression construct as indicated. Two days
post transfection, the binding of phospho-c-Jun to the miR-21
promoter was measured by RT-PCR with a set of specific primers
Cell Migration and Invasion Assay
Rko and HCT-116 cells were transfected with either PM-34a or
A-Src or both. Two days post transfection, cells were washed once
with ice-cold PBS, trypsinized, counted, and 36105 cells were
Figure 1. Overexpression of CD24 activates Src and induces miR-21 expression. (a) Western blot analysis of CD24, p-Src, Src,
phosphorylated c-jun, c-Jun and c-Fos was performed 48 h post transfection. Rko and HCT-116 cells transfected with either with vector control or
with a CD24 expression construct is shown in the left panel. Transfection of HT-29 and Geo cell lines either with negative control siRNA (NC) or siRNA
against CD24 (si-CD24) is shown in the right panel). b-Actin served as an internal control. (b) Luciferase reporter assays of the miR-21 promoter
cotransfected either with a CD24 expression construct in Rko and HCT-116 (left panel) or with siRNA against CD24 (si-CD24) in HT29 and Geo cells (right
panel) along with respective controls. Percent luciferase activity was calculated either with the miR-21 promoter or control samples set as 100%. The
data are presented as the mean 6 S.D. Each bar represents the mean value of three biological replicates (Rko: p = 0.01; HCT116: p = 0.02). (c) miR-21
expression levels were evaluated by RT-PCR 48 h post transfection upon overexpression or knock-down of CD24 in Rko, HCT-116 or HT29, Geo cell
lines, respectively. The data are presented as the mean 6 S.D. Each bar represents the mean value of three biological replicates (Rko: p = 0.009;
HCT116: p = 0.01; HT29: p,0.001; Geo: p = 0.02). Specific p-c-Jun band intensities were normalized relative to b-actin and are represented as fold
change in comparison to the control.
seeded on transwell plates either coated with 10 mg matrigel/well
(for invasion assays) or uncoated (for in vitro migration assays) in
serum-free medium containing 0.1% BSA (Bovine serum
albumin). As a chemoattractant, 10% FBS in the lower chamber was
used. After 14 h, invaded cells were trypsinized and counted using
the ATP-luminiscence-based motility-invasion assay (Promega) as
previously described .
Tissue specimens (tumor, adjacent normal mucosa) from 26
patients with different tumor stages (T1: n = 2; T2: n = 5; T3:
n = 16; T4: n = 3) of colorectal cancer were collected after
informed written consent from all patients and verification of the
samples by a pathologist, and immediately frozen in liquid
nitrogen. Tissue screening and documentation process was
approved by the institutional Medizinische Ethik-Kommission
II ethics committee of Medical Faculty Mannheim, University of
Statistical analysis was performed using SPSS version 14.0
(SPSS). The Wilcoxon Sign Rank Test was used to compare the
expression of Pdcd4, CD24, Src, miR-34a and miR-21 in
colorectal tumors and corresponding normal tissues. Spearman
correlations among continuous variables were computed. In all
tests, p values of #0.05 were considered significant and p values
.0.05 and ,0.1 considered to represent a trend.
Ectopic Expression of CD24 Leads to an Increase in the
Activity of Src and Induces miR-21 Expression
We recently showed that CD24 interacts with Src and promotes
its activity . To investigate further the signalling pathways
addressed by CD24 and their possible implications for cancer
progression, we first investigated whether ectopic expression of
CD24 is able to increase the activity of Src and its downstream
signalling axis in a panel of colorectal cancer cell lines. Initially we
screened for endogenous expression of CD24 and Src to identify
cell lines suitable for loss and gain of function experiments.
Thereby Rko, HCT-116, HT-29 and Geo cells were selected as
cell lines that express low and high endogenous levels of CD24,
respectively, and which differ in their invasive behaviour 
In a first approach, low CD24-expressing Rko and HCT-116
cells were transiently transfected with an expression plasmid for
CD24, and the high CD24-expressing HT-29 and Geo cells were
transiently transfected with a specific siRNA that targets CD24
(Figure S2a). Ectopic expression of CD24 enhanced the
phosphorylation status of Src, and conversely, knock down of CD24
decreased Src phosphorylation (Figure 1a). Next, we investigated
the phosphorylation of c-Jun under these conditions, since it is
known that Src is able to activate AP-1 transcription factors .
Accordingly, we found that ectopic expression of CD24 induced
the phosphorylation of c-Jun, paralleled by an increased
expression of endogenous c-Jun and c-Fos protein (Figure 1a left panel).
The opposite effect was observed upon knock-down of CD24
(Figure 1a right panel). In addition, we screened for miR-21
expression following CD24-overexpression or knockdown, because
we and others have shown that miR-21 is regulated by AP-1 family
members [22,23]. To this end, we investigated the activity of the
miR-21 promoter in luciferase reporter assays, together with the
expression of this micro RNA. Ectopic expression of CD24
significantly induced miR-21 promoter activity as compared to the
vector control, and also significantly increased miR-21 expression.
Consistently, silencing of CD24 expression decreased miR-21
promoter activity and expression (Figure 1b,c).
In a second approach, we investigated the role of Src in the
CD24-mediated induction of miR-21 expression. Consistent with
our previous observations, overexpression of a constitutively
activated Src in Rko and HCT-116 cell lines (Figure S2b) resulted
in an increased phosphorylation of c-Jun and elevated levels of
cJun and c-Fos protein (Figure 2a, left panel), whereas
siRNAmediated silencing of Src in the HT-29 and Geo cell lines led to
the converse effect (Figure 2a, right panel and Figure S2b).
Importantly, we found that Src activity is necessary and sufficient
for miR-21 promoter activity and expression, as evidenced by
ectopic expression of constitutively activated Src in Rko and
HCT116 cells, and specific silencing of Src in HT-29 and Geo cells
(Figure 2b, c).
Induction of miR-21 was first observed 48 h after ectopic
expression of CD24 or Src, and not at 6 h or 24 h
posttransfection. Furthermore, equivalent levels of miR-21 expression
were observed at 48 h and 72 h post-transfection (data not shown).
In further control experiments, we used additional independent
CD24, Src and negative control siRNAs. The results obtain were
equivalent to those described above (data not shown), ruling out
off-target or other non-specific effects.
To determine whether CD24 is able to induce miR-21
expression specifically through the activation of Src, we ectopically
expressed CD24 and silenced Src in Rko and Geo cells, either
alone or in combination. As expected, when Src was silenced,
diminished phosphorylation of c-Jun compared with the control
was observed, which was paralleled by decreased expression of
cJun and c-Fos proteins. (Figure 3a). In contrast, ectopic expression
of CD24 resulted in enhanced phosphorylation of Src and c-Jun,
and elevated c-Jun and c-Fos protein expression (Figure 3a, Figure
S3). However, when Src was silenced in cells ectopically expressing
CD24, phosphorylation of c-Jun and the expression of c-Jun and
cFos was significantly reduced compared to ectopic expression of
CD24 alone (Figure 3a). Additionally, we also observed a
significant reduction in miR-21 promoter activity and expression
following combined ectopic CD24 expression and Src silencing
(p = 0.004) (Figure 3b, c). Similar statistically significant results
were observed for the activity of a 4XAP-1 Luc reporter construct
under similar conditions (Figure S3). Taken together, these results
suggest that the CD24/Src signalling axis phosphorylates c-Jun
and induces the expression of c-Jun and c-Fos, resulting in the
induction of miR-21 expression.
CD24/Src-induced miR-21 Expression Downregulates
Pdcd4 and PTEN
We and others have found that upregulation of miR-21 is
associated with the downregulation of the tumor suppressor genes
Pdcd4 and PTEN [20,21]. To investigate whether
CD24mediated Src activation is able to induce miR-21 and thereby
post-transcriptionally downregulate Pdcd4, we investigated the
Pdcd4 39-UTR activity after silencing of Src and/or ectopic
expression of CD24. After Src silencing, we found a significant
increase in Pdcd4 39-UTR activity (Rko: p = 0.02; Geo: p = 0.01).
Accordingly, ectopic expression of CD24 significantly reduced the
activity of the Pdcd4 39-UTR (Rko: p = 0.03; Geo: p = 0.01)
(Figure 3d). Interestingly, we found only a slight induction of
39UTR activity when we concomitantly knocked down Src and
ectopically expressed CD24 (Figure 3d).
To determine whether AP-1 transcription factors bind to the
miR-21 promoter following CD24/Src signalling, ChIP
experiments were performed using cells transfected with the specific
siRNA against Src and/or the CD24 alone expression construct.
Silencing of Src reduced binding of phosphorylated c-Jun to the
miR-21 promoter as compared to the control. In contrast, in cells
overexpressing CD24 we found an induced binding of
phosphorylated c-jun, and a slightly induced phosphorylated c-jun binding
in combination of both CD24 and si-Src (Figure 3e). Finally, we
assessed the protein expression of Pdcd4 and PTEN [20,21] and
found that CD24-induced expression of miR-21 is able to
downregulate the expression of both tumor suppressor genes
(Figure 3a). These experiments further support the notion that
CD24 is mediating miR-21 expression, and as a consequence
suppresses expression of Pdcd4 and PTEN. However, in addition
to post-transcriptional regulation of PDCD4 and PTEN by CD24,
other mechanisms such as protein degradation/stabilization could
also contribute to the protein expression levels of these genes.
The CD24 and Src 39-UTRs are Targets of miR-34a
Using in silico analysis (miRanda) we found two miR-34a seed
sequences in the CD24 and Src 39UTR. Of these, the seed
sequence that showed the higher mirSVR score was used for
further analysis. We also found a conserved target sequence for
miR-34a in the 39-UTR of Src. These data suggested that CD24
and Src might be targets of miR-34a. To investigate this
hypothesis we first determined the expression of endogenous
miR-34a in different colorectal cell lines and found low
endogenous expression in HT-29 and Geo cells (Figure S1c, top
panel). We then asked whether the 39-UTRs of CD24 and Src are
functional targets of miR-34a in these cells. To this end, we
generated luciferase reporter plasmids driven by the SV40 basal
promoter, and harbouring either the 39-UTR of CD24 or the
39UTR of Src. These constructs were individually co-transfected
together with pre-miR-34a (PM-34a) into the HT-29 and Geo cell
lines. Ectopic expression of miR-34a significantly reduced
luciferase activity for both the CD24 and Src 39-UTRs compared
to appropriate controls was observed (CD24 39-UTR:
HT29:p = 0.05 and Geo: p = 0.02; Src 39-UTR: HT-29: p = 0.028;
Geo: p = 0.01) (Figure 4a,b). To verify the miR-34a-mediated
regulation of the 39UTRs of CD24 and Src, luciferase assay
experiments were performed with co-transfection of either the Src
or CD24 39UTR reporters along with various concentrations of
miR-34a. CD24 and Src 39UTR activity was reduced in a dose
dependent manner (p#0.05) in response to miR-34a (Figure S5).
To demonstrate the specificity of miR-34a in repressing the
CD24 and Src 39-UTRs, we cloned CD24 and Src 39-UTR
reporter constructs in which the conserved miR-34a seed
sequences were mutated for miR-34a interaction (see Figure S4).
These two constructs were then co-transfected with or without
PM-34a into HT-29 and Geo cells. Neither mutated construct
showed any significant change in luciferase activity when
compared with the negative control (Figure 4a,b), confirming the
specificity of miR-34a. Furthermore, transfection of Geo, Rko, and
MDA-MB-231 cells with control miR, PM-34a or AM-34a
resulted in the expected inhibition or induction of CD24 and
Src protein expression in all cell lines (Figure 4c). In addition, Geo
cells were transfected with PM-34a, and the resulting expression of
CD24 and Src mRNA was examined. CD24 and Src mRNA
expression was reduced significantly by PM-34a (Figure S6).
Taken together, our data suggest that miR-34a is a
posttranscriptional regulator of CD24 and Src.
miR-34a Downregulates miR-21 by Targeting CD24 and
To investigate whether the tumor suppressor miR-34a can
regulate miR-21 through CD24/Src signalling, and to elucidate a
possible role for AP-1 family members in such a regulation,
cotransfection experiments were performed either with PM-34a, an
expression construct for constitutively active Src (A-Src), or with a
combination of both. PM-34a inhibited the luciferase activity of
the miR-21 promoter and also significantly reduced
A-Srcinduced luciferase activity of this promoter (Figure 5b; Rko:
p = 0.04; Geo: p = 0.02). These findings encouraged us to
investigate whether miR-34a inhibits CD24/Src-mediated AP-1
activation and miR-21 regulation. We observed that expression of
the c-Jun and c-Fos genes were significantly downregulated at the
mRNA level after PM-34a transfection. Furthermore, this
treatment abolished A-Src-induced expression of c-Jun and c-Fos
mRNA (Figure S7). In contrast, ectopic expression of PM-34a
resulted in upregulation of Pdcd4 mRNA, whereas the ectopic
expression of constitutively active Src reduced mRNA expression
of this protein (Figure S7). Under the same conditions, we
performed luciferase reporter assays using the miR-21 promoter
(Figure 5b), a 4XAP-1 Luc reporter construct as a positive control
for AP-1 transcriptional activity (Figure S6), and the Pdcd4
39UTR (Figure 5d), as well as quantitative PCR for miR-21
(Figure 5c), ChIP analysis for phosphorylated c-jun binding to the
miR-21 promoter (Figure 5e) and Western blot analysis for CD24,
Src, phosphorylated Src, phosphorylated c-jun, c-Jun, c-Fos,
Pdcd4 and PTEN (Figure 5a). Consistent with our previous
observations, transfection with PM-34a reduced CD24, Src,
phosphorylated c-jun, c-Jun and c-Fos protein expression.
Furthermore, diminished miR-21-promoter activity and
expression was observed, which was accompanied by increased
expression of the Pdcd4 and PTEN proteins (Figure 5). Moreover,
ChIP demonstrated inhibition of the binding of phosphorylated
cjun to the miR-21 promoter after pre-miR34a treatment in vivo
(Figure 5e). Taken together, these data suggest that miR-34a
downregulates miR-21 expression by targeting the CD24 and Src
For additional controls, we investigated the role of miR-34 in
the regulation of the expression of other miRNAs (miR-199a and
miR-376). We found no significant differences of these miRNAs at
the expression level, with either miR-34 or ASrc overexpression
(data not shown). These data support the specificity of the effect of
miR-34 and exclude a possible pleotrophic effect of miR-34. In
Figure 4. miR-34a targets the CD24- and Src-39-UTR and regulates their expression. (a,b) Luciferase assays using the CD242 and
Src-39UTR or mutant reporter constructs transfected into HT-29 and Geo cells together with either control-miRNA (NC) or PM-34a. Percent luciferase activity
was calculated either with the CD242 or Src-39-UTR or control-miR samples set as 100%. The data are presented as the mean 6 S.D. Each bar
represents the mean value of three technical replicates. (c) Geo, Rko, HT-29 and MDA-MB-231 cells were transfected either with control miRNA or
PM34a, and 48 h later protein was isolated and western blot analysis for CD24 and Src was performed (left panel). Rko and MDA-MB-231 cells were
transfected either with control miRNA or AM-34a, and 48 h later protein was isolated and western blot analysis for CD24 and Src was performed (right
other experiments, we performed western blot analysis of known
miR-34a targets, such as Axl, c-Myc and b-Catenin (Figure S8)
[25,28,29]. As expected, we observed a clear downregulation of all
proteins investigated upon miR-34 overexpression. Interestingly,
we also observed an induction of Axl protein amounts after
overexpression of ASrc, which is know to be transcriptionally
regulated by AP-1 family members . Finally, we observed a
significant downregulation of Axl protein expression by miR-34a,
which was not affected by the additional overxpression of ASrc.
Based on these data, we conclude that the effect of miR-34a is due
to a direct inactivation of AP-1, leading to downregulation of
miR-34a Inhibits A-Src-induced Migration and Invasion
Several publications have demonstrated a function for CD24,
Src, Pdcd4, miR-21 and miR-34a in migration, invasion and
metastasis [7,14,20,23,25,31]. To determine whether miR-34a
suppresses A-Src-induced tumor progression, we performed
migration and invasion assays. Transfection of Rko and
HCT116 cells with PM-34a significantly inhibited A-Src-dependent
migration and invasion (Figure 6a,b). These data allow us to
conclude that CD24/Src-mediated tumor progression can be
inhibited by miR-34a.
6 S.D. Each bar represents the mean value of three biological replicates. (e) The in vivo association of phosphorylated c-jun with the miR-21
To determine the in vivo relevance of the mechanisms identified,
26 resected colorectal carcinoma tissues of patients and
corresponding normal mucosae were investigated for endogenous
expression of Pdcd4, CD24, Src, miR-34a and miR-21. As shown
in Figure 7, we observed that Pdcd4 and miR-34a were
significantly downregulated, whereas CD24, Src, and miR-21
were significantly upregulated in the tumor tissues as compared to
the respective normal tissues (p = 0.003; p = 0.05; p = 0.001;
p = 0.05 and p = 0.002; respectively). Furthermore, CD24
expression positively and significantly correlated with Src gene
expression in the resected tumor tissues (p = 0.001), and Src expression
showed a trend towards negative association with miR-34a
expression. Additionally, miR-34a expression correlated
significantly with pT stage, demonstrating a significant inversive
correlation (Correlation Coefficient 20.494; p = 0.023),
supporting the relevance of our finding.
In this study we have identified a novel molecular network that
regulates tumor progression, in which miRNAs play a pivotal role
in determining the expression of the tumor suppressor genes
Pdcd4 and PTEN (Figure 7d). Specifically, miRNA-34a suppresses
expression of CD24 and Src, thereby diminishing the tumor
progression-associated functions of these genes, which results in
reduced expression of the oncomir miR-21, and thus relieves
miR21-mediated repression of Pdcd4 and PTEN expression. In
the absence of miRNA-34a, expression of CD24 activates Src,
which in turn induces AP-1 family members and stimulates
expression of miR-21. Expression of Pdcd4 and PTEN is
subsequently suppressed by miR-21. These data suggest that the
balance between oncogenically-acting and tumor-suppressing
miRNAs can determine the course of tumor progression, and cast
new light on how CD24 and Src act to promote tumor growth and
Although we have focused here on expression of miR21, Pdcd4
and PTEN as endpoints of the molecular regulatory network we
describe, it is likely that effects of the network extend beyond these
genes. Src activates AP-1 family members through the MAPK
pathway [18,31], which in turn induces a number of tumor- and
metastasis-promoting molecules including u-PAR .
Furthermore, Src regulates a variety of other signal transduction
pathways, including the PI3K/Akt, STAT and FAK pathways,
and regulates Rho family GTPases, resulting in the regulation of a
variety of cellular processes including cytoskeletal architecture
remodelling, motility, invasion, cell adhesion and the
epithelialmesenchymal transition . Moreover, we show that
CD24/Srcdependent upregulation of miR-21 expression is mediated through
the AP-1 family members c-Jun and c-Fos, confirming the
importance of AP-1 in the regulation of miR-21 expression as
described previously [22,23]. Importantly, miR-21 is a prognostic
marker, and the only microRNA upregulated in all major solid
tumor types screened so far [33,34]. Loss and gain of function
studies in different types of cancer types have established a role for
miR-21 in cell proliferation, inhibition of apoptosis, migration,
invasion and distant metastasis [20,21,35,36,37], and a number of
cancer-relevant miR-21 target genes in addition to Pdcd4 and
PTEN have been identified [38,39,40]. Thus the regulatory
network we describe is likely to affect expression of many
Srcregulated and miR-21 target genes.
Although activating mutations in the Src gene are rarely found
in tumors , Src is often overexpressed in tumors, but high
protein levels do not necessarily correlate with Src kinase activity
. For a number of tumor types including lung, breast and
colon cancers, there is an association between Src activity and
poor clinical prognosis [16,41,42]. We have previously shown that
expression of CD24 is a mechanism that can activate Src kinase
activity in the tumor context , consistent with the observation
that the Ras/ERK/MAPK pathway that is activated by Src  is
downregulated as a consequence of targeting of CD24 . Here
we show a further facet of the tumor-relevant regulation of Src,
namely that the tumor suppressor miRNA-34a inhibits its
expression. Loss of miRNA-34a expression during tumor
progression is therefore likely to contribute to the enhanced levels of Src
protein observed during the progression of many types of tumor.
The tumor suppressor function of miR-34a is reflected in its
epigenetic silencing during tumor progression , its positive
regulation by p53 , and its ability to target and suppress
expression of a number of oncogenes and other cancer relevant
genes, including Axl, c-Met, CD44, MYCN, Notch1, Jagged 1, the
Notch ligand Delta-like1, and the cell cycle regulators CCND1
and CDK6 [25,45,46,47,48,49]. Interestingly, other miR-34a
target genes such as the deacetylase SIRT1 and the transcription
factor YY1 both suppress p53 activity/expression [50,51],
suggesting that a positive feedback loops exists in which p53
induces miR-34a, which in turn suppresses the p53 inhibitors
SIRT1 and YY1. Our study casts further light on the tumor
suppressor function of miR-34a. We demonstrate that it can
suppress the tumor progression genes Src and CD24.
Furthermore, we show it can indirectly diminish expression of the oncomir
miR-21 by targeting CD24 and Src expression
post-transcriptionally. Post-transcriptional regulation of Src by miR-203 and
miR205, two other miRNAs with tumor suppressor functions, has also
been reported in bladder and renal cancer [52,53]. Thus it is likely
that miR-203 and miR-205 also diminish miR-21 expression
through their suppression of Src.
The findings we present here are relevant to human cancer and
have potential therapeutic application. Although we only used a
small patient cohort in this study, nevertheless we saw a clear
correlation between downregulation of Pdcd4 and miR-34a and
enhanced expression of miR-21, CD24 and Src in the resected
colorectal tumor samples compared to normal tissue. Moreover,
CD24 and Src expression positively correlated with each other,
and miR-34a expression was negatively associated with Src in the
tumor tissues. Further work will confirm these findings in a larger
series of patients. Nevertheless, our findings suggest that reversing
the epigenetic silencing of miR-34a could be therapeutically
beneficial for colorectal cancer patients. We note with interest that
treatment of prostate cancer patients with BioResponse
3,39Diindolylmethane (BR-DIM) resulted in re-expression of miR-34a
due to reversal of the methylation-induced silencing of the
miR34a promoter, and suppressed the expression of miR-34a target
genes including the androgen receptor and Notch-1 . These
findings demonstrate the feasibility of reversing epigenetic
silencing of miR-34a for therapeutic purposes in the context of
Figure S1 Endogenous expression of CD24, Src,
miR21, miR-34a and Pdcd4 in a panel of human cancer cell
lines. (a) Relative expression of CD24, Src and Pdcd4 in a panel
of colorectal cell lines and in the MDA-MB-231 breast cancer cell
line was evaluated by Real-Time PCR. b-Actin served as an
internal control. (b) Western blot analysis of CD24, phosphor-Src
(p-Src), Src and Pdcd4 in a panel of colorectal cell lines and in the
MDA-MB-231 breast cancer cell line. b-Actin served as an
internal control. (c) Relative expression of miR-34a and miR-21 in
a panel of colorectal cell lines and in the MDA-MB-231 breast
cancer cell line was evaluated by Real-Time PCR. RNUB6 served
as an internal control.
Figure S2 Expression of CD24 and Src. (a) Rko and
HCT116 cells were transfected either with vector control or a CD24
expression construct. HT-29 and Geo cells were transfected with
negative control siRNA or with siRNA against CD24. After 48 h,
expression levels were determined by Real-Time PCR. (b) Rko
and HCT-116 cells were transfected with either vector control or a
constitutively active Src expression construct (A-Src). HT-29 and
Geo cells were transfected with either negative control siRNA or
with siRNA against Src. After 48 h, expression levels were
determined by Real-Time PCR. Each bar represents the mean
value of three biological replicates.
Figure S3 CD24-dependent regulation of c-Jun, c-Fos
and Pdcd4 is mediated through Src. c-Fos, c-Jun and Pdcd4
mRNA expression was evaluated by RT-PCR 48 h post
transfection of (Top row) or AP-1 4X Luc luciferase activity
(Bottom row) Rko and Geo cells with either empty vector (Vector),
a CD24 expression construct (CD24), Negative control siRNA
(NC) or with an siRNA against Src as indicated. Each bar
represents the mean value of three biological replicates (*p#0.05).
Figure S4 miR-34a target sites within the CD24- and
Src-39-UTR. The location of the putative miR-34a target sites in
CD24 and Src transcripts (WT) and their mutated variants (MT)
are shown with underlined and bold letters, respectively.
Figure S5 Dose dependent expression of miR-34a.
Luciferase assays of the CD24- and Src-39-UTR reporter
constructs transfected into Rko and HCT116 cells together with
either control-miRNA (NC) or increased concentrations of
PM34a as mentioned. Percent luciferase activity was calculated either
with the CD24- or Src-39-UTR or control-miR samples set as
100%. The data are presented as the mean 6 S.D. Each bar
represents the mean value of three technical replicates (NC:
Figure S6 Expression of CD24 and Src mRNA upon
transfection with PM-34a. Geo cells were transfected either
with control miRNA or with PM-34a. After 48 h, expression of
CD24 and Src mRNA was determined by Real-Time PCR. Data
is represented as the mean of three technical replicates. (NC:
Figure S7 Src-induced c-Fos, c-Jun and Pdcd4
expression is antagonised by PM-34a. c-Fos, c-Jun and Pdcd4
mRNA expression levels were evaluated by RT-PCR 48 h post
transfection (Top row) or AP-1 4X Luc luciferase activity (Bottom
row) of Rko and Geo cell lines, with either empty vector (Vector), a
constitutively active Src expression construct (A-Src), PM-34a or a
negative control (NC) as indicated. Each bar represents the mean
value of three biological replicates (*p#0.05).
Figure S8 miR-34a target molecules. Western blot analysis
of known miR-34a target molecules Axl, c-Myc and b-Catenin
was performed 48 h post transfection. Rko cells were transfected
with a constitutively active Src expression construct (A-Src), empty
vector (Vector), PM-34a or scrambled siRNA control (Scrambled)
as indicated. b-Actin served as an internal control.
Oligonucleotides used in this study.
Conceived and designed the experiments: SM GM JL JS HA. Performed
the experiments: SM GM JL. Analyzed the data: SM GM JL JS HA.
Contributed reagents/materials/analysis tools: GM DB JS HA. Wrote the
paper: SM GM JL JS HA.
1. Croce CM ( 2008 ) Oncogenes and cancer . N Engl J Med 358 : 502 - 511 .
2. Sleeman JP ( 2000 ) The lymph node as a bridgehead in the metastatic dissemination of tumors . Recent Results Cancer Res 157 : 55 - 81 .
3. Sporn MB ( 1996 ) The war on cancer . Lancet 347 : 1377 - 1381 .
4. Kristiansen G , Sammar M , Altevogt P ( 2004 ) Tumour biological aspects of CD24, a mucin-like adhesion molecule . J Mol Histol 35 : 255 - 262 .
5. Nestl A , Von Stein OD , Zatloukal K , Thies WG , Herrlich P , et al. ( 2001 ) Gene expression patterns associated with the metastatic phenotype in rodent and human tumors . Cancer Res 61 : 1569 - 1577 .
6. Pedersen MW , Thykjaer T , Orntoft TF , Damstrup L , Poulsen HS ( 2001 ) Profile of differentially expressed genes mediated by the type III epidermal growth factor receptor mutation expressed in a small-cell lung cancer cell line . Br J Cancer 85 : 1211 - 1218 .
7. Baumann P , Cremers N , Kroese F , Orend G , Chiquet-Ehrismann R , et al. ( 2005 ) CD24 expression causes the acquisition of multiple cellular properties associated with tumor growth and metastasis . Cancer Res 65 : 10783 - 10793 .
8. Senner V , Sturm A , Baur I , Schrell UH , Distel L , et al. ( 1999 ) CD24 promotes invasion of glioma cells in vivo . J Neuropathol Exp Neurol 58 : 795 - 802 .
9. Aigner S , Ramos CL , Hafezi-Moghadam A , Lawrence MB , Friederichs J , et al. ( 1998 ) CD24 mediates rolling of breast carcinoma cells on P-selectin . FASEB J 12 : 1241 - 1251 .
10. Celi A , Furie B , Furie BC ( 1991 ) PADGEM: an adhesion receptor for leukocytes on stimulated platelets and endothelial cells . Proc Soc Exp Biol Med 198 : 703 - 709 .
11. McEver RP , Beckstead JH , Moore KL , Marshall-Carlson L , Bainton DF ( 1989 ) GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies . J Clin Invest 84 : 92 - 99 .
12. Bevilacqua MP , Pober JS , Mendrick DL , Cotran RS , Gimbrone MA , Jr. ( 1987 ) Identification of an inducible endothelial-leukocyte adhesion molecule . Proc Natl Acad Sci U S A 84 : 9238 - 9242 .
13. Schabath H , Runz S , Joumaa S , Altevogt P ( 2006 ) CD24 affects CXCR4 function in pre-B lymphocytes and breast carcinoma cells . J Cell Sci 119 : 314 - 325 .
14. Baumann P , Thiele W , Cremers N , Muppala S , Krachulec J , et al. ( 2011 ) CD24 interacts with and promotes the activity of c-src within lipid rafts in breast cancer cells, thereby increasing integrin-dependent adhesion . Cell Mol Life Sci.
15. Brunton VG , Frame MC ( 2008 ) Src and focal adhesion kinase as therapeutic targets in cancer . Curr Opin Pharmacol 8 : 427 - 432 .
16. Aligayer H , Boyd DD , Heiss MM , Abdalla EK , Curley SA , et al. ( 2002 ) Activation of Src kinase in primary colorectal carcinoma: an indicator of poor clinical prognosis . Cancer 94 : 344 - 351 .
17. Alvarez RH , Kantarjian HM , Cortes JE ( 2006 ) The role of Src in solid and hematologic malignancies: development of new-generation Src inhibitors . Cancer 107 : 1918 - 1929 .
18. Luo Y , Liang F , Zhang ZY ( 2009 ) PRL1 promotes cell migration and invasion by increasing MMP2 and MMP9 expression through Src and ERK1/2 pathways . Biochemistry 48 : 1838 - 1846 .
19. Jochum W , Passegue E , Wagner EF ( 2001 ) AP-1 in mouse development and tumorigenesis . Oncogene 20 : 2401 - 2412 .
20. Asangani IA , Rasheed SA , Nikolova DA , Leupold JH , Colburn NH , et al. ( 2008 ) MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer . Oncogene 27 : 2128 - 2136 .
21. Meng F , Henson R , Wehbe-Janek H , Ghoshal K , Jacob ST , et al. ( 2007 ) MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer . Gastroenterology 133 : 647 - 658 .
22. Fujita S , Ito T , Mizutani T , Minoguchi S , Yamamichi N , et al. ( 2008 ) miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism . J Mol Biol 378 : 492 - 504 .
23. Mudduluru G , George-William JN , Muppala S , Asangani IA , Kumarswamy R , et al. ( 2011 ) Curcumin regulates miR-21 expression and inhibits invasion and metastasis in colorectal cancer . Biosci Rep 31 : 185 - 197 .
24. Chang TC , Wentzel EA , Kent OA , Ramachandran K , Mullendore M , et al. ( 2007 ) Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis . Mol Cell 26 : 745 - 752 .
25. Mudduluru G , Ceppi P , Kumarswamy R , Scagliotti GV , Papotti M , et al. ( 2011 ) Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR199a/b in solid cancer . Oncogene 30 : 2888 - 2899 .
26. Chantret I , Barbat A , Dussaulx E , Brattain MG , Zweibaum A ( 1988 ) Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines . Cancer Res 48 : 1936 - 1942 .
27. Sagiv E , Starr A , Rozovski U , Khosravi R , Altevogt P , et al. ( 2008 ) Targeting CD24 for treatment of colorectal and pancreatic cancer by monoclonal antibodies or small interfering RNA . Cancer Res 68 : 2803 - 2812 .
28. Kim NH , Kim HS , Kim NG , Lee I , Choi HS , et al. ( 2011 ) p53 and microRNA34 are suppressors of canonical Wnt signaling . Sci Signal 4 : ra71 .
29. 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 : e29722 .
30. Mudduluru G , Leupold JH , Stroebel P , Allgayer H ( 2011 ) PMA up-regulates the transcription of Axl by AP-1 transcription factor binding to TRE sequences via the MAPK cascade in leukaemia cells . Biol Cell 103 : 21 - 33 .
31. Leupold JH , Asangani I , Maurer GD , Lengyel E , Post S , et al. ( 2007 ) Src induces urokinase receptor gene expression and invasion/intravasation via activator protein-1/p-c-Jun in colorectal cancer . Mol Cancer Res 5 : 485 - 496 .
32. Guarino M ( 2010 ) Src signaling in cancer invasion . J Cell Physiol 223 : 14 - 26 .
33. Krichevsky AM , Gabriely G ( 2009 ) miR-21: a small multi-faceted RNA . J Cell Mol Med 13 : 39 - 53 .
34. Selcuklu SD , Donoghue MT , Spillane C ( 2009 ) miR-21 as a key regulator of oncogenic processes . Biochem Soc Trans 37 : 918 - 925 .
35. Chan JA , Krichevsky AM , Kosik KS ( 2005 ) MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells . Cancer Res 65 : 6029 - 6033 .
36. Sathyan P , Golden HB , Miranda RC ( 2007 ) Competing interactions between micro-RNAs determine neural progenitor survival and proliferation after ethanol exposure: evidence from an ex vivo model of the fetal cerebral cortical neuroepithelium . J Neurosci 27 : 8546 - 8557 .
37. Si ML , Zhu S , Wu H , Lu Z , Wu F , et al. ( 2007 ) miR-21-mediated tumor growth . Oncogene 26 : 2799 - 2803 .
38. Papagiannakopoulos T , Shapiro A , Kosik KS ( 2008 ) MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells . Cancer Res 68 : 8164 - 8172 .
39. Schramedei K , Morbt N , Pfeifer G , Lauter J , Rosolowski M , et al. ( 2011 ) MicroRNA-21 targets tumor suppressor genes ANP32A and SMARCA4 . Oncogene 30 : 2975 - 2985 .
40. Yang XR , Xu Y , Yu B , Zhou J , Li JC , et al. ( 2009 ) CD24 is a novel predictor for poor prognosis of hepatocellular carcinoma after surgery . Clin Cancer Res 15 : 5518 - 5527 .
41. Wilson GR , Cramer A , Welman A , Knox F , Swindell R , et al. ( 2006 ) Activated c-SRC in ductal carcinoma in situ correlates with high tumour grade, high proliferation and HER2 positivity . Br J Cancer 95 : 1410 - 1414 .
42. Zhang J , Kalyankrishna S , Wislez M , Thilaganathan N , Saigal B , et al. ( 2007 ) SRC-family kinases are activated in non-small cell lung cancer and promote the survival of epidermal growth factor receptor-dependent cell lines . Am J Pathol 170 : 366 - 376 .
43. Kim LC , Song L , Haura EB ( 2009 ) Src kinases as therapeutic targets for cancer . Nat Rev Clin Oncol 6 : 587 - 595 .
44. Hermeking H ( 2010 ) The miR-34 family in cancer and apoptosis . Cell Death Differ 17 : 193 - 199 .
45. de Antonellis P , Medaglia C , Cusanelli E , Andolfo I , Liguori L , et al. ( 2011 ) MiR-34a targeting of Notch ligand delta-like 1 impairs CD15+/CD133+ tumorpropagating cells and supports neural differentiation in medulloblastoma . PLoS One 6 : e24584 .
46. Lee JH , Voortman J , Dingemans AM , Voeller DM , Pham T , et al. ( 2011 ) MicroRNA expression and clinical outcome of small cell lung cancer . PLoS One 6 : e21300 .
47. Pang RT , Leung CO , Ye TM , Liu W , Chiu PC , et al. ( 2010 ) MicroRNA-34a suppresses invasion through downregulation of Notch1 and Jagged1 in cervical carcinoma and choriocarcinoma cells . Carcinogenesis 31 : 1037 - 1044 .
48. Sun F , Fu H , Liu Q , Tie Y , Zhu J , et al. ( 2008 ) Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest . FEBS Lett 582 : 1564 - 1568 .
49. Wei JS , Song YK , Durinck S , Chen QR , Cheuk AT , et al. ( 2008 ) The MYCN oncogene is a direct target of miR-34a . Oncogene 27 : 5204 - 5213 .
50. Chen QR , Yu LR , Tsang P , Wei JS , Song YK , et al. ( 2011 ) Systematic proteome analysis identifies transcription factor YY1 as a direct target of miR-34a . J Proteome Res 10 : 479 - 487 .
51. Yamakuchi M , Ferlito M , Lowenstein CJ ( 2008 ) miR-34a repression of SIRT1 regulates apoptosis . Proc Natl Acad Sci U S A 105 : 13421 - 13426 .
52. Majid S , Saini S , Dar AA , Hirata H , Shahryari V , et al. ( 2011 ) MicroRNA-205 inhibits Src-mediated oncogenic pathways in renal cancer . Cancer Res 71 : 2611 - 2621 .
53. Saini S , Arora S , Majid S , Shahryari V , Chen Y , et al. ( 2011 ) Curcumin modulates microRNA-203-mediated regulation of the Src-Akt axis in bladder cancer . Cancer Prev Res (Phila) 4 : 1698 - 1709 .
54. Kong D , Heath E , Chen W , Cher M , Powell I , et al. ( 2012 ) Epigenetic silencing of miR-34a in human prostate cancer cells and tumor tissue specimens can be reversed by BR-DIM treatment . Am J Transl Res 4 : 14 - 23 .