microRNA-21 governs TORC1 activation in renal cancer cell proliferation and invasion.

Abstract

Metastatic renal cancer manifests multiple signatures of gene expression. Deviation in expression of mature miRNAs has been linked to human cancers. Importance of miR-21 in renal cell carcinomas is proposed from profiling studies using tumor tissue samples. However, the role of miR-21 function in causing renal cancer cell proliferation and invasion has not yet been shown. Using cultured renal carcinoma cells, we demonstrate enhanced expression of mature miR-21 along with pre-and pri-miR-21 by increased transcription compared to normal proximal tubular epithelial cells. Overexpression of miR-21 Sponge to quench endogenous miR-21 levels inhibited proliferation, migration and invasion of renal cancer cells. In the absence of mutation in the PTEN tumor suppressor gene, PTEN protein levels are frequently downregulated in renal cancer. We show that miR-21 targets PTEN mRNA 3'untranslated region to decrease PTEN protein expression and augments Akt phosphorylation in renal cancer cells. Downregulation of PTEN as well as overexpression of constitutively active Akt kinase prevented miR-21 Sponge-induced inhibition of renal cancer cell proliferation and migration. Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation. Finally, we demonstrate that expression of constitutively active TORC1 attenuated miR-21 Sponge-mediated suppression of proliferation and migration of renal cancer cells. Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.

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microRNA-21 Governs TORC1 Activation in Renal Cancer
Cell Proliferation and Invasion
Nirmalya Dey1, Falguni Das1, Nandini Ghosh-Choudhury2,4, Chandi Charan Mandal4, Dipen J. Parekh5,
Karen Block1,2, Balakuntalam S. Kasinath1,2, Hanna E. Abboud1,2, Goutam Ghosh Choudhury1,2,3*
1Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America, 2Veterans Administration Research,
South Texas Veterans Health Care System, San Antonio, Texas, United States of America, 3Geriatric Research, Education and Clinical Center, South Texas Veterans Health
Care System, San Antonio, Texas, United States of America, 4Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, Texas,
United States of America, 5Department of Urology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America
Abstract
Metastatic renal cancer manifests multiple signatures of gene expression. Deviation in expression of mature miRNAs has
been linked to human cancers. Importance of miR-21 in renal cell carcinomas is proposed from profiling studies using tumor
tissue samples. However, the role of miR-21 function in causing renal cancer cell proliferation and invasion has not yet been
shown. Using cultured renal carcinoma cells, we demonstrate enhanced expression of mature miR-21 along with pre-and
pri-miR-21 by increased transcription compared to normal proximal tubular epithelial cells. Overexpression of miR-21
Sponge to quench endogenous miR-21 levels inhibited proliferation, migration and invasion of renal cancer cells. In the
absence of mutation in the PTEN tumor suppressor gene, PTEN protein levels are frequently downregulated in renal cancer.
We show that miR-21 targets PTEN mRNA 39untranslated region to decrease PTEN protein expression and augments Akt
phosphorylation in renal cancer cells. Downregulation of PTEN as well as overexpression of constitutively active Akt kinase
prevented miR-21 Sponge-induced inhibition of renal cancer cell proliferation and migration. Moreover, we show that miR-
21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1
activation. Finally, we demonstrate that expression of constitutively active TORC1 attenuated miR-21 Sponge-mediated
suppression of proliferation and migration of renal cancer cells. Our results uncover a layer of post-transcriptional regulation
of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal
cancer cell proliferation and invasion.
Citation: Dey N, Das F, Ghosh-Choudhury N, Mandal CC, Parekh DJ, et al. (2012) microRNA-21 Governs TORC1 Activation in Renal Cancer Cell Proliferation and
Invasion. PLoS ONE 7(6): e37366. doi:10.1371/journal.pone.0037366
Editor: Soumitro Pal, Children’s Hospital Boston & Harvard Medical School, United States of America
Received December 22, 2011; Accepted April 20, 2012; Published June 4, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: A United States National Institutes of Health (NIH) RO1 DK50190 grant to GGC supported this work. Part of this work was also supported by VA
Research Service Merit Review grant to GGC. GGC is also supported by Juvenile Diabetes Research Foundation 1-2008-185 grants and is recipient of VA Senior
Research Career Scientist Award. NGC is supported by VA Merit Review, NIH RO1 AR 52425 grants and Ronald P. Williams Orthopedic Oncology Developmental
Research Award from Cancer Therapy and Research Center, San Antonio, Texas. KB, BSK and HEA are supported by grants, NIH RO1 CA 131272 (KB), NIH RO1 DK
077295 (BSK), NIH RO1 DK078971 (HEA), NIH RC2A 036613 (BSK) and VA Research Service Career Scientist Award (KB) and Merit Review Awards (KB, BSK and HEA).
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: choudhuryg@uthscsa.edu
Introduction
Renal cell carcinoma represents the most common kidney
malignancy; about 70,000 new cases have been reported in the
year 2011 (www.cancer.gov). Among the five subtypes, clear cell
renal carcinoma (RCC) accounts for about 70% of the cases [1].
About 30% of patients with RCC develop invasive disease
commonly metastasizing to bone, lung, brain and liver [2,3]. Loss
of VHL (von Hippel-Lindau) protein expression due to germline
mutation, biallellic somatic mutation or hypermethylation of its
gene locus poses a high risk for clear cell renal carcinoma,
hemangiomas and pheochromocytomas [4,5]. Defective VHL
expression causes stabilization of Hifa transcription factors, which
contribute to the increased expression of vascular endothelial
growth factor (VEGF) to maintain vascular nature of the tumor.
Also, Hifa regulates anaerobic respiration often found in RCC [5].
Hifa-independent function of VHL has been reported in driving
kidney carcinoma, including regulation of senescence [5,6].
Furthermore, VHL positive kidney tumors utilize alternative
mechanisms to increase Hifa transcription factors for VEGF
expression, and, Hifa-independent growth factor receptor upre-
gulation [5,7].
miRNAs are short noncoding oligonucleotides with imperfect
complementarity predominantly to the 39untranslated region
(UTR) of target mRNAs [8,9,10]. Nearly 1000 miRNAs in
humans regulate the expression of one third of the total protein
coding transcriptome at the posttranscriptional and translational
level [9]. miRNAs predominantly act by inhibiting mRNA
translation although mRNA degradation and mRNA cleavage
may also contribute to downregulation of protein levels. Inappro-
priate expression of miRNAs have been linked to oncogenesis
[10,11]. miRNAs are coded by the intronic and intergenic as well
as exon sequences in the genome [12]. They are synthesized
predominantly by the RNA polymerase II-dependent transcription
to produce pri-miRNA hairpin, which binds Drosha/DGCR8
complex. The double stranded RNA-binding protein DGCR8
recognizes the proximal bases (, 10 bp) of the pri-miRNA stem
followed by its cleavage by the RNase III enzyme Drosha to
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release the pre-miRNA short hairpin [13]. Exportin-5 and its
partner Ran-GTP induce nuclear export of the pre-miR to the
cytoplasm where it is processed by the dicer RNase III/TRBP to
yield ,22 nucleotide small RNA duplex. The guide strand then is
incorporated into effector Argonaute complex to form RISC
(RNA-induced silencing complex) and to bind with imperfect
complementarity to the mRNA for translational repression [12].
Recent reports established a firm role of specific miRNA
signature in renal tumorigenesis. Profiling experiments showed
that more miRNAs are downregulated in RCC than upregulated
[14,15,16,17]. For example, in an initial screen of 470 miRNAs,
only six miRNAs were found to be upregulated in RCC while 15
were downregulated [16]. In another study, only 2 miRNAs were
increased in RCC including miR-21 whereas the expression of 17
miRNAs was decreased [17]. Similarly, a more recent report
showed increased expression of miR-21 among 9 miRNAs while
the expression of 26 miRNAs was suppressed [14]. Recently, an
extensive study using a large number of cancer samples from 31
different solid tumors described a significant increase in miR-21
suggesting its function in oncogenesis [18]. However, its functional
role in many cancers including renal carcinoma has not been
elucidated. In the present study, we find increased expression of
mature, pre- and pri-miR-21 in renal cancer cells as compared to
normal proximal tubular epithelial cells. This increase in miR-21
was associated with decreased PTEN levels. Neutralization of
miR-21 prevented proliferation, migration and invasion of these
cells concomitant with increased PTEN and reduced tuberin
phosphorylation and mTORC1 activation.
Results
Increased Expression of miR-21 in Renal Cell Carcinoma
miRNA expression profiling using tumor samples from patients
with renal carcinoma has been reported. In three studies, the
expression of miR-21 was found to be increased [14,17,19].
However, in another study in 16 clear cell renal carcinoma and 4
chromophobe renal carcinoma samples, miR-21 was not detected
[16]. We used a panel of tumor tissues from clear cell renal
carcinomas to detect the expression of mature miR-21. Real time
qRT-PCR revealed markedly increased expression of mature
miR-21 in all grade 2 (3 out of 3) and grade 3 (3 out of 3) renal cell
carcinoma samples (Fig. S1A and S1B). Next, we investigated the
expression of miR-21 in ACHN renal carcinoma cells. We found
significantly increased expression of mature miR-21 in ACHN
cells as compared to the HK2 normal proximal tubular epithelial
cells (Fig. 1A). Similar results were obtained in another renal
cancer cell line Caki-2 (Fig. S2). Recent reports showed that miR-
21 is regulated at the level of maturation [20]. Therefore, we
examined the levels of pre-miR-21. Real time qRT-PCR showed
marked expression of pre-miR-21 in ACHN cells compared to
HK2 cells (Fig. 1B). Similarly, increased expression of pri-miR-21
was also detected in the ACHN cells (Fig. 1C). This increase in pri-
miR suggested a transcriptional mechanism of miR-21 expression.
To directly examine the transcriptional regulation of miR-21
expression, we used a miR-21 promoter-driven firefly luciferase
reporter construct (miR-21-Luc). Transient transfection assays in
ACHN cells revealed significantly increased transcription of the
reporter gene (Fig. 1D). These results indicate that enhanced levels
of mature miR-21 in renal carcinoma cells may result from the
increased transcription of miR-21 gene to produce pri-mir-21.
Transcription of miR-21 gene has recently been shown to be
regulated by NFkB [21]. NFkB is upregulated in renal cancer
[22,23]. The transcriptional activity of the p65 subunit of NFkB is
dependent upon its phosphorylation at Ser-536 [24]. Therefore,
we examined phosphorylation of p65. As shown in Fig. 1E,
phosphorylation of p65 at Ser-536 was significantly increased in
the ACHN renal cancer cells compared to the HK2 cells.
Furthermore, enhanced p65 levels were also observed (Fig. 1E,
middle panel). Next, we tested the involvement of NFkB in
transcription of miR-21 in renal cancer cells. miR-21-Luc reporter
was cotransfected with either the S536A mutant of p65 or with a
vector plasmid. Expression of p65 S536A significantly inhibited
the reporter activity in ACHN cells. These data indicate that
upregulation of miR-21 may partly be due to increased NFkB
activity in renal cancer cells.
miR-21 Regulates Proliferation and Invasion of Renal
Cancer Cells
miR-21 is considered to be an onco-miR in many cancers.
However, its role in renal cell carcinoma has not been explored.
Therefore, we tested the involvement of miR-21 in mitogenesis of
ACHN cells, using a plasmid vector expressing seven copies of the
bulged miR-21 binding site placed in the 39 end of CMV
promoter-driven GFP mRNA (Fig. S3) [25]. Expression of this
construct serves as a ‘‘Sponge’’ that quenches the levels of
endogenous miR-21 [25,26]. ACHN cells were transfected with
this construct (miR-21 Sponge). DNA synthesis was measured as
3H-thymidine incorporation. Expression of miR-21 Sponge
significantly inhibited DNA synthesis in ACHN cells (Fig. 2A
and Fig. S4A). To confirm this observation, we performed
proliferation assay by counting the number of cells after
transfecting miR-21 Sponge. Expression of miR-21 Sponge
suppressed cell growth (Fig. 2B and Fig. S4B).
Renal cell carcinoma is often highly metastatic. We tested
whether miR-21 controls ACHN cell migration using trans-well
chamber assay. Cells cultured in a serum-free medium on the top
of the membrane migrated to the bottom of the membrane
(Fig. 2C, panel a). Expression of miR-21 Sponge prevented the
migration of ACHN cells (Fig. 2C and Fig. S4C). Quantification of
these results shows significant inhibition of migration of renal
cancer cells in response to miR-21 Sponge (Fig. 2D).
The initial step in metastasis consists of local invasion by cancer
cells (intravasation). To examine the metastatic potential of ACHN
renal cancer cells, we employed an invasion assay using collagen-
embedded membranes in trans-wells. Vector-transfected ACHN
cells showed marked invasion (Fig. 2E, panel a). Expression of
miR-21 Sponge blocked the invasion of tumor cells (Fig. 2E and
Fig. S4D). Quantification showed significant inhibition of invasion
of ACHN cells by miR-21 Sponge (Fig. 2F).
miR-21 Targets PTEN in Renal Cancer Cells
Mutation in PTEN tumor suppressor gene or its reduced
expression contribute to tumorigenesis and metastasis of many
cancers [27,28]. However, PTEN mutation is not frequently found
in renal cell carcinoma [29]. PTEN has been validated to be a
target of miR-21 [30]. We investigated the levels of PTEN in
ACHN renal cancer cells. As shown in Fig. 3A, the abundance of
PTEN in ACHN was significantly reduced as compared to that in
HK2 cells. Identical results were obtained in Caki-2 renal
carcinoma cell line (Fig. S5A). PTEN reduces the levels of PIP3,
which controls the phosphorylation/activation of Akt [27,31,32].
Reduced PTEN levels in ACHN and Caki-2 cells were associated
with increased phosphorylation of Akt at Thr-308 and Ser-473
(Fig. 3B and Fig. S5B), indicating its activation [32].
Next, we examined whether miR-21 targets PTEN. We used a
reporter construct in which the 39UTR of PTEN mRNA is cloned
downstream of firefly luciferase gene (PTEN 39UTR-Luc) [25].
ACHN cells were transfected with this reporter and vector or miR-
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21 expression plasmid. Expression of miR-21 significantly
inhibited the reporter activity (Fig. 4A and Fig. S6A). To confirm
this observation, we used miR-21 Sponge. Expression of miR-21
Sponge markedly increased the luciferase activity of PTEN
39UTR-Luc (Fig. 4B and Fig. S6B). These results suggest that in
renal cancer cells, PTEN may be a downstream target of miR-21.
To test this, we examined PTEN protein level in miR-21 Sponge-
transfected ACHN cells. miR-21 Sponge increased the abundance
of PTEN (Fig. 4C and Fig. S6C), concomitant with decreased
phosphorylation of Akt at Thr-308 and Ser-473 (Fig. 4D).
Figure 1. Expression of miR-21 in renal cancer cells. (A – C) Total RNAs from HK2 proximal tubular epithelial cells and ACHN renal cancer cells
were used to detect mature miR-21 (panel A), pre-miR-21 (panel B) and pri-miR-21 (panel C) by real time qRT-PCR as described in the Materials and
Methods. Mean 6 SE of 4 measurements is shown. In panels A and B, *p =0.004 vs HK2 cells. In panel C, *p =0.02 vs HK2 cells. (D) HK2 and ACHN
cells were transfected with miR-21-Luc reporter along with Renilla null plasmid. The cell lysates were used to determine luciferase activity as
described in the Materials and Methods. Mean 6 SE of 6 measurements is shown. *p =0.001 vs HK2 cells. (E) Increased phosphorylation of p65 NFkB
in renal cancer cells. Lysates of HK2 and ACHN cells were immunoblotted with phospho-p65 (Ser-536), p65 and actin antibodies. (F) Mutant p65
S536A inhibits miR-21 transcription. ACHN renal cancer cells were cotransfected with miR-21-Luc and p65 S536A expression vector. The luciferase
activity was determined in the cell lysates. Mean 6 SE of quadruplicate measurements is shown. *p =0.02 vs vector. Bottom panels show expression
of the HA-tagged p65 S536A and actin.
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miR-21 Regulates Renal Cancer Cell Proliferation and
Migration via PTEN/Akt Axis
Our results above suggest that miR-21 promotes Akt activation
by decreasing PTEN levels in renal cancer cells (Fig. 4). In order to
directly investigate the role of this signaling pathway in renal
cancer cell proliferation, we transfected ACHN and Caki-2 cells
with miR-21 Sponge and siRNAs targeting PTEN (siPTEN). As
expected, miR-21 Sponge inhibited DNA synthesis (Fig. 5A and
Fig. S7A). In contrast, transfection of siPTEN significantly
prevented miR-21 Sponge-induced inhibition of DNA synthesis
Figure 2. Effect of miR-21 Sponge on proliferation, migration and invasion of renal cancer cells. ACHN cells were transfected with miR-
21 Sponge or vector plasmids. (A) Serum-starved cells were incubated with
described in the Materials and Methods [115]. Mean 6 SE of 12 measurements is shown. *p =0.004 vs vector alone. (B) The cells were counted at
indicated time periods as described in the Materials and Methods. Diamond and square symbols represent vector and miR-21 Sponge-transfected
cells, respectively. Means 6 SE of triplicate measurements are shown. *p,0.05 vs vector alone. (C) Serum-starved cells were seeded on to membrane
in a trans-well chamber. Migration assay was performed and the migrated cells at the opposite side of the membrane were stained as described in
the Materials and Methods [111]. (D) The stains in the migrated cells in panel C were eluted as described in the Materials and Methods [111]. Mean 6
SE of triplicate measurements is shown. *p,0.0001 vs vector-transfected cells. (E) Transfected serum-starved cells were seeded on to collagen-
embedded membrane in a trans-well chamber. Invasion assay was performed and the invaded cells at the opposite side of the membrane were
stained as described in the Materials and Methods [111]. (F) The stains in the invaded cells in panel E were eluted as described in the Materials and
Methods [111]. Mean 6 SE of triplicate measurements is shown. *p,0.001 vs vector-transfected cells.
doi:10.1371/journal.pone.0037366.g002
3H-thymidine and its incorporation into DNA was determined as
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(Fig. 5A and Figs. S7A, S7B and S7C). Similarly, siPTEN reversed
the decrease in proliferation of ACHN cells induced by miR-21
Sponge (Fig. 5B and Fig. S8). Next, we determined the effect of
siPTEN on ACHN and Caki-2 cell migration. Expression of miR-
21 Sponge decreased the migration of both these renal cancer cell
lines. siPTEN significantly suppressed the inhibitory effect of miR-
21 Sponge on cell migration (Figs. 5C, 5D and Figs. S9 and S10).
The lipid phosphatase activity of PTEN is known to regulate the
Akt phosphorylation [27]. We have shown above that reduced
PTEN level in renal cancer cells is associated with increased
phosphorylation of Akt (Fig. 3 and Fig. S5). Since miR-21
regulates PTEN protein level, which in turn activates Akt, we
tested the role of this kinase in renal cancer cell proliferation in
relation to miR-21. We transfected ACHN and Caki-2 cells with
constitutively active Gag-Akt along with miR-21 Sponge [33].
Expression of Gag Akt significantly reversed the inhibition of DNA
synthesis induced by miR-21 Sponge (Fig. 6A, Fig. S11A and Fig.
S12). Expression of Gag Akt also reversed the cell proliferation by
miR-21 Sponge (6B and Figs. S13A). Similarly, constitutively
active Akt significantly prevented the inhibition of migration of
ACHN cells in response to miR-21 Sponge (Figs. 6C, 6D and Fig.
S13B). Identical results were obtained with Caki-2 renal cancer
cells (Figs. S14A, S14B and S14C). These results indicate that
miR-21 targets PTEN mRNA to suppress its protein expression,
which leads to Akt-dependent proliferation and migration of renal
cancer cells.
Figure 3. Expression of PTEN in HK2 and ACHN cells. (A) The lysates of cells from three independent wells of HK2 and ACHN cells were
immunoblotted with PTEN and actin antibodies. (B) The same cell lysates were immunoblotted with phospho-Akt (Thr-308), phospho-Akt (Ser-473)
and Akt antibodies.
doi:10.1371/journal.pone.0037366.g003
Figure 4. miR-21 regulates expression of PTEN in renal cancer cells. (A and B) ACHN cells were transfected with pCMV-miR-21 expression
plasmid (A) or miR-21 Sponge (B) and vector along with PTEN 39UTR-Luc reporter construct. The cell lysates were used to determine luciferase activity
as described in the Materials and Methods. Mean 6 SE of 6 measurements is shown. In panel A, *p =0.02 vs vector alone. In panel B, *p =0.002 vs
vector alone. (C and D) Lysates of miR-21 Sponge-transfected ACHN cells were immunoblotted with PTEN and actin antibodies (panel C), and
phospho-Akt (Ser-473), phospho-Akt (Thr-308) and Akt antibodies (panel D).
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miR-21 Modulates TORC1 Activity to Induce Renal Cancer
Cell Proliferation and Migration
Activated Akt phosphorylates the tumor suppressor protein
tuberin at Thr-1462 leading to its inactivation, and Rheb-
mediated activation of TORC1 [34,35,36]. We have established
that downregulation of PTEN due to increased miR-21 expression
in renal cancer cells activates Akt (Fig. 3), which contributes to
proliferation and migration of these cells (Figs. 5 and 6). We tested
the role of miR-21 in tuberin phosphorylation. ACHN cells were
transfected with miR-21 Sponge. Due to increased Akt activation,
ACHN cells display enhanced phosphorylation of tuberin (Fig. 7A,
lane 1). Expression of miR-21 Sponge inhibited the phosphory-
Figure 5. miR-21 targets PTEN to induce proliferation and migration of renal cancer cells. ACHN cells were transfected with miR-21
Sponge along with siRNA pools targeting PTEN mRNA as indicated. (A)3H-thymidine incorporation was determined as described in the Materials and
Methods [115]. Mean 6 SE of 6 measurements is shown. *p,0.05 vs vector; **p,0.05 vs miR-21 Sponge-transfected cells. (B) Transfected cells were
counted at indicated time periods. The symbols diamond, square and cross represent vector, miR-21 Sponge and miR-21 Sponge plus siRNA pool
against PTEN, respectively. Mean 6 SE of triplicate measurements is shown. *p,0.05 vs vector alone; **p,0.01 vs miR-21 Sponge alone. (C)
Transfected cells were seeded onto membrane in trans-well chambers and the migrated cells were stained as described in the Materials and Methods
[111]. (D) Stains from the membranes in panel C were eluted and absorbance at 590 nm was measured. Mean 6 SE of 3 independent chambers is
shown. *p,0.001 vs vector alone; **p,0.001 vs miR-21 Sponge.
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lation of tuberin (Fig. 7A and Fig. S15A). Similar to increased
tuberin phosphorylation, ACHN cells showed augmented phos-
phorylation of S6 kinase at Thr-389 (Fig. 7B, lane 1), measured as
a surrogate for TORC1 activity. miR-21 Sponge blocked S6
kinase phosphorylation (Fig. 7B). This reduction in S6 kinase
phosphorylation by miR-21 Sponge was associated with inhibition
of mTOR phosphorylation at Ser-2448 (Fig. 7C). These results
suggest that miR-21 regulates TORC1 activity in the renal cancer
cells. To examine whether miR-21 Sponge-dependent mTOR
inhibition is due to inactivation of Rheb, we cotransfected ACHN
cells with miR-21 Sponge and a constitutively active Rheb
expression plasmid and the results were compared with that in
miR-21 Sponge-transfected cells. mTOR activation was examined
in the cell lysates. Expression of constitutively active Rheb reversed
the inhibitory effect of miR-21 Sponge on S6 kinase phosphor-
ylation (Fig. 7D and Fig. S15B). Similarly, the reduced phosphor-
ylation of mTOR by miR-21 Sponge was also prevented by
constitutively active Rheb (Fig. 7E and Fig. S15C). These results
Figure 6. miR-21 stimulates Akt kinase to induce proliferation and migration of renal cancer cells. ACHN cells were transfected with
miR-21 Sponge along with constitutively active Gag Akt plasmids as indicated. (A)3H-thymidine incorporation was determined as described in the
Materials and Methods [115]. Mean 6 SE of 6 measurements is shown. *p,0.01 vs vector; **p,0.001 vs miR-21 Sponge alone. (B) Transfected cells
were counted at indicated time periods. The symbols diamond, square and cross represent vector, miR-21 Sponge and miR-21 Sponge plus Gag Akt
expression plasmids, respectively. *p,0.01 vs vector alone; **p,0.001 vs miR-21 Sponge alone. (C) Transfected cells were seeded onto membrane in
trans-well chambers and the migrated cells were stained as described in the Materials and Methods [111]. (D) Stains from the membranes in panel C
were eluted and absorbance was measured. Mean 6 SE of 3 independent chambers is shown. *p,0.001 vs vector alone; **p,0.001 vs miR-21
Sponge.
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conclusively demonstrate that miR-21-mediated phosphorylation/
inactivation of tuberin activates Rheb to increase mTOR activity.
To elucidate whether miR-21-activated TORC1 contributes to
proliferation of renal cancer cells, we cotransfected ACHN and
Caki-2 cells with miR-21 Sponge and a constitutively active
mTOR expression vector.
determined. Expression of constitutively active mTOR signifi-
cantly prevented the inhibition of DNA synthesis by miR-21
Sponge (Fig. 8A, Fig. S16 and Fig. S17). Similarly, constitutively
active mTOR inhibited the miR-21 Sponge-mediated decrease in
ACHN cell proliferation (Fig. 8B and Fig. S18A). Next we
examined the effect of constitutively active mTOR on renal cancer
cell migration. miR-21 Sponge abolished migration of ACHN and
Caki-2 cells (Fig. 8C, Fig. S18B and Fig. S19). However,
coexpression of constitutively active mTOR with miR-21 Sponge
significantly reversed the inhibition in migration of both renal
cancer cell lines in response to miR-21 Sponge (Figs. 8C, 8D and
Fig. S19). Taken together these results demonstrate that miR-21
contributes to renal cancer cell proliferation and migration via
TORC1.
3H-thymidine incorporation was
Discussion
In renal cancer cells, we provide evidence for the expression of
pri-miR-21 to produce pre- and mature miR-21, which contrib-
utes to proliferation and migration/invasion. We show an inverse
correlation between miR-21 levels and PTEN abundance. We
demonstrate that miR-21-sensitive PTEN regulates proliferation
and migration of renal cancer cells via activation of Akt. Finally,
we establish a role for miR-21-stimulated TORC1 in renal cancer
cell proliferation and migration (Fig. 9).
Altered gene expression controls signaling networks to regulate
the biological outputs, which contribute to cell transformation and
metastasis. Although transcriptional regulation of genes to produce
mRNAs mediates a substantial contribution to oncogenesis, recent
discovery of miRNAs playing a role similar to DNA binding
transcription factors is established in regulating expression of
target genes by post-transcriptional mechanism. Many miRNAs
have been identified and characterized as tumor promoters as well
as tumor suppressors. For example, the ubiquitously expressed
miRNA, miR-21, is frequently upregulated in many cancers
including breast, lung, neuroblastoma, glioblastoma, leukemia,
gastric cancer, cholangiocarcinoma, colon cancer and hepatocel-
lular cancer [37,38,39,40,41,42]. However, information about the
functional role of miR-21 is available only from in vitro studies.
Only recently the in vivo role of miR-21 in development of cancer
has been demonstrated in pre-B-cell lymphoma and non-small cell
lung carcinoma. In both these cancers, expression of miR-21 is
significantly increased [43,44,45]. Using mouse models of overex-
pression as well as deletion of miR-21, Medina et al and Hatley et
al recently showed a highly specific role of this miRNA in the
development of pre-B-cell lymphoma and non-small cell lung
carcinoma, respectively [46,47].
miR-21 regulates proliferation and mitochondrial apoptotic
pathways controlled by tumor suppressor proteins, cell cycle
regulators and growth factors [48,49]. miR-21 is expressed
Figure 7. miR-21 regulates phosphorylation of tuberin and activation of mTOR in renal cancer cells. ACHN cells were transfected with
miR-21 Sponge or vector. The cell lysates were immunoblotted with phospho-tuberin (Thr-1462), tuberin (panel A), phospho-S6 kinase (Thr-389), S6
kinase (panel B), phospho-mTOR (Ser-2448) and mTOR (panel C). miR-21 uses Rheb activation for TORC1 activity. ACHN cells were cotransfected with
miR-21 Sponge and CA Rheb plasmid. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel D), phospho-mTOR
(Ser-2448) and mTOR (panel E).
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abundantly in kidney proximal tubules [50]. In animal models,
increased expression of miR-21 is associated with fibrotic disorders
of kidney and renal ischemic injury [51,52,53,54]. Recently, we
have reported increased expression of miR-21 in a model of type 1
diabetic nephropathy [25]. We showed that miR-21 regulates
hypertrophy of proximal tubular epithelial cells, indicating the
pathological role of this miRNA in renal diseases [25]. Similarly,
miRNA profiling studies identified increased expression of miR-21
in both clear cell and papillary renal cell carcinomas [14,19,55]. In
contrast, in a recent microarray analysis of 30 grades 1 and 2 clear
cell renal carcinoma tissue, miR-21 was reported to be downreg-
ulated by more than 2-fold [56]. Interestingly, a weak reciprocal
correlation was detected between survival of patients with clear
cell renal carcinoma and miR-21 expression [55]. In this report
Figure 8. miR-21 regulates proliferation and migration of renal cancer cells through activation of TORC1. ACHN cells were transfected
with miR-21 Sponge along with constitutively active mTOR plasmids as indicated. (A)3H-thymidine incorporation was determined as described in the
Materials and Methods [115]. Mean 6 SE of 6 measurements is shown. *p,0.001 vs vector; **p,0.001 vs miR-21 Sponge alone. (B) Transfected cells
were counted at indicated time periods. The symbols diamond, square and cross represent vector, miR-21 Sponge and miR-21 Sponge plus
constitutively active (CA) mTOR expression plasmids, respectively. *p,0.01 vs vector alone; **p,0.001 vs miR-21 Sponge alone. (C) Transfected cells
were seeded onto membrane in trans-well chambers and the migrated cells were stained as described in the Materials and Methods [111]. (D) Stains
from the membranes in panel C were eluted and absorbance at 590 nm was measured. Mean 6 SE of 3 independent chambers is shown. *p,0.001
vs vector alone; **p,0.001 vs miR-21 Sponge.
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downregulation of miR-21 was found in VHL deficient RCC4
renal cancer cell line [55]. Reconstitution of VHL resulted in
increased expression of miR-21 in a Hifa-independent manner. It
should be noted that 42% of the clear cell tumors show VHL
mutation and 10% of the tumors carry methylation in the VHL
promoter [57,58,59]. In the present study, we show a significant
increase in miR-21 expression in the VHL positive ACHN and
Caki-2 renal cancer cells compared to normal proximal tubular
epithelial cells (Fig. 1 and Fig. S2) [60]. Furthermore, our results
demonstrate significantly enhanced miR-21 expression in 100% of
grade 2 and grade 3 renal tumor tissues compared to matched
control tissue (Fig. S1). Additionally, using a VHL negative renal
cancer cell line, 786-O, we found significantly increased expression
of miR-21 as compared to HK2 proximal tubular epithelial cells
(Fig. S20). These results demonstrate that irrespective of VHL
status, renal cancer cells express high levels of miR-21.
Metastatic renal carcinoma cells display increased TGFb-
dependent Smad 2/3 signaling [61]. Recently a role of TGFb in
increasing the levels of mature miR-21 has been reported [62].
These authors described a transcription-independent function of
TGFb-specific Smad proteins, which are recruited into the RNase
III Drosha through interaction with RNA helicase p68. This
microprocessor complex recruits pri-miR-21 to facilitate produc-
tion of pre-miR-21, thus increasing the levels of mature miR-21
[62]. This study did not find any increase in pri-miR-21 in
response to TGFb. More recently, the same group reported the
presence of TGFb-specific Smad binding element in the stem
region of pri-miR-21, which recruits Smad directly to the Drosha
microprocessor/pri-miR-21 complex for increased processing to
produce pre-miR-21, providing another mechanism for more
miR-21 production in response to TGFb [20]. In contrast to these
observations, we found increased expression of pri-miR-21 in the
metastatic ACHN renal cancer cells (Fig. 1). In addition, we
identified increased transcription of miR-21 gene by constitutively
active NFkB present in these cells (Fig. 1). This observation
provides a mechanism for enhanced levels of miR-21 detected in
renal carcinoma cells. Furthermore, in all of grade 2 and grade 3
renal tumor samples examined, we detected increased levels of pri-
miR-21 (data not shown). Thus in renal cell carcinomas, our
results demonstrate the existence of a regulatory mechanism for
increased mature miR-21 expression through increased transcrip-
tional activation of pri-miR-21 locus.
Since miRNAs essentially downregulate the expression of target
proteins, their increased expression in relation to tumorigenesis
and metastasis may possibly counteract the negative regulatory
mechanisms that operate in the proliferation/invasion signaling
pathways. We found enhanced levels of miR-21 in the renal
cancer cells. Inhibition of miR-21 significantly abrogated DNA
synthesis, proliferation, migration and invasion of these cells.
These results are in line with the recent demonstration of a positive
correlation between miR-21 levels and tumor size in patients with
renal carcinoma [55].
PI 3 kinase/Akt signaling pathway contributes to carcinogenesis
and metastasis of various organs including kidney. Activation of
these enzymes occurs due to increased growth factor receptor
activation as well as gain of function mutations in these two
enzymes. In fact 3000 different somatic mutations have been
identified in the p110a catalytic subunit of PI 3 kinase in various
cancers [63]. However, its mutation in the renal cancer is
extremely rare. Similarly, although activating mutation in Akt
occurs in various cancers, it has not been reported in renal
carcinoma [64]. Mutation in the PTEN gene, which codes for a
protein phosphatase as well as a lipid phosphatase and regulates
Akt activity, has been identified in many cancers [65]. Similar to
PI 3 kinase and Akt, PTEN mutation is rare in renal cell
carcinoma. However, 25% of clear cell renal carcinomas display
about 25% reduction in PTEN protein levels [66,67]. PTEN is
known to be transcriptionally downregulated in many cancer cells,
e.g., by NFkB-mediated transcriptional repression [68]. PTEN is
also regulated at post-translational level by phosphorylation,
acetylation, oxidation, ubiquitinylation and proteasomal degrada-
tion [69]. Furthermore, PTEN expression is lost in many human
tumors due to its promoter methylation [70]. Although PTEN
promoter methylation was detected in the renal cancer cells, it did
not affect PTEN transcription [71]. An additional mode of post-
transcriptional regulation of PTEN protein expression includes a
host of miRNAs, including miR-21 [72,73]. The half lives of
mature miRNAs are controlled by extracellular signals and cell
cycle status [74]. In renal tumor tissue and in metastatic ACHN
and Caki-2 renal cancer cells, we demonstrate constitutive increase
in the levels of mature miR-21 (Fig.1 and Fig. S2). Furthermore, in
renal cancer cells, a reciprocal relationship exists between miR-21
and PTEN protein expression (Fig. 3 and Fig. S5). Also, we
validated functional activity of miR-21 in ACHN cells in
negatively maintaining the PTEN protein abundance (Fig. 4).
PTEN prevents cell cycle arrest and apoptosis in a cell-specific
manner and also suppresses cell invasion [65,75]. Our results
demonstrate that inhibition of PTEN reversed miR-21 Sponge-
mediated decrease in DNA synthesis and proliferation (Figs. 5A,
5B and Fig. S7A). These data indicate that miR-21 regulates cell
cycle progression of renal cancer cells via PTEN. Importantly,
Figure 9. Schematic demonstrating our results. Enhanced miR-21
attenuates PTEN protein levels, resulting in activation of Akt, which
inactivates tuberin to increase TORC1 activity leading to proliferation,
migration and invasion of renal cancer cells.
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miR-21-targeted PTEN contributes to the migration of renal
cancer cells (Figs. 5C, 5D and Fig. S10).
Along with its lipid phosphatase activity, PTEN also possesses
phosphatase activity towards specific tyrosine phosphorylated
proteins including focal adhesion kinase, c-Src and platelet-
derived growth factor receptor [76,77,78]. The protein phospha-
tase activity of PTEN is required for cell proliferation, migration
and invasion [78,79,80]. Our results showing a role for miR-21-
targeted PTEN in renal cancer cell migration do not discriminate
between these two enzymatic activities of PTEN (Fig. 5 and Fig.
S10). Furthermore, a study in Drosophila showed that a PH-domain
mutant of Akt, which does not bind PIP3but retains 80% of wild
type kinase activity, reverses the lethality caused by absence of
PTEN [81]. These results conclusively demonstrate that the only
function of PTEN loss is to translocate Akt to the membrane to
enhance its kinase activity. In our study, we used constitutively
active Gag Akt, which localizes to the plasma membrane and
bypasses the PTEN-mediated regulatory loop [33]. Our results
using constitutively active Gag Akt to prevent the inhibitory action
of miR-21 Sponge on DNA synthesis, proliferation and migration
of ACHN and Caki-2 cells demonstrate directly the requirement
of PIP3phosphatase activity of PTEN for these biological functions
in renal cancer cells (Fig. 6 and Figs. S12, S14). Thus, our results
demonstrate that miR-21 predominantly targets the lipid phos-
phatase activity of PTEN to regulate Akt kinase in favor of renal
cancer cell proliferation and migration.
Reduced expression of PTEN resulting in Akt activation is
associated with cancer progression in many organs including
breast tumor [82,83]. In addition, miR-21 is upregulated in many
solid tumors [18]. To test the generality of our observation, we
examined the involvement of miR-21 in regulation of PTEN-
mediated proliferation of BT-20 human breast cancer cells.
Expression of miR-21 in these cells was significantly increased as
compared to the MCF10A normal breast epithelial cells (Fig. S21).
Expression of miR-21 Sponge markedly inhibited the proliferation
of BT-20 cells (Fig. S22A). Cotransfection of siPTEN reversed the
inhibition of proliferation induced by miR-21 Sponge (Fig. S22A,
S22B). Similarly, siPTEN prevented the reduction of BT-20 breast
cancer cell migration induced by miR-21 Sponge (Figs. S23A,
S23B). Next, we determined the involvement of Akt in the effect of
miR-21 Sponge in these breast cancer cells. Similar to the results
observed in renal cancer cells, expression of constitutively active
Gag-Akt reversed the inhibitory effect of miR-21 Sponge on BT-
20 cell proliferation and migration (Figs. S24 and S25). These
results indicate that miR-21 targeting of PTEN to activate Akt
contributes to proliferation and migration of other cancer cells
along with the renal cancer cells.
PTENheterozygous mice
cancer with 50% penetrence, while mice with p27 homozygous
deletion do not develop prostate cancer [84,85]. Interestingly,
all PTEN+/2p272/2
micedevelop
Similarly, in a recent study absence of both PTEN and the
cell cycle inhibitor p27 was shown to be associated with renal
cancer progression [86]. Furthermore, in these patients phos-
phorylation of ribosomal protein s6 was significantly correlated
with tumor stage and grade [86]. Since s6 is a substrate of
TORC1, these results predict high activity of this kinase
complex in renal cancer cells.
mTOR exists in two independent complexes each containing
shared and unshared protein subunits with non-overlapping
substrate specificity [87,88,89,90]. Both TORC1 and TORC2
regulate cell proliferation and apoptosis of tumor cells by
regulating distinct kinases [88,91,92]. For example, TORC1
augments inactivating phosphorylation of the translational repres-
producenoninvasive prostate
prostate cancer [85].
sors 4EBPs and activating phosphorylation of translational
activator S6 kinase [91]. Increased mRNA translation is a
prerequisite for cell proliferation [93]. On the other hand,
TORC2 phosphorylates Akt at the hydrophobic motif site Ser-
473 [92]. Akt plays a pivotal role in mTOR-mediated signaling as
it acts both upstream of TORC1 and downstream of TORC2
[91]. Thus Akt phosphorylates tuberin at Thr-1462 to inactivate
its suppressive action on Rheb, resulting in TORC1 activation
[35,94]. Inactivation of endogenous miR-21 in renal cancer cells
blocked phosphorylation of tuberin (Fig. 7A). This inhibition of
tuberin phosphorylation was associated with decreased phosphor-
ylations of S6 kinase and mTOR (Fig. 7B and 7C) [90,91].
Furthermore, our results demonstrate that miR-21 utilizes Rheb to
increase TORC1 activity (Figs. 7D and 7E). Expression of
constitutively active mTOR, which increases only TORC1 activity
without affecting TORC2, reversed the inhibitory effect of miR-21
Sponge on DNA synthesis and proliferation (Figs. 8A, 8B and Fig.
S17) [95]. These results thus indicate that in renal cancer cells,
miR-21 regulates the TORC1 activity by acting upstream of Akt,
presumably through targeting PTEN. PTEN as a PIP3phospha-
tase maintains the level of this lipid in cells and regulates TORC2
activity [96,97]. In fact we demonstrate inhibition of phosphor-
ylation of Akt at the TORC2 site Ser-473 by miR-21 Sponge
(Fig. 4D). Thus, our results show that miR-21 acts through both
TORC1 and TORC2.
TORC2 regulation of Rho GTPase, paxillin phosphorylation,
cytoskeletal organization and cell migration was initially reported
[89,98]. More recently TORC1-mediated S6 kinase pathway has
been shown to regulate phosphorylation of paxillin, p130CASand
focal adhesion kinase, which contribute to lamillapodia formation
and cell migration [99,100]. Rapamycin, which predominantly
inhibits TORC1 activity, abrogates invasion of normal as well as
many cancer cells in vitro and metastasis of implanted tumor cells in
mouse models, including pulmonary metastasis of human renal
cancer cells [101,102]. In the present study, we show that
expression of constitutively active TORC1 reversed the inhibition
of renal cancer cell migration in response to miR-21 Sponge
(Figs. 8C and 8D). Thus our results demonstrate a role of miR-21-
induced TORC1 activity in renal cancer cell invasion.
Although many rapalogs including temsirolimus and everolimus
with increased solubility and bioavailability are approved for
treatment of metastatic renal cancer, clinical trials showed limited
efficacy [103,104,105,106]. This is thought to be due to the release
of the negative feedback loop involving IGF-1 receptor signaling
on phosphorylation of Akt at Ser-473 by TORC2 [107,108]. In
fact use of rapamycin in a small clinical trial with 15 recurrent
glioblastoma patients lacking PTEN expression showed increased
phosphorylation of Akt at Ser-473 in 50% of the patients,
indicating the importance of relief of the negative feed back loop in
vivo [109]. In our study here, we demonstrate reduced PTEN levels
in renal cancer cell due to increased miR-21 expression, which
causes TORC1-mediated proliferation and invasion. Quenching
of miR-21 blocked proliferation and migration, which result from
attenuation of Akt phosphorylation at Thr-308 and Ser-473. Thus,
this strategy of miR-21 inhibition does not evoke the release of
negative feedback loop in these cells. Therefore, use of anti-miR-
21 in preclinical model of renal cell carcinoma holds promise to
provide a therapeutic option for this devastating cancer.
Materials and Methods
Materials
b-actin antibody, phenylmethylsulfonylfluoride, Na3VO4, NP-
40 and protease inhibitor cocktail were obtained from Sigma, St.
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Louis, MO. Antibodies against phospho-Akt (Ser-473 and Thr-
308), phospho-S6 kinase (Thr-389), phospho-tuberin (Thr-1462),
phospho-mTOR (Ser-2448), phospho-p65 (Ser-536), Akt, S6
kinase, mTOR and tuberin were purchased from Cell Signaling,
Boston, MA. p65 and PTEN antibodies and siRNA pool of three
oligonucleotides against human PTEN were obtained from Santa
Cruz, Delaware, CA. Trans-well migration chambers were
purchased from Greiner Bioone, Monroe, NC. Invasion chambers
were obtained from Millipore, MA. Fugene-HD transfection
reagent was purchased from Roche Molecular Biology, Indianap-
olis, IN. TRIZol reagent for RNA isolation was purchased from
Invitrogen, Carlsbad, CA. RT2real-time SYBR green/ROX PCR
master mix, RT2miRNA first strand kit, GAPDH RT-PCR
primers for human and primers for detection of mature miR-21
were obtained from SuperArray Biosciences, Frederick, MD. U6
primers (for normalization) were obtained from Ambion, Austin,
TX. Luciferase Reporter Assay System kit was purchased from
Promega, Madison, WI.
PerkinElmer, Boston, MA. pCMV-miR-21 expression plasmid,
PTEN 39 UTR-Luc reporter plasmid and miR-21 Sponge vector
have been described previously [25]. Constitutively active (CA)
Rheb (phage-CMV-Rheb (S16H) was obtained from Addgene.
Constitutively active mTOR expression vector was provided by
Dr. Tatsuya Maeda, The University of Tokyo, Japan [95]. miR-21
promoter driven luciferase reporter (-332 to +1957 bp) plasmid
(miR-21-Luc) was a kind gift from Dr. X-M Chen, Creighton
University Medical Center, Nebraska [21]. The constitutively
active Akt (Gag Akt) was a kind gift from Dr. J. Downward, Signal
Transduction Laboratory, London, UK [33].
3H-thymidine was purchased from
Human Tumor Specimens/Ethics Statement
Tumor samples and normal corresponding tissue from patients
with renal cancer were obtained from the Department of Urology
at the University of Texas Health Science Center at San Antonio.
The collection and handling of human samples was carried out
according to a protocol approved by the University of Texas
Health Science Center at San Antonio, Institutional Review Board
(IRB protocol #HSC 20070777N). This is a ‘‘Non Human/
NonResearch’’ protocol. Tissue is collected under non-identifiers
from Pathology using "unwanted tissue". Therefore, patient
consent is not necessary. The tumors for this study were
histologically classified as clear cell renal carcinoma and staged
according to the TNM classification.
Cell Culture
The ACHN renal carcinoma cell line was purchased from
American Type Culture Collection, Manassas, VA, and grown in
RPMI 1640 medium containing 10% fetal bovine serum and
penicillin/streptomycin. Caki-2 cells were kind gift of Dr. Sunil
Sudarsan, Department of Urology, The University of Texas Health
Science Center at San Antonio. These cells were grown in DMEM
containing low glucose with 10% fetal bovine serum. HK2 normal
human proximal tubular cells were grown in DMEM/F12 in the
presence of 10% fetal bovine serum [110] BT-20 human breast
cancer cells have been described previously [111].
Real Time Quantitative RT-PCR (qRT-PCR)
Total RNA was prepared from HK2 and ACHN cells and from
human tissue using TRIZol reagent as described previously
[25,68]. One mg of RNA was used to synthesize cDNA using
RT2miRNA first strand kit according to the manufacturer’s
instructions. qRT-PCR was performed using a real-time PCR
machine (7900HT, Applied Biosystems). Each sample was
analyzed in duplicate. PCR conditions for pre-miR-21 were:
94uC for 10 minutes, followed by 40 cycles at 94uC for 30 seconds,
56uC for 30 seconds, 72uC for 40 seconds. The primers used for
detection of pre-miR-21 are as follows: Forward primer: 59-
TGTCGGGTAGCTTATCAGAC-39;
TTCAGACAGCCCATCGACTG-39. Primers for pri-miR-21
are: Forward primer: 59- ACAGGCCAGAAATGCCTGGG-39;
Reverse primer: 59- GATGGTCAGATGAAAGATAC-39. For
mature miR-21, qRT-PCR primer sets for hsa-mir-21 (Super-
array) were used according to the manufacturer’s protocol. The
PCR conditions for amplifying pri-miR-21 were identical to those
for pre-miR-21 except annealing was performed at 54uC.
mirVana qRT-PCR primer sets for U6 (Ambion) were used for
normalization. Data analyses were done by the comparative Ct
method as described previously [25].
Reverseprimer:59-
Immunoblotting
Cells were lysed in RIPA buffer (20 mM Tris–HCl, pH 7.5,
150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1 mM PMSF,
0.1% protease inhibitor cocktail and 1% NP-40) at 4uC for half an
hour as described previously [112,113,114]. The cell extracts were
centrifuged at 10,0006g for 20 min at 4uC. Protein was estimated
in the supernatant using BioRad reagent. Equal amounts of cell
lysates were separated by SDS polyacrylamide gel electrophoresis
and transferred to PVDF membrane. Proteins present in the
membrane were immunoblotted with the indicated antibodies as
described previously [25,113,114].
DNA Synthesis and Proliferation Assay
Eighteen hours post-transfection, cells were serum deprived for
24 hours and incubated with
thymidine incorporation was determined as a measure of DNA
synthesis as described previously [115]. For proliferation assay,
eighteen hours post-transfection the cells were serum deprived for
indicated time periods. The starting time of serum free medium
was considered as time zero. The cells were trypsinized and
counted in a hemocytometer as described [115].
3H-thymidine for 18 hours.
3H-
Transient Transfection
The cells were transfected with the indicated plasmids using
Fugene HD as described previously [25,111,113,114,116].
Migration and Invasion Assays
ACHN cells were transfected with vector or miR-21 Sponge
plasmids. Both migration and invasion assays were performed
essentially as described previously [111]. Briefly, 256104ACHN
cells were seeded in trans-well chambers containing a membrane
with 8 mm pore size. The migration chambers placed in a 24-well
plate were incubated for 14 hours at 37uC. The migrated cells
were stained with the reagent using a kit and photographed
followed by elution of the stains according to the vendor’s
instruction. The absorbance of the eluted stain was measured at
590 nm and used arbitrarily as a measure of number of cells
migrated. For the invasion assay, the cells were seeded in trans-
well chambers with a membrane embedded with collagen and
invasion measured as described above.
Luciferase Assay
The cells were transfected with the miR-21-Luc reporter along
with Renilla null reporter plasmid. Luciferase activities were
determined in the cell lysate using a dual luciferase assay kit as
described previously [68,116]. Mean 6 SE of triplicate measure-
ments are presented as ratio of firefly and Renilla luciferase
activity as described previously [68,113,114,116,117].
Mechanism of Renal Tumorigenesis
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Page 13

Statistics
The significance of the data was analyzed by paired t-test.
Where necessary ANOVA followed by Student–Newman–Keuls
analysis was used as described previously [25,113,114,118]. A p
value less than 0.05 was considered as significant.
Supporting Information
Figure S1
Grade 3 clear cell renal carcinomas. Total RNAs from three
renal tumor samples and from the normal portion of three kidneys
were used for real time qRT-PCR to detect mature miR-21 as
described in the text. The expression levels were normalized to
U6. Each panel represents one subject. N, normal tissues; T,
tumor tissues.
(TIF)
Expression of mature miR-21 in Grade 2 and
Figure S2
cells. Total RNA from HK2 normal proximal tubular epithelial
cells and Caki-2 cells was used for real time qRT-PCR to detect
mature miR-21 as described in the text. The expression levels were
normalized to U6. Mean 6 SE of quadruplicate measurements is
shown. *p =0.03 vs HK2.
(TIF)
Expression of miR-21 in Caki-2 renal cancer
Figure S3
plasmid. Consecutive 7 copies of anti-miR-21 sequence with a
bulge in each were introduced downstream of green fluorescence
protein (GFP) cDNA. Sequence of the anti-miR-21 bulge is shown
at the bottom. The GFP sequence is driven by RNA Pol II from
the human cytomegalovirus (CMV) early promoter.
(TIF)
Structure of miR-21 Sponge expression of
Figure S4
described in Fig. 2A – 2F. ACHN cells were transfected with
miR-21 Sponge or vector plasmids as indicated in the Figure 2.
Total RNAs were used in RT-PCR for the detection of GFP
mRNA, which serves as the surrogate for the expression of miR-21
neutralizing ‘‘Sponge’’ sequence. Detection of GAPDH mRNA
was used as control. Panels A and B represent data for Fig. 2A and
2B respectively. Panels C represents data for Fig. 2C and 2D.
Panel D shows data for Fig. 2E and 2F, respectively.
(TIF)
Expression of miR-21 Sponge for the results
Figure S5
Caki-2 renal cancer cells. (A) Lysates of HK2 proximal
tubular epithelial cells and Caki-2 cells were immunoblotted with
PTEN and actin antibodies. (B) The same lyssates were
immunoblotted with phospho-Akt (Ser-473 and Thr-308) and
Akt antibodies as indicated.
(TIF)
Expression of PTEN and activation of Akt in
Figure S6
described in Fig. 4A in the text. ACHN cells were transfected with
CMV-miR-21 or Vector. The total RNAs were used to detect
mature miR-21. The level of miR-21 was corrected for U6 RNA
expression. (B) Expression of miR-21 Sponge for the results
presented in Fig. 4B. (C) Expression of miR-21 Sponge for the
results presented in Figs. 4C and 4D. Total RNAs were used to
detect GFP mRNA and GAPDH as described in the Fig. S4.
(TIF)
(A) Expression of mature miR-21 for the results
Figure S7
Sponge-induced inhibition of DNA synthesis in Caki-2 renal
cancer cells.3H-thymidine incorporation was used as a measure of
DNA synthesis as described in the legend of Fig. 2A. Mean 6 SE
of 6 measurements is shown. *p,0.05 vs control; **p,0.01 vs
(A) Downregulation of PTEN reversed mir-21
miR-21 Sponge. (B) Expression of miR-21 Sponge and PTEN for
the results described in panel A. Total RNAs and cell lysates were
used from miR-21 Sponge and PTEN siRNA-transfected Caki-2
cells. GFP mRNA and GAPDH were detected as described in the
Supplemental Fig. S4. Cell lysates were immunoblotted with
PTEN and actin antibodies. (C) Expression of miR-21 Sponge and
PTEN for the results described in Fig. 5A. Total RNAs and cell
lysates were used from miR-21 Sponge and PTEN siRNA-
transfected ACHN cells. GFP and GAPDH mRNAs were detected
as described in the Supplemental Fig. S4. Cell lysates were
immunoblotted with PTEN and actin antibodies.
(TIF)
Figure S8
the results described in Fig. 5B. Expression of GFP and
GAPDH mRNAs and PTEN and actin proteins were determined
as described in the legend of Fig. S7C. Expression of miR-21
Sponge for the results described in Figs. 5C and 5D. Total RNAs
were used to detect GFP and GAPDH and cell lysates were used
for immunoblotting with PTEN and actin antibodies.
(TIF)
Expression of miR-21 Sponge and PTEN for
Figure S9
described in Figs. 5C and 5D. Total RNAs were used to
detect GFP and GAPDH and cell lysates were used for
immunoblotting with PTEN and actin antibodies.
(TIF)
Expression of miR-21 Sponge for the results
Figure S10
Sponge-induced inhibition of migration of Caki-2 renal cancer
cells. Caki-2 cells were transfected either with miR-21 Sponge
alone or along with siRNAs against PTEN. Migration of the
transfected cells were measured as described in the legend of
Fig. 2C. (B) The absorbance of the stain of the migrated cells in
panel A was determined. Mean 6 SE of 3 measurements is shown.
*p,0.001 vs control; **p,0.001 vs miR-21 Sponge. (C)
Expression of miR-21 Sponge and PTEN for the results described
in panels A and B. Total RNAs and cell lysates were prepared
from Caki-2 cells plated independently. GFP mRNA was detected
as a surrogate for miR-21 Sponge expression. GAPDH was used
as control. Cell lysates were immunoblotted with PTEN and actin
antibodies.
(TIF)
(A) Downregulation of PTEN reversed mir-21
Figure S11
results described in Fig. 6A. Total RNAs and cell lysates were
used from miR-21 Sponge and Gag Akt-transfected ACHN cells.
GFP mRNA and GAPDH were detected.
(TIF)
Expression of miR-21 Sponge and Akt for the
Figure S12
reversed mir-21 Sponge-induced inhibition of DNA synthesis in
Caki-2 renal cancer cells.3H-thymidine incorporation was used as
a measure of DNA synthesis as described in the legend of Fig. 2A.
Mean 6 SE of 6 measurements is shown. *p,0.01 vs control;
**p,0.001 vs miR-21 Sponge. (B) Expression of miR-21 Sponge
and Akt for the results described in panel A. Total RNAs and cell
lysates were used from miR-21 Sponge and Gag-Akt-transfected
Caki-2 cells. GFP mRNA was used as a surrogate for miR-21
epxression. Expression of GAPDH mRNA was used as control.
Cell lysates were immunoblotted with Akt and actin antibodies.
(TIF)
(A) Expression of constitutively active Gag-Akt
Figure S13
the results described in Fig. 6B. Total RNAs and cell lysates were
used from miR-21 Sponge and Gag-Akt-transfectedACHNcells.
GFP mRNA was used as a surrogate for miR-21 epxression.
(A) Expression of miR-21 Sponge and Gag Akt for
Mechanism of Renal Tumorigenesis
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Page 14

Expression of GAPDH mRNA was used as control. Cell lysates
were immunoblotted with Akt and actin antibodies. (B) Expression
of miR-21 Sponge and Gag Akt for the results described in Fig. 6C.
Expression of GFP mRNA and Akt protein was determined as
described above.
(TIF)
Figure S14
reversed mir-21 Sponge-induced inhibition of migration of Caki-2
renal cancer cells. Caki-2 cells were transfected either with miR-21
Sponge alone or along with Gag-Akt expression plasmid.
Migration of the transfected cells were measured as described in
the legend of Fig. 2C. (B) The absorbance of the stain of the
migrated cells in panel A was determined. Mean 6 SE of 3
measurements is shown. *p,0.001 vs control; **p,0.001 vs miR-
21 Sponge. (C) Expression of miR-21 Sponge and Akt for the
results described in panels A and B. Total RNAs and cell lysates
were prepared from Caki-2 cells plated independently. GFP
mRNA was detected as a surrogate for miR-21 Sponge expression.
GAPDH was used as control. Cell lysates were immunoblotted
with Akt and actin antibodies.
(TIF)
(A) Expression of constitutively active Gag-Akt
Figure S15
presented in Fig. 7. (A) ACHN cells were transfected with miR-
21 Sponge or vector as indicated in the Figure 7A–7C. (B and C)
ACHN cells were transfected either with miR-21 Sponge alone or
along with CA Rheb expression plasmid as indicated in Fig. D and
7E, respectively. Total RNAs were used in RT-PCR for the
detection of GFP mRNA, which serves as a surrogate for the
expression of miR-21-neutralizing ‘‘Sponge’’ sequence. Detection
of GAPDH mRNA was used as control.
(TIF)
Expression of miR-21 Sponge for the results
Figure S16
the results described in Fig. 8A. Total RNAs and cell lysates
were used from miR-21 Sponge and constitutively active (CA)
mTOR-transfected ACHN cells. GFP mRNA and GAPDH were
detected. Cell lysates were immunoblotted with mTOR and actin
antibodies.
(TIF)
Expression of miR-21 Sponge and mTOR for
Figure S17
reversed mir-21 Sponge-induced inhibition of DNA synthesis in
Caki-2 renal cancer cells.3H-thymidine incorporation was used as
a measure of DNA synthesis as described in the legend of Fig. 2A.
Mean 6 SE of 6 measurements is shown. *p,0.05 vs control;
**p,0.001 vs miR-21 Sponge. (B) Expression of miR-21 Sponge
and mTOR for the results described in panel A. Total RNAs and
cell lysates were used from miR-21 Sponge and CA mTOR-
transfected Caki-2 cells. GFP mRNA was used as a surrogate for
miR-21 expression. Expression of GAPDH mRNA was used as
control. Cell lysates were immunoblotted with mTOR and actin
antibodies.
(TIF)
(A) Expression of constitutively active mTOR
Figure S18
the results described in Fig. 8B. Total RNAs and cell lysates were
used from miR-21 Sponge and constitutively active (CA) mTOR-
transfected ACHN cells. GFP mRNA and GAPDH were detected.
Cell lysates were immunoblotted with mTOR and actin
antibodies. (B) Expression of miR-21 Sponge and mTOR for the
results described in Figs. 8C and 8D. Total RNAs were used to
detect GFP and GAPDH and cell lysates were used for
immunoblotting with mTOR and actin antibodies.
(TIF)
(A) Expression of miR-21 Sponge and mTOR for
Figure S19
reversed mir-21 Sponge-induced inhibition of migration of Caki-
2 renal cancer cells. Caki-2 cells were transfected either with miR-
21 Sponge alone or along with CA mTOR expression plasmid.
Migration of the transfected cells were measured as described in
the legend of Fig. 2C. (B) The absorbance of the stain of the
migrated cells in panel A was determined. Mean 6 SE of 3
measurements is shown. *p,0.001 vs control; **p,0.001 vs miR-
21 Sponge. (C) Expression of miR-21 Sponge and mTOR for the
results described in panels A and B. Total RNAs and cell lysates
were prepared from Caki-2 cells plated independently. GFP
mRNA was detected as a surrogate for miR-21 Sponge expression.
GAPDH was used as control. Cell lysates were immunoblotted
with mTOR and actin antibodies.
(TIF)
(A) Expression of constitutively active mTOR
Figure S20
renal cell carcinoma cells. Total RNAs from HK2 normal
proximal tubular epithelial cells and 786-O renal carcinoma cells
were used to detect mature miR-21 as described in the text.
Expression of U6 RNA was used to normalize the data. Mean 6
SE of 4 measurements is shown. *p,0.0001 vs HK2.
(TIF)
Expression of miR-21 in VHL negative 786-O
Figure S21
cells. Total RNAs from MCF-10A normal breast epithelial cells
and BT-20 mammary carcinoma cells were used to detect mature
miR-21 as described in the text. Expression of U6 RNA was used
to normalize the data. Mean 6 SE of 4 measurements is shown.
*p =0.0006 vs MCF10A.
(TIF)
Expression of miR-21 in BT-20 breast cancer
Figure S22
Sponge-induced inhibition of BT-20 human breast cancer cell
proliferation. BT-20 cells were transfected either with miR-21
Sponge alone or along with siRNAs against PTEN. The cells were
trypsinized and counted using hemocytometer at indicated times.
Mean 6 SE of 3 measurements is shown. *p,0.01 vs vector;
**p,0.01 vs miR-21 Sponge. (B) Expression of miR-21 Sponge
and PTEN for the results described in panel A. Total RNAs and
cell lysates were used from miR-21 Sponge and PTEN siRNA-
transfected BT-20 cells. GFP mRNA and GAPDH were detected
as described. Cell lysates were immunoblotted with PTEN and
actin antibodies.
(TIF)
(A) Downregulation of PTEN reversed miR-21
Figure S23
Sponge-induced inhibition of migration of BT-20 human breast
cancer cells. BT-20 cells were transfected either with miR-21
Sponge alone or along with siRNAs against PTEN. Migration of
the transfected cells were measured as described in the legend of
Fig. 2C. (B) The absorbance of the stain of the migrated cells in
panel A was determined. Mean 6 SE of 3 measurements is shown.
*p,0.05 vs vector; **p,0.05 vs miR-21 Sponge. (C) Expression of
miR-21 Sponge and PTEN for the results described in panels A
and B. Total RNAs and cell lysates were prepared from BT-20
cells plated independently. GFP mRNA was detected as a
surrogate for miR-21 Sponge expression. GAPDH was used as
control. Cell lysates were immunoblotted with PTEN and actin
antibodies.
(TIF)
(A) Downregulation of PTEN reversed miR-21
Figure S24
reversed miR-21 Sponge-induced inhibition of BT-20 human
breast cancer cell proliferation. BT-20 cells were transfected either
with miR-21 Sponge alone or along with Gag-Akt expression
plasmid. The cells were trypsinized and counted using hemocy-
(A) Expression of constitutively active Gag-Akt
Mechanism of Renal Tumorigenesis
PLoS ONE | www.plosone.org 14June 2012 | Volume 7 | Issue 6 | e37366

Page 15

tometer at indicated times. Mean 6 SE of 3 measurements is
shown. *p,0.05 vs vector; **p,0.01 vs miR-21 Sponge. (B)
Expression of miR-21 Sponge and Akt for the results described in
panel A. Total RNAs and cell lysates were used from miR-21
Sponge and Gag-Akt-transfected BT-20 cells. GFP mRNA and
GAPDH were detected as described. Cell lysates were immuno-
blotted with Akt and actin antibodies.
(TIF)
Figure S25
reversed miR-21 Sponge-induced inhibition of migration of BT-20
human breast cancer cells. BT-20 cells were transfected either with
miR-21 Sponge alone or along with Gag-Akt expression plasmid.
Migration of the transfected cells were measured as described in
the legend of Fig. 2C. (B) The absorbance of the stain of the
migrated cells in panel A was determined. Mean 6 SE of 3
measurements is shown. *p,0.01 vs vector; **p,0.05 vs miR-21
Sponge. (C) Expression of miR-21 Sponge and Akt for the results
(A) Expression of constitutively active Gag-Akt
described in panels A and B. Total RNAs and cell lysates were
prepared from BT-20 cells plated independently. GFP mRNA was
detected as a surrogate for miR-21 Sponge expression. GAPDH
was used as control. Cell lysates were immunoblotted with Akt and
actin antibodies.
(TIF)
Acknowledgments
We thank Xiaonan Li for isolation of plasmid DNAs.
Author Contributions
Conceived and designed the experiments: GGC NGC ND. Performed the
experiments: ND FD CCM. Analyzed the data: ND FD GGC BSK.
Contributed reagents/materials/analysis tools: KB DJP. Wrote the paper:
GGC. Provided intellectual inputs to analyze the data and write the
manuscript: HEA BSK.
References
1.Novick AC (2007) Kidney cancer: past, present, and future. Urol Oncol 25:
188–195.
Rouviere O, Bouvier R, Negrier S, Badet L, Lyonnet D (2006) Nonmetastatic
renal-cell carcinoma: is it really possible to define rational guidelines for post-
treatment follow-up? Nat Clin Pract Oncol 3: 200–213.
Milowsky MI, Nanus DM (2003) Chemotherapeutic strategies for renal cell
carcinoma. Urol Clin North Am 30: 601–609, x.
Kaelin WG Jr. (2002) Molecular basis of the VHL hereditary cancer syndrome.
Nat Rev Cancer 2: 673–682.
Kaelin WG Jr. (2009) Treatment of kidney cancer: insights provided by the
VHL tumor-suppressor protein. Cancer 115: 2262–2272.
Young AP, Schlisio S, Minamishima YA, Zhang Q, Li L, et al. (2008) VHL loss
actuates a HIF-independent senescence programme mediated by Rb and p400.
Nat Cell Biol 10: 361–369.
Kaelin WG Jr. (2008) The von Hippel-Lindau tumour suppressor protein: O2
sensing and cancer. Nat Rev Cancer 8: 865–873.
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function.
Cell 116: 281–297.
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell
136: 215–233.
Couzin J (2008) MicroRNAs make big impression in disease after disease.
Science 319: 1782–1784.
He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, et al. (2005) A
microRNA polycistron as a potential human oncogene. Nature 435: 828–833.
Fabian MR, Sonenberg N, Filipowicz W (2010) Regulation of mRNA
translation and stability by microRNAs. Annu Rev Biochem 79: 351–379.
Yang JS, Lai EC (2011) Alternative miRNA Biogenesis Pathways and the
Interpretation of Core miRNA Pathway Mutants. Mol Cell 43: 892–903.
Juan D, Alexe G, Antes T, Liu H, Madabhushi A, et al. (2010) Identification of
a microRNA panel for clear-cell kidney cancer. Urology 75: 835–841.
Petillo D, Kort EJ, Anema J, Furge KA, Yang XJ, et al. (2009) MicroRNA
profiling of human kidney cancer subtypes. Int J Oncol 35: 109–114.
Nakada C, Matsuura K, Tsukamoto Y, Tanigawa M, Yoshimoto T, et al.
(2008) Genome-wide microRNA expression profiling in renal cell carcinoma:
significant down-regulation of miR-141 and miR-200c. J Pathol 216: 418–427.
Huang Y, Dai Y, Yang J, Chen T, Yin Y, et al. (2009) Microarray analysis of
microRNA expression in renal clear cell carcinoma. Eur J Surg Oncol 35:
1119–1123.
Volinia S, Galasso M, Costinean S, Tagliavini L, Gamberoni G, et al. (2010)
Reprogramming of miRNA networks in cancer and leukemia. Genome Res 20:
589–599.
Powers MP, Alvarez K, Kim HJ, Monzon FA (2011) Molecular classification of
adult renal epithelial neoplasms using microRNA expression and virtual
karyotyping. Diagn Mol Pathol 20: 63–70.
Davis BN, Hilyard AC, Nguyen PH, Lagna G, Hata A (2011) Smad proteins
bind a conserved RNA sequence to promote microRNA maturation by
Drosha. Mol Cell 39: 373–384.
Zhou R, Hu G, Gong AY, Chen XM (2010) Binding of NF-kappaB p65
subunit to the promoter elements is involved in LPS-induced transactivation of
miRNA genes in human biliary epithelial cells. Nucleic Acids Res 38:
3222–3232.
Oya M, Takayanagi A, Horiguchi A, Mizuno R, Ohtsubo M, et al. (2003)
Increased nuclear factor-kappa B activation is related to the tumor
development of renal cell carcinoma. Carcinogenesis 24: 377–384.
Oya M, Ohtsubo M, Takayanagi A, Tachibana M, Shimizu N, et al. (2001)
Constitutive activation of nuclear factor-kappaB prevents TRAIL-induced
apoptosis in renal cancer cells. Oncogene 20: 3888–3896.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.Madrid LV, Mayo MW, Reuther JY, Baldwin AS Jr. (2001) Akt stimulates the
transactivation potential of the RelA/p65 Subunit of NF-kappa B through
utilization of the Ikappa B kinase and activation of the mitogen-activated
protein kinase p38. J Biol Chem 276: 18934–18940.
Dey N, Das F, Mariappan MM, Mandal CC, Ghosh-Choudhury N, et al.
(2011) MicroRNA-21 orchestrates high glucose-induced signals to TOR
complex 1, resulting in renal cell pathology in diabetes. J Biol Chem 286:
25586–25603.
Ebert MS, Neilson JR, Sharp PA (2007) MicroRNA sponges: competitive
inhibitors of small RNAs in mammalian cells. Nat Methods 4: 721–726.
Cully M, You H, Levine AJ, Mak TW (2006) Beyond PTEN mutations: the
PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat
Rev Cancer 6: 184–192.
Salmena L, Carracedo A, Pandolfi PP (2008) Tenets of PTEN tumor
suppression. Cell 133: 403–414.
Abou Youssif T, Fahmy MA, Koumakpayi IH, Ayala F, Al Marzooqi S, et al.
(2010) The mammalian target of rapamycin pathway is widely activated
without PTEN deletion in renal cell carcinoma metastases. Cancer 117:
290–300.
Meng F, Henson R, Lang M, Wehbe H, Maheshwari S, et al. (2006)
Involvement of human micro-RNA in growth and response to chemotherapy
in human cholangiocarcinoma cell lines. Gastroenterology 130: 2113–2129.
Maehama T, Dixon JE (1998) The tumor suppressor, PTEN/MMAC1,
dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-
trisphosphate. J Biol Chem 273: 13375–13378.
Cantley LC, Neel BG (1999) New insights into tumor suppression: PTEN
suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT
pathway. Proc Natl Acad Sci U S A 96: 4240–4245.
van Weering DH, de Rooij J, Marte B, Downward J, Bos JL, et al. (1998)
Protein kinase B activation and lamellipodium formation are independent
phosphoinositide 3-kinase-mediated events differentially regulated by endog-
enous Ras. Mol Cell Biol 18: 1802–1811.
Manning BD, Logsdon MN, Lipovsky AI, Abbott D, Kwiatkowski DJ, et al.
(2005) Feedback inhibition of Akt signaling limits the growth of tumors lacking
Tsc2. Genes Dev 19: 1773–1778.
Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC (2002)
Identification of the tuberous sclerosis complex-2 tumor suppressor gene
product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol
Cell 10: 151–162.
Tee AR, Manning BD, Roux PP, Cantley LC, Blenis J (2003) Tuberous
sclerosis complex gene products, Tuberin and Hamartin, control mTOR
signaling by acting as a GTPase-activating protein complex toward Rheb. Curr
Biol 13: 1259–1268.
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.
Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, et al. (2005) MicroRNA
expression profiles classify human cancers. Nature 435: 834–838.
Si ML, Zhu S, Wu H, Lu Z, Wu F, et al. (2007) miR-21-mediated tumor
growth. Oncogene 26: 2799–2803.
Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, et al. (2005)
MicroRNA gene expression deregulation in human breast cancer. Cancer Res
65: 7065–7070.
Chan JA, Krichevsky AM, Kosik KS (2005) MicroRNA-21 is an antiapoptotic
factor in human glioblastoma cells. Cancer Res 65: 6029–6033.
Schetter AJ, Leung SY, Sohn JJ, Zanetti KA, Bowman ED, et al. (2008)
MicroRNA expression profiles associated with prognosis and therapeutic
outcome in colon adenocarcinoma. JAMA 299: 425–436.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
Mechanism of Renal Tumorigenesis
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