The MicroRNA let-7a Modulates Interleukin-6-dependent
STAT-3 Survival Signaling in Malignant
Fanyin Meng‡, Roger Henson‡, Hania Wehbe-Janek‡, Heather Smith‡, Yoshiyuki Ueno§, and Tushar Patel‡1
Medicine,Temple,Texas76508andthe§DivisionofGastroenterology,TohokuUniversity,Sendai 980-8577, Japan
The inflammation-associated cytokine interleukin-6 (IL-6)
activation of survival mechanisms. In several human cancers,
IL-6-activated survival signaling involves the signal transducers
and activators of transcription (Stat) factors or protein kinase
cascades. microRNAs (miRNAs) are endogenous regulators of
gene expression that are altered in expression in many cancers.
However, the effect of inflammatory cytokines on miRNA
cell survival are unknown. We investigated the involvement of
miRNA in malignant cholangiocytes stably transfected to over-
of apoptosis. We provide evidence that (i) miRNA expression
both in vitro and in vivo is altered by overexpression of IL-6; (ii)
contributes to the constitutively increased phosphorylation of
Stat-3 by a mechanism involving the neurofibromatosis 2 (NF2)
gene. These findings reveal a novel mechanism by which IL-6
mediates tumor cell survival that may be therapeutically tar-
between deregulated expression of miRNA and transcription
factors in human cancers.
Increased expression of the inflammation-associated cyto-
kine interleukin-6 (IL-6)2occurs in chronic inflammatory con-
ditions and in several human cancers such as multiple
myeloma, prostate cancer, and cholangiocarcinoma. IL-6 has
been implicated in tumor growth in many of these tumors, and
elevated IL-6 expression has been associated with poor out-
comes and resistance to chemotherapy (1). Experimentally,
growth of prostate cancer and cholangiocarcinoma xenografts
in athymic mice has been shown to be increased by enforced
expression of IL-6 by activation of cell survival signaling (2, 3).
The mechanisms by which IL-6 promotes cell survival in can-
IL-6-activated survival signaling has been shown to involve
tors or various protein kinase cascades (4, 5). Although consti-
precise mechanisms involved are incompletely understood.
Cholangiocarcinomas are highly resistant to chemotherapy.
However, inhibition of IL-6-dependent pathways such as the
Jak-Stat pathway, phosphatidylinositol 3-kinase, or the p38
MAPK pathways can enhance chemotherapy-induced cell
death. Thus, aberrant IL-6-dependent survival signaling may
We sought to understand the role of microRNAs (miRNAs)
in IL-6-mediated tumor cell survival. miRNAs are endogenous
regulators of gene expression that are altered in expression in
many cancers (6-8). The expression of several miRNAs in
cholangiocarcinoma xenografts in athymic mice is altered dur-
ing in vivo treatment with gemcitabine (9). miRNAs are small
endogenous molecules that can regulate gene expression in a
sequence complementary manner. Several hundred miRNAs
have been identified, and details of the mechanisms by which
they regulate gene expression are being unraveled (10, 11).
Less is known about the mechanism by which miRNAs con-
tribute to cellular behavior and function. Alterations in
miRNA expression occur in many different cancers (8).
Thus, individual miRNAs may play contributory or regula-
tory roles in tumor cell pathogenesis or behavior. Potential
downstream targets of miRNA include oncogenes or tumor
tumor biology have been described such as the Ras oncogene
altered miRNA regulatory networks may contribute to tumor
cell response and resistance to chemotherapy.
Cell Lines and Cultures—Mz-1 and KMCH-1 human malig-
nant cholangiocytes and their respective IL-6-overexpressing
stable transfectants, Mz-IL-6 and KM-IL-6, were obtained and
tion of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in accord-
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
a supplemental figure.
1To whom correspondence should be addressed: Division of Gastroenterol-
Hall, 410 West 10thAve., Columbus, OH 43210. Tel.: 614-293-6202; Fax:
614-293-0861; E-mail: email@example.com.
rofibromatosis 2; Stat-3, signal transducers and activators of transcrip-
PARP, poly(ADP-ribose) polymerase-1; TUNEL, terminal deoxynucleotidyl-
transferase-mediated dUTP nick end-labeling; HRS, hepatocyte growth
factor regulated tyrosine kinase substrate.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 11, pp. 8256–8264, March 16, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
8256 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 282•NUMBER 11•MARCH 16, 2007
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cultured as described previously (2). Basal IL-6 expression was
increased by ?1.5-fold in KM-IL-6 and ?3-fold in Mz-IL-6
cells relative to their respective controls.
Transfections—20 ?l of 100 nM microRNA precursor, anti-
sense inhibitor, or controls were added to 1 ? 106cells sus-
pended in 80 ?l of Nucleofector solution (Amaxa Biosystems,
formed using the Nucleofector system (Amaxa Biosystems).
Transfected cells were then resuspended in regular culture
medium containing 10% serum for 48–72 h prior to study.
MicroRNA Isolation and Expression Profiling—miRNA was
isolated by PAGE purification of total RNA, and expression
profiling was performed using a custom-generated microarray
as described previously (9). Microarrays were scanned using a
GenePix 4000A array scanner (Axon Instruments, Union City,
replicate relative to a control miRNA (Ambion, Austin, TX)
added to each sample, thus allowing comparisons between
chips. Data were analyzed using GeneSpring 7.0 Software (Sil-
icon Genetics, Redwood City, CA), and an average value of the
MicroRNA expression levels were clustered using a self-orga-
nizing tree algorithm using the MultiExperiment Viewer Ver-
sion 3.1 from The Institute for Genomic Research (13).
Quantitative Real-time PCR—RNA was isolated using the
ToTALLY RNA isolation kit (Ambion), and cDNA was gener-
ated by reverse transcription using 1 ?g of total RNA and the
reverse transcription kit (Invitro-
gen). Mature let-7a miRNA expres-
sion was assessed using a TaqMan?
(Applied Biosystems, Foster City,
CA). Real-time PCR was performed
using a MX 3000PTMPCR instru-
ment (Stratagene, San Diego, CA).
Cytotoxicity Assay—Cell viability
was assessed using a commercially
available tetrazolium bio-reduction
assay as described previously (14).
10,000 viable cells/well were seeded
into 96-well plates and incubated
with gemcitabine, 5-fluorouracil,
camptothecin, or appropriate dilu-
ent controls in a final volume of 200
?l of medium containing 0.5% fetal
in 96-well plates (20,000 cells/well)
and incubated with different che-
motherapeutic agents or diluent
control. Caspase 3/7 activity was
assayed using the fluorometric
Apo-ONE homogenous caspase
3/7 assay (Promega, Madison, WI)
and a Cytofluor microplate fluo-
rescence plate reader.
activity was assessed in cell lysates
after immunoprecipitation using monoclonal P-Stat-3-Tyr705
antibody (Cell Signaling Technology) and using a tyrosine
kinase activity assay kit (Chemicon, Temecula, CA).
Luciferase Reporter Vectors—The pMIR-NF2-luc and pMIR-
the 3?-UTR of NF2, respectively, cloned downstream of the
firefly luciferase gene were constructed as follows. Synthetic
oligonucleotides encompassing the intact or mutated let-7a
recognition sequence, sticky ends for HindIII and SpeI to elim-
inate digestion of the insert, and a unique BlpI site to test for
positive clones were synthesized and annealed. The oligonu-
cleotides used were 5?-CTAGTGCTCAGCTACAAGAGAT-
TCTCCTGCCTCAA-3? (sense) and 5?-AGCTTTGAGG-
CAGGAGAATCTCTTGTAGCTGAGCA-3? (antisense) for
pMIR-NF2-luc, or 5?-CTAGTGCTCAGCTATAGGCGCTT-
TGCTCGATGGAA-3?(sense) and 5?-AGCTTTCCATCGAG-
CAAAGCGCCTATAGCTGAGCA-3? (antisense) for pMIR-
NF2-MUT-luc. The recognition sequence is underlined in the
former sequence, and the random mutations introduced are
(30 mM HEPES, pH 7.4, 100 nM potassium acetate, 2 mM mag-
nesium acetate) for a final volume of 50 ?l and incubated at
90 °C for 3 min and then at 37 °C for 1 h. The annealed insert
of the pMIR-REPORT luciferase expression vector (Ambion).
FIGURE 1. Enforced expression of IL-6 decreases apoptosis in vivo. A, IL-6-overexpressing Mz-IL-6 cell
xenografts in athymic nude mice grow faster than control Mz-1 tumor cell xenografts. B, Mz-1 and Mz-IL-6
tumor cell xenografts (n ? 4 each) were treated with gemcitabine (120 mg/kg intraperitoneally) every 3 days
for a total of five doses. Tumor volume was assessed at baseline and at the end of treatment. The starting size
of tumors was similar in both groups, and the studies were performed with tumors ranging from 200 to 230
mm3. The data represent the mean and 95% confidence intervals from four tumors. p ? 0.05 for differences
between change in volume from baseline for Mz-IL-6 when compared with Mz-1 xenografts. C, xenograft
sections were obtained, and TUNEL staining for apoptotic cells was performed. The data represent mean and
standard deviation of the number of TUNEL-positive cells from eight random high power fields (HPF). There
was a decrease in chemosensitivity in vivo and in the numbers of TUNEL-positive cells in Mz-IL6 xenografts.
Thus, enhanced IL-6 expression decreases chemotherapy-induced apoptosis in vivo.
MARCH 16, 2007•VOLUME 282•NUMBER 11JOURNAL OF BIOLOGICAL CHEMISTRY 8257
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Ueno and Tushar Patel
J. Biol. Chem.
Wehbe-Janek, Heather Smith, Yoshiyuki
Fanyin Meng, Roger Henson, Hania
Signaling in Malignant Human
Interleukin-6-dependent STAT-3 Survival
The MicroRNA let-7a Modulates
Mechanisms of Signal Transduction:
doi: 10.1074/jbc.M607712200 originally published online January 12, 2007
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