An HNF4a-miRNA Inflammatory
Feedback Circuit Regulates
Maria Hatziapostolou,1,2Christos Polytarchou,1,2Eleni Aggelidou,4Alexandra Drakaki,5George A. Poultsides,6
Savina A. Jaeger,7Hisanobu Ogata,8Michael Karin,8Kevin Struhl,3Margarita Hadzopoulou-Cladaras,4,9
and Dimitrios Iliopoulos1,2,9,*
1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute
2Department of Microbiology and Immunobiology
3Department of Biological Chemistry and Molecular Pharmacology
Harvard Medical School, Boston, MA 02115, USA
of Thessaloniki, Thessaloniki 54124, Greece
5Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA
6Department of Surgery, Stanford University Medical Center, Stanford, CA 94305, USA
7Department of Developmental Molecular Pathways, Novartis Institute for Biomedical Research, Cambridge, MA 02139, USA
8Laboratory of Gene Regulation and Signal Transduction, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
9These authors contributed equally to this work
Hepatocyte nuclear factor 4a (HNF4a) is essential for
liver development and hepatocyte function. Here, we
show that transient inhibition of HNF4a initiates
hepatocellular transformation through a microRNA-
inflammatory feedback loop circuit consisting of
miR-124, IL6R, STAT3, miR-24, and miR-629. More-
over, we show that, once this circuit is activated,
it maintains suppression of HNF4a and sustains
oncogenesis. Systemic administration of miR-124,
which modulates inflammatory signaling, prevents
and suppresses hepatocellular carcinogenesis by
inducing tumor-specific apoptosis without toxic
side effects. As we also show that this HNF4a circuit
data raise the possibility that manipulation of this
microRNA feedback-inflammatory loop has thera-
peutic potential for treating liver cancer.
Hepatocellular carcinoma (HCC) is the main type of liver cancer
and the third most common cause of cancer mortality world-
wide. The major risk factor for HCC is chronic hepatitis, due
to hepatotropic viruses (HBV, HCV) (El-Serag and Rudolph,
2007), but the molecular mechanisms leading to HCC have not
been well characterized. Hepatocellular carcinogenesis involves
many genetic and epigenetic alterations and is influenced by
environmental factors. Genes such as c-myc, cyclin D1, p53,
p16, E-cadherin, and PTEN have been linked to hepatocarcino-
genesis (Villanueva et al., 2007). Persistent inflammation also
impacts the course of liver tumor development (Coussens and
Werb, 2002), and chronic inflammatory stimuli and increased
STAT3 activation recapitulate hepatic oncogenesis in various
animal models (He et al., 2010). In addition, the inflammatory
responses induced by obesity or administration of the diethylni-
trosamine (DEN) are known to promote HCC in mice (Park et al.,
2010; Maeda et al., 2005).
HNF4a is a member of the nuclear receptor superfamily of
ligand-dependent transcription factors (NR2A1) that is enriched
in liver tissue (Zhong et al., 1993). HNF4a is indispensable for
development and maintenance of the hepatic epithelium (Parviz
et al., 2003) and also has links to a variety of human diseases,
including diabetes, colitis, and cancer. A number of mutations
within the HNF4A gene are considered to contribute to several
forms of maturity-onset diabetes in children (Gupta and Kaest-
ner, 2004). Suggesting a potential link between HNF4a and
inflammation, genome-wide association studies have identified
HNF4A as a susceptibility locus for ulcerative colitis (Barrett
et al., 2009), and recent evidence supports an oncogenic role
for HNF4a in intestinal cancer (Darsigny et al., 2010). But con-
flicting reports have assigned HNF4a both tumor-promoting
and tumor-suppressing roles in liver cancer (Xu et al., 2001;
Yin et al., 2008).
Here, we show that HNF4a is a key regulator of hepatocellular
carcinogenesis. During hepatocellular transformation, transient
inhibition of HNF4a becomes a stable event, with a feedback
loop consisting of miR-124, IL6R, STAT3, miR-24, and miR-
629 maintaining the hepatocyte-transformed phenotype in vitro
and in vivo. Perturbation of this network, through miR-124
systemic administration, prevents and suppresses HCC devel-
opment in a murine liver cancer model. Components of the
HNF4a feedback loop circuit are differentially expressed in
Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc. 1233
Figure 1. HNF4a Suppression through miR-24 and miR-629 Induces Hepatocellular Transformation
(A) Soft agar colony assay of nontransformed immortalizedhepatocytes (IMH1, IMH2) treated for 48 hrwithsiRNA-negative control (siNC) or two different siRNAs
against HNF4a (siHNF4a#1, siHNF4a#2). Colonies (mean ± SD) 50 mm were counted using a microscope 20 days later.
(B) Tumor volume (mean ± SD) in mice injected with IMH1 cells untreated or treated for 48 hr with siRNA NC, siHNF4a#1, or siHNF4a#2.
(C) Effects of microRNAs (primary screen) on HNF4a luciferase activity in HepG2 cells (top). The top nine hits identified from the primary microRNA library screen
were tested in secondary screen in HepG2 and Hep3B cells (bottom).
1234 Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc.
Though the epigenetic switch described here resembles an
epigenetic switch that converts a nontransformed breast cell
line into a stably transformed line that relies on an inflammatory
feedback look involving STAT3 (Iliopoulos et al., 2009, 2010), the
microRNA, transcription factors, and target genes mediating
these epigenetic switches differ considerably in the breast and
livercontexts. Overall, ourdata suggest that epigenetic switches
are regulatory events that are essential for cancer initiation and
maintenance in addition to mutational events.
Transient Inhibition of HNF4a Induces Hepatocellular
To elucidate the role and function of HNF4a in liver cancer initi-
ation, we modulated its expression in nontransformed immortal-
ized human hepatocytes (IMH). We found that HNF4a inhibition
transformed IMH cells and increased their invasiveness (Figures
1A and 1B and Figures S1A–S1C available online). Strikingly,
transient inhibition of HNF4a was sufficient to induce transfor-
mation of IMH cells and promote tumor formation in immunode-
mRNA expression was still suppressed (Figure S1D), suggesting
that inhibition of HNF4a initiates a feedback loop that continu-
ously suppresses HNF4a expression and induces a stable trans-
formedphenotype. Inaccordance withthe datafromourprimary
IMH cells, transient inhibition of HNF4a increased colony for-
mation and invasiveness of HepG2 and SNU-449 cancer
cells (Figures S1E and S1F) and decreased expression levels
of HNF4a direct metabolic target genes (Figure S1G). Overall,
thesedata suggest thatHNF4ainhibitioninduces transformation
of immortalized hepatocytes through a feedback regulatory
miR-24 and miR-629 Suppress Directly HNF4a
Expression during Hepatocellular Transformation
How is HNF4a suppression triggered and maintained during
hepatocellular transformation? Recently, we and others have
described the existence of dynamic microRNA-transcription
factor networks in a variety of cancers (Iliopoulos et al., 2009,
2010; Kent et al., 2010). To identify microRNAs that regulate
directly HNF4a expression, we performed a microRNA-based
genetic screen (Figure 1C, top). MicroRNAs that inhibited
HNF4a 30UTR luciferase activity by more than 75% were scored
as positive hits. These were further validated in HepG2 and
Hep3B cells, seeded in 6-well plates, according to the same
criteria (Figure 1C, bottom). Our approach resulted in the identi-
fication of two microRNAs, miR-24 and miR-629, as direct regu-
lators of HNF4a expression.
Several lines of evidence indicate that miR-24 and miR-629
target HNF4a directly, binding to its 30UTR. Sequence comple-
mentarity analysis revealed that HNF4a is a gene target of
miR-24 and miR-629, and upon overexpression of miR-24 or
miR-629, HNF4a mRNA levels are reduced 5-fold and 2-fold,
respectively (Figure 1D). In addition, HNF4a protein levels drop
(Figure 1E), and the direct downstream targets are downregu-
lated by miR-24 and miR-629 (Figure 1F). In addition, combined
knockdown (Figure 1F).
Transient inhibition of HNF4a by siRNA resulted in upregula-
tion of both miR-24 and miR-629 in IMH cells (Figure 1G). We
also identified increased expression of miR-24 and miR-629
in tumors derived from IMH cells treated with two different
siRNAs against HNF4a (Figure 1H). Taken together, these data
suggest that both microRNAs regulate directly HNF4a expres-
sion and are part of the feedback loop circuit.
miR-24 and miR-629 Play a Key Role in Hepatocellular
Cancer Initiation and Growth
nicity,wetestedwhethertheir overexpression can transformtwo
and/or miR-629 is sufficient for hepatocellular transformation
and colony formation in soft agar (Figure 2A). Though miR-24
has a stronger effect than miR-629, the combination of the two
microRNAs closely resembles HNF4a knockdown. The ability
of miR-24 or miR-629 to induce transformation in vitro led us
to extend our results and examine their ability to regulate tumor
initiation in vivo. Overexpression of miR-24 or miR-629, to a
lesser extent, was sufficient for the induction of tumor initiation
and growth (Figure 2B). These observations indicate that tran-
sient expression of either miR-24 or miR-629 is sufficient to
induce stable transformation of hepatocytes in vitro and in vivo.
Reduced HNF4a expression in miR-24/miR-629-treated tumors
(Figure 2C) also indicates that both microRNAs cooperatively
To address the functional role of miR-24 and miR-629 in the
maintenance of the transformed phenotype, we tested the
effects of their upregulation on the tumorigenicity of hepatocel-
lular cancer cells. Overexpression of miR-24 or miR-629 in
(Figure 2D) and their invasive capacity (Figure 2E). As expected,
a combination of the two microRNAs exhibited the same effects
with HNF4a inhibition. To delineate the role of miR-24 and miR-
629 in HCC growth in vivo, we performed xenograft experiments
(D) HNF4a mRNA levels (mean ± SD of three independent experiments) assessed by real-time RT-PCR analysis in HepG2, Hep3B, and SNU-449 cells untreated
or treated with 100 nM miR NC or miR-24 and/or miR-629 for 48 hr.
(E) HNF4a protein levels in HepG2 cells untreated or treated with 100 nM miR NC or miR-24 and/or miR-629 for 48 hr.
(F) mRNA levels of HNF4a direct targets (mean ± SD of three independent experiments) assessed by real-time RT-PCR analysis in HepG2 cells untreated or
treated with 100 nM miR NC or miR-24 and/or miR-629 or siRNA NC or siRNA or siHNF4a#1 for 48 hr.
(G) miR-24 and miR-629 expression levels (mean ± SD of three independent experiments) assessed by real-time RT-PCR analysis in IMH1 cells that were
untreated or treated for 48 hr with siRNA NC or siHNF4a#1.
(H)miR-24and miR-629expression levels(mean ±SD ofthreeindependentexperiments)assessedbyreal-timeRT-PCR analysisintumors derived from injected
IMH1 cells that were untreated or treated for 48 hr with siRNA NC or siHNF4a#1 or siHNF4a#2.
Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc. 1235
in which SNU-449 cells were injected subcutaneously in immu-
nodeficient mice (Figure 2F). We found that overexpression of
miR-24 and miR-629 increased the growth of SNU-449 xeno-
graft tumors (Figure 2F), whereas simultaneous inhibition of
both microRNAs completely suppressed tumor growth. Are the
effects of miR-24 and miR-629 on tumor growth related to
HNF4a expression? We tested HNF4a mRNA levels in xenograft
tumors (day 30) from the same mice, as described above.
Tumors treated with the antisense microRNAs are smaller,
contain many apoptotic cells (Figure S2), and exhibit elevated
HNF4a mRNA levels (Figure 2G).
STAT3 Is a Direct Regulator of miR-24 and miR-629
According to our data, both miR-24 and miR-629 directly
suppressHNF4a expression, and they are activated byinhibition
circuit. We found that miR-24 and miR-629 are coordinately
Tumor Volume (mm3)
5 101520 2530
# invading cells
Tumor Volume (mm3)
05 10 15 20 25 30 35 40 45 50 55
as-miR NC -
miR NC -
Figure 2. miR-24 and miR-629 Regulate the Induction and Stability of the Hepatocellular Transformed Phenotype
(A) Number of colonies (>50 mm) (mean ± SD) of IMH1 and IMH2 cells treated with 100 nM miR NC, miR-24, and/or miR-629 or siHNF4a#1 for 48 hr.
(B) Tumor volume (mean ± SD) in mice injected with IMH1 cells untreated or treated for 48 hr with 100 nM miR NC or miR-24 and/or miR-629.
(C) HNF4amRNA levelsassessedbyreal-timeRT-PCR analysisintumors(day30)derived fromIMH1cellsuntreatedortreatedfor48hrwith100nMmiR-24and/
(D) Soft agar colony assay (mean ± SD) and (E) invasion assay (mean ± SD) of HepG2 and SNU-449 cells treated with 100 nM miR NC, miR-24, miR-629, or
siHNF4a#1 for 24 hr.
(F) Tumor volume (mean ± SD) in mice injected with SNU-449 cells and treated with as-miR NC or as-miR-24 and/or as-miR-629, or miR-24 and miR-629.
(G) HNF4a mRNA levels (mean ± SD) in tumors (day 30) derived from mice treated with as-miR NC or as-miR-24 and/or as-miR-629 or miR-24 and miR-629.
1236 Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc.
upregulated in both hepatocellular cell lines and human tumors
(Figure 3A). Examination of potential common transcription
factor binding sites in miR-24 and miR-629 promoter areas re-
vealed a highly conserved STAT3 binding motif in miR-24
promoter and a moderately conserved STAT3 motif in miR-629
promoter (Table S1). Chromatin immunoprecipitation (ChIP)
analysis in SNU-449 cells revealed that, upon IL6 stimulation,
STAT3 binds in miR-24 and miR-629 promoter regions, with
binding to the highly conserved miR-24 site being stronger
(Figure 3B). STAT3 activation by IL6 treatment resulted in upre-
gulationofboth miR-24 andmiR-629levels, whereas pharmaco-
logical inhibition of STAT3 (JSI-124) strongly reduced miR-24
and miR-629 expression levels (Figure 3C).
To determine whether STAT3 is a member of the HNF4a feed-
back loop circuit, we measured STAT3 phosphorylation levels
upon overexpression of miR-24 and/or miR-629 or inhibition of
HNF4a in SNU-449 cells (Figure 3D). Strikingly, all treatments
significantly induced STAT3 phosphorylation when compared
to the negative control samples. In accordance with our data
above, miR-24 had a more pronounced effect (compared to
miR-629), similar to that of HNF4a knockdown and the combina-
torial expression of the two microRNAs. These results strongly
SNU-449 SNU-387 SNU-398 SNU-475
CA CA CA CA CA CA CA CA CA CA CA CA
siRNA NC -
- - - +
Figure 3. STAT3 Regulates miR-24 and miR-629 during Hepatocellular Transformation
(A) HNF4a, miR-24, and miR-629 levels (mean ± SD) in nontransformed immortalized hepatocytes (IMH2), different HCC lines, two normal liver tissues (N), and 12
hepatocellular cancer tissues (CA).
(B) STAT3 occupancy (fold enrichment) at the miR-24 and miR-629 loci, as determined by chromatin immunoprecipitation of crosslinked SNU-449 cells treated
with IL6 (20 ng/ml) for 6, 12, or 24 hr.
(C) miR-24 and miR-629 expression levels (mean ± SD) in SNU-449 cells treated with IL6 (10 ng/ml) for 24 hr or JSI-124 (5 mg/ml) for 24 hr and then IL6 for 24 hr.
(D) STAT3 phosphorylation status (Tyr 705) assessed by ELISA in SNU-449 cells treated with 1 nM siRNA NC, siHNF4a#1, miR-24, and/or miR-629 for 24 hr. The
data are presented as mean ± SD of three independent experiments.
Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc. 1237
suggest thatthesemicroRNAs,STAT3, andHNF4aarepartofan
inflammatory feedback loop and not simply downstream effec-
tors of IL6.
miR-124 Is a Direct Downstream Effector of HNF4a
Activity and Part of the Feedback Loop Network
Recent studies have identified microRNA-transcription factor
feedback loops in cancer cells (Fabbri et al., 2011; Iliopoulos
et al., 2010). To further unravel the mechanism by which inhibi-
tion of HNF4a expression induces hepatocellular transformation
through a feedback loop, we looked for HNF4a-binding sites in
miRNA promoters. Lever algorithm analysis revealed HNF4a-
binding sites in eight microRNA promoter areas (Table S2).
ChIP analysis showed that HNF4a binds strongly (15- to 25-
fold enrichment) to miR-124 promoter in HepG2 and SNU-449
cells (Figure 4A), and inhibition of HNF4a expression resulted
in significant reduction of miR-124 levels (?5-fold) (Figure 4B).
Similarly, miR-124 expression is significantly inhibited upon the
combined overexpression of miR-24 and miR-629, and this inhi-
bition is comparable with the one caused byHNF4a knockdown.
Based on our observation of an inverse correlation between
STAT3 activation and HNF4a expression, we examined how
IL6 treatment influenced activity of a luciferase reporter con-
struct containing the miR-124 promoter (Figure 4C). Treatment
of HepG2 cells with IL6 significantly inhibited the activity of the
miR-124 luciferase reporter, whereas there was no effect when
the HNF4a site was mutated.
As HNF4a directly regulates miR-124 expression in HCC
lines, we tested the possibility that miR-124 may mediate the
HNF4a-regulated inhibition of STAT3. Interestingly, STAT3 acti-
vationwas induced upon miR-124suppression when compared
to the respective negative controls (Figure 4D). The above ex-
periments suggest that miR-124 participates also in the
HNF4a feedback loop. To further show that miR-124 is a
member of this loop, we examined IMH1 transformation effi-
ciency upon inhibition of miR-124 expression. As expected,
inhibition of miR-124 expression strongly induces colony forma-
tion, and this effect is reversed by STAT3 knockdown or
combined suppression of miR-24 expression (Figure 4E). Like-
wise, suppression of miR-124 or knockdown of HNF4a induces
colony formation and invasiveness of HepG2 and SNU-449
cells, whereas overexpression of miR-124 in these cell lines
reverses the phenotype (Figure S3). Taken together, these
vation inhibits HNF4a expression, which leads to suppressed
expression of miR-124 and establishes an inflammatory feed-
back loop that is necessary and sufficient for human hepatocyte
miR-124 Targets IL6R and Consequently Modulates
IL6R/STAT3 Pathway during Hepatocellular
Because STAT3 activation is suppressed by miR-124, we
hypothesized that miR-124 might target one of the components
of the IL6-STAT3 pathway. In support of this hypothesis,
sequence complementarity and conservation analysis revealed
that interleukin 6 receptor (IL6R) is a potential direct gene target
of miR-124. Furthermore, miR-124 and IL6R expression levels
are inversely correlated in IMH1 cells and five hepatocellular
cancer cell lines (Figure 4F). In addition, suppression of miR-
124expression, either directlybyantisensemiR-124or indirectly
by knockdown of HNF4a, leads to induced expression of IL6R
(Figure 4G). Conversely, overexpression of miR-124 significantly
reduced IL6R mRNA and protein levels (Figures 4G and 4H).
Also, miR-124 overexpression inhibits the activity of a luciferase
reporter construct containing the IL6R 30UTR and vice versa
(Figure 4I). Next, phosphorylation of STAT3, a downstream
target of IL6R, is induced by inhibition of miR-124 expression
or knockdown of HNF4a (Figure 4J). In addition to IL6R, we
found that inhibition of miR-124 expression results in increased
IL6 production (Figure S4A), suggesting that miR-124 regulates
STAT3 activity by affecting the IL6-IL6R levels and pathway.
Similar effects were identified when HNF4a was suppressed.
Specifically, HNF4a inhibition resulted in increased levels of
soluble IL6 and IL6R (Figures S4B and S4C), which, in turn,
increased liver tumorigenicity (Figure S4D). These experiments
support a central role for HNF4a in regulating the IL6-STAT3
The Feedback Loop Involving HNF4a, miR-124, IL6R,
STAT3, miR-24, and miR-629 Is Required for the
Induction and Maintenance of the Transformed
Phenotype in Hepatocytes
of hepatocytes, IMH1 cells were transiently transfected with the
were plated in soft agar and injected in mice (Figures S5A
and S5B). Suppression of miR-124 or HNF4a or overexpression
of miR-24 or miR-629 induced hepatocellular transformation.
We also find that the kinetics of STAT3 activation along with
expression levels of miR-124, miR-24, miR-629, and HNF4a
demonstrate the establishment and maintenance of the regula-
tory loop even 480 hr after transfection (Figures S5C–S5G). In
addition to transcriptional activation, we show that suppression
of HNF4a led to increased soluble IL6 and IL6R levels (Fig-
ure S5H), hepatocyte hyperproliferation, and decreased apo-
ptosis (Figure S6). On the other hand, breaking the regulatory
circuit by manipulation of different members of the loop blocked
the stable transformed phenotype of human hepatocytes
(Figures S5I and S5J). Overall, these data indicate that HNF4a
is a central regulator of hepatocyte growth and transformation.
HNF4a-miRNA Inflammatory Circuit Is Perturbed during
HCC Development in Mice
Building on our in vitro findings, we asked whether the HNF4a
circuit is perturbed during development of chemical-induced
hepatocellular carcinogenesis in vivo. To exclude the possibility
that the IL6/STAT3 pathway is activated by Kupffer cells, we
examined the expression levels of HNF4a, miR-124, IL6R, and
miR-24 in purified hepatocytes derived from DEN-treated mice
(Figure 5A). In accordance with our in vitro data, we identified
HCC development in mice. Interestingly, HNF4a suppression
started on week 4, whereas miR-24 was upregulated on week
24, when the tumors have already been formed. These data
are consistent with the idea that early suppression of HNF4a
1238 Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc.
Figure 4. HNF4a Binds and Regulates miR-124, which Controls Directly IL6R Expression in Hepatocytes
(A) HNF4a occupancy (fold enrichment) (mean ± SD) in ApoCIII, miR-7-1, and miR-124 promoter areas.
(B) miR-124 levels (mean ± SD) in HepG2 and SNU-449 treated with siRNA NC, siHNF4a#1, miR-24, and miR-629 for 24 hr.
(C) Luciferase activity (mean ± SD) of a reporter construct harboring miR-124 promoter (wild-type or deletion mutant in the HNF4a-binding site) 12 and 24 hr
posttreatment with IL6 (10 ng/ml) in HepG2 cells.
(D) STAT3 phosphorylation status (Tyr 705) (mean ± SD) evaluated by ELISA and western blot analyses after treatment with as-miR-NC or as-miR-124 for 24 hr in
(E)Numberof colonies(mean ± SD) of nontransformedimmortalizedhepatocytes(IMH1) treated with as-miRNC oras-miR-124together withsiRNANC orsiRNA
against STAT3 (siSTAT3) or as-miR-24 for 24 hr. The data are presented as mean ± SD of three independent experiments.
(F) miR-124 and IL6R levels in the indicated cell lines and a correlation coefficient (r) are shown.
(G) IL6R mRNA levels (mean ± SD) in HepG2 and SNU-449 cells treated for 24 hr with miR-NC, miR-124, as-miR NC, as-miR-124, siRNA NC, and siHNF4a.
(H) IL6R protein levels in HepG2 and SNU-449 cells treated for 24 hr with miR-NC or miR-124.
(I)Luciferase assayusingareporter constructcontainingthe30UTRof IL6R,24hrafter transfectionwithmiR-NC, miR-124,as-miR NC,and as-miR-124. Thedata
are presented as (mean ± SD).
(J) STAT3 phosphorylation status (Tyr 705) evaluated by ELISA in HepG2 and SNU-449 cells treated for 24 hr with as-miR NC, as-miR-124, siRNA NC, and
siHNF4a. The data are presented as mean ± SD of three independent experiments.
Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc. 1239
leadstoactivation ofthemiRNAinflammatory circuitduringHCC
In addition, we tested whether the HNF4a-miRNA circuit is
(STAT3f/f/Alb-Cre = STAT3Dhep). It is known that the DEN-treated
STAT3Dhepmice develop fewer and much smaller tumors in
comparison to the DEN-treated STAT3f/fmice (He et al., 2010).
Consistent with our hypothesis, we identified that tumors
derived from DEN-treated STAT3Dhepmice had increased
HNF4a and miR-124 levels and decreased miR-24 and miR-629
levels in comparison to DEN-treated STAT3f/fmice (Figure 5B).
These data show that suppression of the inflammatory response
be affected at any step.
Perturbation of the HNF4a Circuit Has Therapeutic
and Preventive Effects in Different Murine Liver Cancer
asked how perturbation of this circuit would affect tumor growth
Tumor Volume (mm3)
0 5 10 15 20 25 30 35 40 45 50 55
0 5 10 15 20 25 30 35 40 45 50 55
Tumor Volume (mm3)
1 4 8 12 24 32 weeks
1 4 8 12 24 32 weeks
Figure 5. The HNF4a Circuit Is Perturbed during HCC Development
(A) Assessment of HNF4a, miR-124, IL6R, and miR-24 levels (mean ± SD) in purified hepatocytes during DEN-induced liver carcinogenesis in mice.
(B) Evaluation of HNF4a mRNA levels and miR-124, miR-24, and miR-629 levels derived from DEN-treated male STAT3f/fand STAT3Dhepmice. The experiments
have been performed in triplicate, and data show mean ± SD.
(C) Tumor volume (mean ± SD) in mice injected with HepG2 and SNU-449 cells treated with miR NC or miR-124. Treatments were repeated every 5 days, and
tumor volume was monitored every 5 days for 55 days.
(D) IL6R, miR-24, miR-629, and HNF4a levels (mean ± SD) assessed by real-time RT-PCR analysis, in tumors (day 30) derived from mice treated with miR NC or
1240 Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc.
miR-124 on hepatocellular neoplastic transformation suggested
the possibility that HCC-derived tumors could be eradicated
efficiently by interference with the feedback loop on the level of
miR-124. We found that miR-124 treatment suppressed HepG2
and SNU-449 xenograft tumor growth (Figure 5C) by reducing
IL6R, miR-24, and miR-629 expression levels and significantly
increasing HNF4a expression (Figure 5D).
In addition to the subcutaneous HCC mouse model, we tested
whether systemic administration of miR-124 is able to suppress
HCC tumor growth in DEN-treated mice. According to our treat-
ment protocol, miR-NC or miR-124 was systemically adminis-
tered in DEN-treated mice on a weekly basis (first day of the
week) for four cycles (weeks 32, 33, 34, and 35) (Figure 6A). On
week 36, the mice were sacrificed, and we assessed the tumor
burden. We found that miR-124 suppressed > 80% HCC tumor
34 35 36
miR NC miR-124
HCCs / liver
NT miR NC
Tumor size (mm)
NT miR NC miR-124
Cl. casp-3 Cl. PARP
miR NC miR-124
miR NC miR-124
ALT levels (U/L)
AST levels (U/L)
spleen pancreas lung
Urea levels (mg/dL)
Figure 6. Modulation of the HNF4a Circuit Prevents and Suppresses HCC Development in Mice
(A) Timeline of miR-124 therapeutic delivery experiment.
(B) Number of HCC tumors/liver and tumor size (mm3) (mean ± SD) in nontreated (NT), miR-NC-, and miR-124-treated mice (week 36).
(C) Levels of cleaved PARP and caspase-3 in untreated, miR NC-, and miR-124-treated mice (week 36) assessed by ELISA and western blot analyses.
(D) Evaluation of miR-124 levels, HNF4a levels, and IL6R levels by real-time PCR and STAT3 phosphorylation status (Tyr705) by western blot in tumors derived
from nontreated (NT), miR-NC-, and miR-124-treated mice (week 36). The data are shown as mean ± SD.
(E and F) Levels of (E) alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, and (F) urea were assessed in the serum of mice treated
with 10 mg/kg miR-NC or miR-124 for 48 hr. Each bar represents a different mouse. The experiment was performed in triplicate, and the data show mean ± SD.
Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc. 1241
growth and size (Figure 6B) through induction of apoptosis
(Figure 6C), and actually, miR-124 administration resulted in
restoration of physiological miR-124 expression, whereas miR-
miR-124 delivery perturbed the HNF4a circuit through upregula-
tion of HNF4a mRNA levels, IL6R suppression, and inhibition of
STAT3 activation (phosphorylation). Importantly, we found that
systemic delivery of miR-NC or miR-124 did not affect liver
and kidney function (Figures 6E and 6F) and did not have any
toxicity effects on essential organs (Figure 6G). These data
demonstrate that miR-124 administration does not affect the
physiology of mice through induction of cytotoxic effects.
bation of the HNF4a circuit can prevent HCC development in
mice. Weidentified that miR-124deliveryrestoredthephysiolog-
ical levels of this microRNA in liver tumors, even 2 weeks postin-
jection (Figure S7A). According to these data, miR-124 was
administered systemically on week 12 every 2 weeks until week
30, and at week 32, we assessed tumor burden (Figure S7B).
We found that miR-124 delivery prevented efficiently HCC tumor
growth in DEN-treated mice (Figure S7C), suggesting that the
ment in vivo.
The HNF4a Regulatory Circuit Is Perturbed in Human
We examined the expression levels of miR-24, IL6R, miR-124,
and HNF4a in total RNA extracted from 12 normal liver tissues
and 45 hepatocellular carcinomas (HCCs). We found that
HNF4a and miR-124 were downregulated, whereas miR-24
and IL6R mRNA levels were increased in liver cancers relative
to normal tissues (Figure 7A). In addition, immunohistochemical
(IHC) analysis for HNF4a and phosphorylated STAT3 and in situ
hybridization for miR-124, miR-24, and miR-629 revealed that, in
13/30 (43.3%) of HCC tumors, the circuit is perturbed
Due to the fact that our in vitro data suggest that activation of
an inflammatory response through suppression of HNF4a levels
is cell autonomous, we examined the activation of the inflamma-
tory circuit in the absence of Kupffer cells. We tested expression
levels of each member of the HNF4a circuit in RNA samples
derived from laser capture microdissected hepatocytes, which
were negative for CD45 expression. Specifically, in all (8/8)
human normal liver tissues, we found high HNF4a and miR-
124 levels and low IL6R, miR-24, and miR-629 levels. On the
other hand, we identified that the HNF4a circuit is perturbed
(HNF4a and miR-124 low levels; IL6R, miR-24, and miR-629)
in 18/31 of human hepatocellular carcinomas (Figure 7C).
Furthermore, in the same samples, we tested whether there is
any correlation between the RNA expression levels of the
different members of this circuit. We found an inverse correla-
tion between HNF4a and miR-24 or miR-629 levels, an inverse
correlation between miR-124 and IL6R levels, and a positive
correlation between HNF4a and miR-124 levels (Figure 7D).
Also, in the same human tissue samples, we examined IL6
and IL6R protein levels and STAT3 phosphorylation status and
identified that the HCC samples (n = 18) with perturbed
HNF4a circuit have higher levels in comparison to the HCC
samples (n = 13) with nonperturbed HNF4a circuit or normal liver
tissues (n = 8) (Figure 7E).
Furthermore, we were interested in identifying whether the
HNF4a circuit is perturbed not only during liver cancer initiation,
but also during liver cancer progression. Thus, we examined the
mRNA expression levels of the different members of the circuit in
different stages of HCC oncogenesis. We found that HNF4a and
miR-124 levels were decreased, whereas IL6R and miR-24
levels were increased, during HCC progression (Figure 8A).
Interestingly, the activity of this circuit correlated to HCC grade
(Figure 8B). Overall, these data strongly suggest that, in addition
to tumor initiation, the HNF4a-miRNA inflammatory feedback
circuit is important for the progression of human cancer.
An HNF4a Circuit Is Essential for the Transformation
of Immortalized Hepatocytes
Our data reveal the dynamics of a complex molecular self-
reinforcing circuit that involves HNF4a, miR-124, IL6R, STAT3,
and miR-24/miR-629 in the regulation of hepatocellular transfor-
mation and liver cancer (Figure 7F). The first component of the
circuit links HNF4a to STAT3 activation, with HNF4a controlling
IL6R expression through transcriptional regulation of miR-124.
Although miR-124 has been identified as a cancer-associated
tumor suppressive microRNA (Lujambio et al., 2007), its regula-
tion and mode of action has been elusive. Here, we show that
HNF4a binding and transcriptional regulation of miR-124 are
comparable to the bona fide HNF4a target ApoCIII (Kardassis
et al., 1997; Ladias et al., 1992). The second component of the
circuit connects STAT3 activity to HNF4a expression via regula-
tion of miR-24 and miR-629. Perturbations of the STAT3-HNF4a
axis interfere with processes that govern hepatic transformation
and oncogenesis, mechanistically linking inflammation and liver
An Epigenetic Switch Regulates Hepatocyte
The main characteristic of the HNF4a feedback circuit is that it
transforms immortalized human hepatocytes by converting
a transient signal (e.g., acute HNF4a inhibition) into a stable
signal. Overexpression of any positive factor (miR-24, miR-629)
or inhibition of any negative factor (HNF4a, miR-124) transforms
immortalized hepatocytes, indicating that the loop can be
affected at any step. Thus, the initiating event in different HCC
mouse models and patients can be different. It is not necessary
that the loop begins with reduction of HNF4a. According to our
data, suppression of HNF4a expression is the first event in
DEN-treated mice, followed by perturbation of the other
members of the loop. In other scenarios, the IL6-STAT3 axis
may activate the loop by different extracellular stimuli. Specifi-
cally, secreted IL6 from different immune cells in the tumor
microenvironment, including Kupffer cells, could initiate this
axis. For example, recent studies show that IL-22, a cytokine
secreted by Th17 cells, controls hepatocellular oncogenesis
via upregulation of STAT3 activity (Jiang et al., 2011), and hepa-
titis C viral infection can promote STAT3 activation (Tacke et al.,
2011). Interestingly, miR-124 has been found epigenetically
1242 Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc.
silenced through tumor-specific methylation both in human HCC
cell lines and tissues (Furuta et al., 2010), and therefore miR-124
downregulation may be the first event that triggers hepatic carci-
nogenesis. Together, all of these data suggest that the initial
event that activates this circuit could differ from patient to
Due to the fact that the epigenetic switch in immortalized
hepatocytes occurs within a few days, it is extremely unlikely
HCC Normal liver
3 6 9 12
0 3 6 9 12
0 3 6 9 12
0 3 6 9
Normal HCC Normal HCC
Normal HCC Normal HCC
Relative HNF4A levels
Relative miR-24 levels
Relative IL6R levels
Relative miR-124 levels
Normal CA circuit CA non circuit
Figure 7. HNF4a Circuit Is Perturbed in Human Hepatocellular Carcinomas
(A) Assessment of HNF4a, IL6R, miR-24, and miR-124 levels (mean ± SD) by real-time PCR analysis in total RNAs derived from 12 normal liver tissues and
45 hepatocellular carcinomas.
(B) Immunohistochemistry for HNF4a, pSTAT3, and in situ hybridization for miR-124 and miR-24 in FFPE sections of hepatocellular carcinomas and normal liver
tissues. Sections were subjected to immunohistochemistry for HNF4a (DAB staining, brown) and phospho-STAT3 (Tyr705) (DAB staining) and counterstained
with hematoxylin (blue) and in situ hybridization for miR-124, miR-24, and miR-629 and counterstained with nuclear fast red. Scale bar, 100 mm.
normal liver tissues and 31 hepatocellular carcinomas.
(D) Correlation between the expression levels of different members of the HNF4a circuit (same samples as in Figure 7C). Each data point represents an individual
liver tissue sample, and a correlation coefficient (r) is shown.
circuit [CA circuit] and 13 liver cancer tissues without activation of the HNF4a circuit [CA noncircuit]). The data are presented as mean ± SD of three independent
(F) Schematic representation of the proposed HNF4a feedback circuit in hepatocellular oncogenesis.
Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc. 1243
to involve changes in the DNA sequence, which is consistent
with the definition of a true epigenetic switch (Ptashne, 2009).
This notion of a self-reinforcing feedback loop that controls
hepatocellular transformation comes in line with our previous
observation of an epigenetic switch that mediated transforma-
tion of immortalized mammary epithelial (MCF10A) cells to a
stably transformed cell (Iliopoulos et al., 2009, 2010). Further-
more, the identification of an epigenetic switch in hepatocellular
transformation indicates that it is not a rare mechanism involved
cells of diverse developmental origin might share a common
mechanism for the establishment of the transformed state.
Our data also suggest that microRNA transcription factor
regulatory circuits mediate epigenetic switches that induce
transformation of immortalized cells. Recent reports posit that
transcriptional (TFs) and nontranscriptional elements (micro-
RNAs) may cooperate to tune gene expression in various biolog-
ical processes (Chen et al., 1994; Gerstein et al., 2010; Martinez
et al., 2008), including oncogenesis (Fabbri et al., 2011; Kent
et al., 2010). Various network motifs have been proposed, but
miRNA-TF feedforward and feedback loops predominate. For
example, a feedforward regulatory circuit (KRAS-miR-143/miR-
145) plays an essential role in pancreatic tumorigenesis (Kent
et al., 2010). Taken together, these observations demonstrate
that transcription factors participate in similar circuits that regu-
late induction and maintenance of stable transformation pro-
grams, suggesting that use of analogous regulatory loops may
be a widespread property of oncogenic processes.
Role of HNF4a and Its Downstream Effectors
in Hepatocellular Oncogenesis
HNF4a has long been considered a key transcription factor
during liver embryonic development (Kyrmizi et al., 2006; Parviz
et al., 2003). In the adult liver, HNF4a is expressed at high levels
and binds to the promoter of 12% of genes expressed (Odom
and the mechanisms involved are far from clear. It has been
shown that HNF4a is upregulated in human hepatocellular carci-
noma (Xu et al., 2001) and, on the other hand, impedes the
formation of liver tumors in mice by inducing differentiation of
tocytes (Yin et al., 2008). Recent findings that the Wnt/b-catenin
pathway interacts with HNF4a in intestinal epithelial cells (Cattin
et al., 2009) and hepatocytes (Colletti et al., 2009) strengthen the
notion that HNF4a acts as a tumor suppressor gene in both
cancer types. Our study refines the repressive role of HNF4a in
hepatic neoplasia, suggesting that HNF4a inhibition mediates
an epigenetic switch that is essential for the transformation of
Inflammation is one of the downstream mechanisms linking
HNF4a to hepatocellular carcinogenesis. The protective action
of HNF4a against inflammatory bowel diseases (Ahn et al.,
2008; Darsigny et al., 2010) and the potential associations
between the HNF4A locus and ulcerative colitis (Barrett et al.,
2009) raise the possibility that this multifaceted transcription
factor is a potent mediator of inflammatory responses (Marcil
et al., 2010). Several studies have identified STAT3 as an onco-
genic transcription factor activated by inflammatory responses
Figure 8. HNF4a Circuit Is Perturbed during HCC Progression
(A) Assessment of HNF4a, IL6R, miR-24, and miR-124 levels in total RNAs
derived from 45 hepatocellular carcinomas, according to their tumor stage.
The experiments have been performed in triplicate, and data are shown as
mean ± SD.
(B) HCC sections were subjected to immunohistochemistry for HNF4a and
phospho-STAT3 (Tyr705) (DAB staining, brown) and counterstained with
hematoxylin (blue) and in situ hybridization for miR-124 and miR-24 and
counterstained with nuclear fast red. Representative pictures are shown from
normal, HCC grade I, and HCC grade III tissues.
Scale bar, 50 mm.
1244 Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc.
(Bromberg et al., 1999; Grivennikov et al., 2009; Iliopoulos et al.,
STAT3 (Zhong et al., 1994). STAT3 activity has been correlated
with poor prognosis in HCC patients (Calvisi et al., 2006), and
STAT3 inhibitors inhibit the growth of several human cancers
(Hedvat et al., 2009), including HCC development and growth
in mice (He et al., 2010).
As genetic alterations that result in constitutive STAT3 activa-
tion in hepatocytes only cause benign hepatic adenomas unless
combined with oncogenic mutations (Rebouissou et al., 2009), it
will be important to discover the parameters that distinguish
primary hepatocytes from nontransformed immortalized cells.
Although a transient inflammatory signal is insufficient to trigger
suggest that the epigenetic switch described here is relevant to
human cancer. The epigenetic switch requires that a transient
inflammatory response is converted to a chronic inflammatory
of the inflammatory signal. Thus, the results presented here pro-
videa paradigm in which akey step in transformation involves an
epigenetic switch in response to an inflammatory signal, as
opposed to a mutational change in a tumor suppressor or onco-
gene. In support of this idea, recent data suggest that chronic
activation of the IL6-STAT3 axis contributes to the transforma-
tion of hepatocytes that have acquired oncogenic mutations
upon exposure to environmental and dietary carcinogens (Park
et al., 2010).
Therapeutic and Preventive Effects of miR-124 Delivery
in Hepatocellular Carcinogenesis
We show that miR-124 administration restored miR-124 expres-
sion to physiological levels in the liver, inhibiting and preventing
DEN-induced hepatocellular carcinogenesis in mice. Previous
studies have aimed to suppress microRNA expression in animal
models through delivery of antagomirs or locked nucleic acid
(LNA) oligomers (Elme ´n et al., 2008; Esau et al., 2006). Few
studies have investigated the therapeutic delivery of microRNAs
in vivo. A recent study has shown that restoration of miR-26a
liver tumorigenesis in liver-specific MYC transgenic mice (Kota
et al., 2009) without any cytotoxic effects.
Our data suggest that systemic delivery of miR-124 may be
a clinically viable anticancer therapeutic approach for liver
cancer. Delivery of microRNAs in the liver is more efficient in
comparison to other tissues, and a recent study revealed that
delivery of the antisense-microRNA-122 suppressed hepatitis
C viremia in primates, with no evidence of viral resistance or
side effects (Lanford et al., 2010), leading to the initiation of
phase I clinical trials in HCV-infected patients. This work lays
the ground for testing whether miR-124 can also exert tumor-
suppressive effects in human liver cancers. Together, our find-
ings elucidate a molecular mechanism that is responsible for
the initiation and maintenance of the hepatocyte-transformed
phenotype, which enhances our understanding of liver cancer
pathogenesis and provides a microRNA therapeutic strategy
for prevention and treatment of liver cancer. Though we have
identified a novel molecular circuit that is essential for the trans-
formation of hepatocytes and is found to be perturbed in
different HCC models and in human hepatocellular carcinomas,
significant work remains to identify the driver signaling pathways
involved in hepatocellular carcinogenesis.
Extensive details for all experimental procedures are provided in the Extended
Human nontransformed immortalized hepatocytes (IMH1, IMH2) were
purchased from ATCC (cat no. CRL-4020) and from Xenotech LLC (cat. no.
IFH15), respectively. Detailed description of the origin of these immortalized
hepatocytes and their culture conditions can be found in the Extended Exper-
imental Procedures. In addition, human liver cancer cell lines (HepG2, Hep3B,
SNU-449, SNU-398, and SNU-387) were purchased from ATCC. Human liver
cancer cell lines HepG2 and Hep3B were maintained in DMEM medium
(GIBCO) supplemented with 10% FBS and 10 units/ml penicillin and
100 mg/ml streptomycin. SNU-449, SNU-398, and SNU-387 were maintained
in RPMI-1640 medium (GIBCO) supplemented with 10% FBS and 10 units/ml
penicillin and 100 mg/ml streptomycin.
MicroRNA Library HNF4a Screening
A microRNA library consisting of 317 microRNA mimics and two microRNA
negative control mimics (100 nM) (Dharmacon Inc) was transfected in
HepG2 cells plated in 96-well plates. At 24 hr posttransfection, the cells
were transfected with a firefly luciferase vector harboring the 30UTR of
erase ReporterAssay System(Promega,WI,USA). MicroRNAs thatinhibited>
experimental description can be found in the Extended Experimental
Apoptosis was determined using the DeadEnd Fluorometric TUNEL System
(G3250, Promega), as previously described (Polytarchou et al., 2008).
Real-Time PCR Analysis
RNA purified from IMH, HepG2, Hep3B, SNU-449, SNU-398, SNU-387, and
SNU-475 cells under different transfection conditions with siRNAs or micro-
RNAs was reverse transcribed to form cDNA, which was subjected to SYBR
Green-basedreal-timePCRanalysis.MicroRNA expression levelsweretested
using the Exiqon PCR Primer Sets, according to the manufacturer’s instruc-
tions (Exiqon Inc, Denmark). Primer sequences can be found in the Extended
Identification of Transcription Factor Sites in MicroRNA Regulatory
The Lever and PhylCRM algorithms have been used to identify STAT3 and
RNAs.Adetaileddescription ofthismethod hasbeenincludedintheExtended
All of the experiments in xenografts and DEN-treated mice are described
analytically in the Extended Experimental Procedures.
RNA Expression Studies from Patient Samples
RNAs from 45 hepatocellular carcinomas and 12 normal tissues were
purchased from Biochain (Hayward, CA) and Origene (Rockville, MD). The
expression levels of miR-24, IL6R, miR-124, and HNF4a were analyzed by
real-time RT-PCR in all of the tissue described above. Each sample was run
in triplicate, and the data represent the mean ± SD.
Cell 147, 1233–1247, December 9, 2011 ª2011 Elsevier Inc. 1245
figures,and two tables and can be found with this article online at doi:10.1016/
This work was supported by DFCI CIA start-up funds to D.I. and research
grants to M.K. from the National Institutes of Health (RO1-CA118165 and
P42-ES0100337). We would like to thank the Nikon Imaging Center at Harvard
Medical School for their help with light microscopy. Also, we would like to
thank Hye-Jung Kim for her assistance with the flow cytometry analysis and
Virgilio Garcia for his technical support.
Received: February 16, 2011
Revised: July 22, 2011
Accepted: October 11, 2011
Published: December 8, 2011
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