Liver International ISSN 1478-3223

BASIC STUDIES

Delta-like 1contributes to cell growth by increasing the interferon-

inducible protein 16 expression in hepatocellular carcinoma
Feng Yu1,2, Xiangfang Hao2, Heng Zhao2, Chao Ge2, Ming Yao2, Shengli Yang1,2 and Jinjun Li2
1 Institute of Life Sciences, Jiangsu University, Zhenjiang, China
2 State Key Laboratory of Oncogenes and Related Genes, Cancer Institute of Shanghai Jiao Tong University School of Medicine, Shanghai, China

Keywords Abstract
DLK1 – hepatocellular carcinoma – IFI16 Background: Delta-like 1 (DLK1), a fetal liver stem cell marker, is strongly
expressed in human and rodent fetal liver, but not in adult liver. Notably,
dysregulation of DLK1 was found in some human hepatocellular carcinomas
Correspondence
(HCC). However, the effect of DLK1 on HCC cell growth and its underlying
Jinjun Li, State Key Laboratory of Oncogenes
mechanism are still largely unknown. Aims: To (i) assess the expression of
and Related Genes, Cancer Institute of
DLK1 in human HCC and adjacent liver tissues and human HCC cell lines; (ii)
Shanghai Jiao Tong University School of
Medicine, 25/Ln 2200 Xietu Road, Shanghai
evaluate the effect of DLK1 on SMMC-7721, Huh7 HCC cell growth in vitro
200032, China
and in vivo; and (iii) explore the potential mechanism of DLK1 that regulates
Tel: 186 21 64432140 HCC cell growth. Methods: The expression of DLK1 mRNA and protein were
Fax: 186 21 64432140 detected using reverse transcriptase-polymerase chain reaction and immuno-
e-mail: jjli@shsci.org histochemistry respectively. The effect of DLK1 on the proliferation of
SMMC-7721 and Huh7 cells was evaluated by colony formation and tumour
Received 15 July 2009 xenograft assay. The differential expression profiles of DLK1-overexpressing
Accepted 19 January 2010 SMMC-7721 cells and control cells were compared using HG-U133 Plus 2
Genechip. The cell cycle distribution of DLK1 forced expressing cells was
DOI:10.1111/j.1478-3231.2010.02214.x comparatively analysed. Results: Upregulation of DLK1 was observed in 41 of
57 (71.9%) human HCC samples. Ectopic expression of DLK1 promoted cell
proliferation, colony formation and tumorigenicity in SMMC-7721 and Huh7
cells. DLK1 upregulated the expression of interferon-inducible protein 16
(IFI16) and its promoter transcriptional activity, decreased p21waf1/cip1 and
induced cell cycle acceleration. However, silencing of IFI16 using small
interfering RNA abrogated DLK1-induced proliferation in these cells.
Conclusions: IFI16 may be an essential downstream target of DLK1 in HCC
cells and required for DLK1-induced cell proliferation.

Delta-like 1 homologue (DLK1), also known as Pref-1
(1), is widely expressed in a variety of murine and human
embryonic tissues, but it is expressed in only a small
number of adult tissues, such as the adrenal gland and
the pituitary (2, 3). In fetal mouse fetal liver, DLK1 is
highly expressed from embryonic day 12.5 (e12.5) to
e16.5, but its expression dramatically decreases in later
gestation and is absent in normal adult liver (4). DLK1
was found to participate in the regeneration of adult rat
liver oval cells in both retrorsine/partial hepatectomy
(PHx) and 2-acetylaminofluorine-treated/PHx rat mod-
els (5). Moreover, DLK1 may be involved in the transfor-
mation of hepatic stellate cells from fat-storing cells to
myofibroblasts (6). DLK1 is closely linked to fibrogenesis
and even cirrhosis (7). Currently, DLK1 has been used as
a hepatic stem cell (HSC) marker to enrich mouse or rat
Contributed equally.
hepatoblasts (8, 9). This suggests that DLK1 may be
involved in the modulation of liver stem cell differentia-
tion and liver development.
Human hepatocellular carcinoma (HCC) is the third
leading cause of cancer death and the fifth most common
malignancy worldwide (10, 11). The development of
HCC is accompanied by accumulated genetic changes,
which result in alterations in gene expression, including
an increase in oncogenic gene expression and a decrease
in tumour suppressor gene expression. Therefore, ex-
ploring the signalling pathways modulated by these genes
is helpful in understanding the mechanisms of hepato-
carcinogenesis. Currently, the upregulation of DLK1 has
been described in various cancers, including small cell
lung carcinomas (12), neuroendocrine tumours (13) and
liver cancers (14–16), suggesting that it may contribute to
tumorigenesis. Despite the fact that DLK1 possesses
oncogenic activities, little is known about its underlying
mechanisms and target genes.

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DLK1 upregulates IFI16 in HCC Yu et al.

The present study focused on the upregulation and
oncogenic activities of DLK1 in HCC and the mechanisms
underlying the DLK1-induced HCC cell proliferation.
Materials and methods
Hepatocellular carcinoma specimens and cell lines
Fifty-seven human HCC tissue specimens were obtained
from patients who underwent surgical resection at Qi-
dong Liver Cancer Institute (Qidong, China) or at the
First Affiliated Hospital of Zhejiang University (Hang-
zhou, China). The 57 HCC patients without preoperative
chemotherapy included 49 males and eight females,
median age of 50 years (range 31–74), whose clinical
pathological data were summarized in Table 1. The
procedures for the investigation were performed in
accordance with the China Ethical Review Committee.
All samples were fixed in 4% phosphate-buffered for-
malin (pH 7.4) and embedded in paraffin. Tissue arrays
were constructed as described previously (17). The HCC
cell lines (HepG2, Huh7, Hep3B, MHCC-97L, MHCC-
LM3, SMMC-7721 and BEL-7402), and the fetal liver-
derived cell line L-02 from the Cell Bank of the Institute
of Biochemistry and Cell Biology, at the China Academy
of Sciences, were used in this study.
Cell culture, transfection and stable cell lines
All cell lines were grown in DMEM (Sigma, St Louis,
MO, USA) supplemented with 10% heat-inactivated fetal
bovine serum (FBS; Hyclone, Logan, UT, USA), 100 U/ml
penicillin-G, and 100 mg/ml streptomycin at 37 1C under
a humidified atmosphere with 5% CO2. SMMC-7721 and
Huh7 cells were transfected with the DLK1-EGFP or
pEGFP-N1 plasmid using the Lipofectamine 2000 trans-
fection reagent (Invitrogen, Carlsbad, CA, USA), accord-
ing to the manufacturer’s instructions. The transfectants
were selected in 750 mg/ml G418 medium for 3 weeks and
sorted using an Epics Altra flow cell sorter (Beckman
Coulter, Fullerton, CA, USA), then confirmed by reverse
transciptase-polymerase chain reaction (RT-PCR) and
Western blot.
Immunostaining of hepatocellular carcinoma tissue
arrays and cultured cells
Paraffin-embedded HCC sections (5 mm thick) were
dewaxed, dehydrated and then incubated in methanol
(with 0.3% H2O2) at room temperature (RT) for 30 min
to inactivate the endogenous peroxidase. Antigen retrie-
val was performed by heating slides in sodium citrate
buffer (10 mM, pH 6.0) at 94 1C for 10 min. The slides
were incubated with a rabbit anti-DLK1 antibody (H-
118, 1:25 dilution, Santa Cruz, Santa Cruz, CA, USA) at
RT for 1 h and then with a HRP-conjugated anti-rabbit
antibody (Dako Japan Ltd, Kyoto, Japan) at RT for 1 h.
The signal was detected using the DAB Substrate Kit
(Dako, Glostrup, Denmark), and counterstaining was
performed with haematoxylin. Immunofluorescence was
performed to detect the subcellular localization of DLK1,

Table 1. Correlation between delta-like 1 protein staining and clinical pathology in 57 hepatocellular carcinoma tissues
Clinical DLK1 negative, DLK1 weakly DLK1 moderately DLK1 strongly
pathology n (%) positive, n (%) positive, n (%) positive, n (%) P value
Gender
Male 15 (93.8) 18 (85.7) 12 (75.0) 4 (100.0)
Female 1 (6.2) 3 (14.3) 4 (25.0) 0 (0.0) 0.384
Age
o = 50 8 (50.0) 15 (71.4) 6 (37.5) 0 (0.0)
4 50 8 (50.0) 6 (28.6) 10 (62.5) 4 (100.0) 0.031
AFP (ng/ml)
o = 20 4 (25.0) 9 (42.9) 3 (18.8) 0 (0.0)
4 20 12 (75.0) 12 (57.1) 13 (81.2) 4 (100.0) 0.204
HBV infection
Absent 13 (81.2) 15 (71.4) 14 (87.5) 3 (75.0)
Present 3 (18.8) 6 (28.6) 2 (12.5) 1 (25.0) 0.681
Tumour size (cm)
o3 4 (25.0) 1 (4.8) 2 (0.0) 0 (0.0)
3–5 6 (37.5) 9 (42.8) 3 (50.0) 0 (0.0)
45 6 (37.5) 11 (52.4) 11 (50.0) 4 (100.0) 0.145
Histological grade
I 0 (0.0) 3 (15.8) 2 (13.3) 0 (0.0)
II 3 (20.0) 2 (10.5) 7 (46.7) 2 (50.0)
III 7 (46.7) 9 (47.4) 3 (20.0) 1 (25.0)
IV 5 (33.3) 5 (26.3) 3 (20.0) 1 (25.0) 0.291
Cirrhosis
Absent 10 (62.5) 11 (52.4) 10 (62.5) 3 (75.0)
Present 6 (37.5) 10 (47.6) 6 (37.5) 1 (25.0) 0.811

AFP, a-fetoprotein; DLK1, delta-like 1; HBV, hepatitis B virus.

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Yu et al.
as described previously (18). Briefly, established HCC
stable cells fixed on slide were blocked with the SuperB-
locking solution (Pierce, Rockford, IL, USA) at RT for
30 min, then were stained with the primary antibodies
(mouse anti-GFP, clone B-2, 1:25 dilution; anti-DLK1,
clone H-118, 1:25 dilution) at RT for 1 h. The cells were
then incubated with a FITC-conjugated goat anti-mouse
IgG (1:100 dilution, Sigma) or a Alexa Fluor 594-
conjugated donkey anti-rabbit IgG (1:100 dilution, In-
vitrogen) at RT for 1 h. The nuclei were counterstained
with DAPI. The slides were observed and photographed
using an Axioskop 2 microscope (Carl Zeiss, Germany)
with a DP70 CCD system (Olympus, Japan) and a laser
confocal microscope (FV1000, Olympus, Japan).
Colony formation assay
SMMC-7721 and Huh7 cells (1  105 per well) in six-well
plates were transfected either with DLK1-EGFP (2 mg) or
with pEGFP-N1 (2 mg) using Lipofectamine 2000 (Invi-
trogen). Each transfection was performed in triplicate.
The transfected cells were selected in a medium contain-
ing G418 (750 mg/ml) for 3–4 weeks. The resistant cells
were fixed in 4% phosphate-buffered formalin (pH 7.4)
for 15 min and stained with Giemsa solution and then
scored.
Tumorigenicity in nude mice
Six- to eight-week old male athymic BALB/c nude mice
were housed in a specific pathogen-free facility. A total of
2  106 cells from each stable cell line were injected
subcutaneously in the right flank of the nude mice, with
six mice in each treatment group. The average tumour
weight of each group was obtained by weighing each
tumour at the time of euthanasia, 4 weeks after the
injection.
Microarray analysis
The gene expression profile determination and data
analysis were performed at Shanghai Biochip Co. Ltd
using a Human Genome U133 Plus 2.0 oligonucleotide
microarray from Affymetrix (Santa Clara, CA, USA).
Quantitative real-time polymerase chain reaction and
semi-quantitative real-time reverse transcriptase-
polymerase chain reaction
One microgram of RNA was reverse transcribed into
first-strand cDNA using oligo (dT) primers and MMLV
reverse transcriptase (TaKaRa Biotechnology, Dalian,
China). Quantitative real-time RT-PCR (qRT-PCR) was
performed in an ABI Prism 7300 Sequence Detection
System (ABI, Applied Biosystems, Foster city, CA, USA)
with optimized reaction conditions to verify the micro-
array data. The primer pairs are listed in supporting
information Table S1.
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DLK1 upregulates IFI16 in HCC
RT-PCR was carried out using the following primers
(forward and reverse): DLK1: 5 0 -AAGAAGCGCGCGC
TGAGCC-3 and 5-GTGGTCATGTCGATCTTCT C-3;
IFI16: 5-ACTGAGTACAACAAAGCCATT-3 and 5-TCC
TGAAGCTTGTTT GTGAAG-3; b-actin: 5-GGACTTC
GAGCAAGAGATGG-3, and 5-AGCACTGTG TTGGCG
TACAG-3. The cycling parameters were 30 cycles of
denaturation at 94 1C for 30 s, annealing at 58 1C for 30 s
and extension at 72 1C for 30 s.
Western blot analysis
Cells were lysed in RIPA buffer (Santa Cruz Biotechnol-
ogy) with a cocktail of phosphatase inhibitors (Roche
Applied Science, Indianapolis, IN, USA) and a cocktail of
proteinase inhibitors (Roche Applied Science), and the
total protein concentration was determined using a BCA
reagent (Sigma). The protein (30–60 mg) was resolved by
a 10% SDS-PAGE electrophoresis and transferred to
nitrocellulose membranes (Bio-Rad, Hercules, CA,
USA). The membranes were blocked with PBS contain-
ing 5% non-fat milk (Santa Cruz) for 1 h and then
probed with the appropriate antibodies: DLK1 (clone:
H-118, 1:600, Santa Cruz), IFI16 (clone: 1G7, 1:600,
Santa Cruz), GFP (clone: B-2, 1:1000, Santa Cruz), p53
(clone: DO-7, 1:400, Neomarkers, Fremont, CA, USA),
MDM2 (clone: SMP14, 1:500, Santa Cruz), p21waf1/cip1
(clone: EA10, 1:400, Calbiochem, San Diego, CA, USA),
Rb (clone: SMP441, 1:600, Santa Cruz), phospho-Rb
(1:300, MBL), phospho-p53 (Ser20) (1:100, CST), Bcl-2
(SC7382, 1:200, Santa Cruz), Bcl-xL (1:200, CST), Bax
(clone: 2D2,1:100, Neomarkers) and b-actin (clone: AC-
15, 1:10 000, Sigma). Following incubation at 4 1C over-
night, the immune complex was incubated with relevant
HRP-conjugated secondary antibodies (Sigma) at RT for
1 h. The SuperSignal West Femto maximum sensitivity
substrate kit (Pierce) was used for the visualization of the
signal.
RNAi-based gene silencing
Small interfering RNA oligonucleotides specific for
DLK1 or IFI16 and a general negative control (Cat. No.
B01001; siNC) were chemically synthesized by Gene-
Pharma (Shanghai, China): siDLK1-1 (5 0 -CCUGGACG
AUGGCCUCUAUtt-3 0 and 5 0 -AUAGAGGC CAUCGUC
CAGGtt-3 0 ), siDLK1-2 (5 0 -GGGAAAGGACUGCCAGA
AAtt-3 0 and 5 0 -UUUCUGGCAGUCCUUUCCCtt-3 0 );
siIFI16-1(5 0 -AGAACAUUGUUCUACUAAAtt-3 0 and
5 0 -UUUAGUAGAACAAUGUUCUTG-3 0 ), siIFI16-2 (5 0 -
AGGAGAAAUUCAAUGGAAAtt-3 0 and 5 0 -UUUCCAU
UGAAUUUCUCCUtt-3 0 ), and siIFI16-3 (5-UCAGAA
GACCACAAUCUACtt-3 and 5-GUAGAUUGUGGUCU
UCUGAtt-3 0 ). RNA interference was performed using
the reverse transfection method as described previously
(18). After transfection, the cells were cultured at 37 1C
for an additional 48 h and then harvested for further
analysis.
705
DLK1 upregulates IFI16 in HCC
Cell cycle analysis
The trypsinized cells were collected by centrifugation,
and then fixed in 70% ethanol at  20 1C overnight.
After fixation, the cells were resuspended in PBS contain-
ing 2 mg/ml propidium iodide (Sigma). Flow cytometry
was performed using an Epics Altra flow cell sorter
(Beckman Coulter).
BrdU incorporation
SMMC-7721-DLK1 and SMMC-7721-GFP cells, trans-
fected with or without siIFI16-3 or siNC for 48 h, were
incubated with BrdU (10 mM) for 4 h, and BrdU incor-
poration was measured using the cell proliferation en-
zyme-linked immuno sorbent assay, according to the
manufacturer’s specifications (Roche Applied Science).
Similar experiments were performed using Huh7-DLK1
and Huh7-GFP cells.
Drug treatment
To further analyse the difference in the S-phase check-
point control, the cells were treated with a genotoxin,
doxorubicin (Sigma). SMMC-7721-DLK1, SMMC-7721-
GFP and Huh7-DLK1, Huh7-GFP cells were exposed to
5 mg/ml doxorubicin in DMEM containing 10% FBS at
37 1C for 4 h, and then changed with fresh complete
medium. After 4 h incubation, the cells were harvested
for cell cycle distribution analysis.
Collection of conditioned media
SMMC-7721-DLK1-GFP and empty vector control cells
were cultured in 100 mm Petri dishes in DMEM com-
plete growth medium. At 70–80% confluence, the med-
ium was replaced by fresh DMEM containing 2% FBS,
and the cells were cultured for an additional 48 h.
Conditioned media were then collected and centrifuged
at 1500g for 5 min at 4 1C to remove cell debris and
subjected to secreted DLK1 detection.
Luciferase reporter gene assay
The IFI16 promoter reporter recombinant plasmid used
in this study was constructed as described previously
(19). The DNA sequences between nt 230 976 and
232 592 of the GenBank accession AP002534, were
cloned upstream of the firefly luciferase gene in the
pGL3-basic vector (Promega, Madison, WI, USA). Tran-
sient co-transfection was performed with the Renilla
luciferase plasmid (Dual-Luciferase Reporter Assay Sys-
tem, Promega) as an internal control using Lipofecta-
mine 2000 transfection reagent in 96-well plates. The
dual luciferase activities were measured after 48 h.
Ultraviolet treatment and apoptosis analysis
One day before treatment, the cells were subcultured in
six-well plates. ultraviolet (UV) exposure (at 254 nm)
706
Yu et al.
was carried out using a UVC 500 crosslinker (Amersham
Biosciences, San Francisco, CA, USA). Before UV irradia-
tion, the medium was removed. The UV irradiation was
300 J/m2 for SMMC-7721 cells and 30 J/m2 for Huh7 cells.
The cells were harvested at 12 h and 24 h after UV
irradiation for apoptosis, cell cycle distribution and Wes-
tern blot analysis.
Statistical analysis
The data were statistically analysed with Student’s t-test
and w2 using SPSS 16.0 software (SPSS Inc., Chicago, IL,
USA). P o 0.05 was considered statistically significant.
Results
Delta-like 1 is upregulated in hepatocellular carcinoma
Immunohistochemistry detection result showed that
DLK1 was significantly upregulated in 41 (71.9%) of the
57 HCC specimens as compared with their matched non-
cancerous liver tissue, where DLK1 was weakly or not
expressed. Among the 41 DLK1-positive HCC tissues, four
(10%) HCC samples showed high DLK1 expression, 16
(39%) and 21 (51%) displayed moderate and weak reac-
tions respectively (Fig. 1A). RT-PCR analysis confirmed
that DLK1 expression was increased in these cancers
compared with the matched adjacent liver tissues (Fig.
1B). The overexpression of DLK1 in HCC tissues was not
correlated with the gender, a-fetoprotein, hepatitis B
infection, tumour size, histological grade or cirrhosis
(P 4 0.05) (Table 1). Unexpectedly, the expression of
DLK1 was correlated with the age of HCC patients. The
four HCC samples with a strong DLK1 expression were all
from older patients (4 50 years). Furthermore, HepG2,
Hep3B and BEL-7402 cells expressed a high level of DLK1,
while SMMC-7721 and Huh7 cells expressed a low level
(Fig. 1C). These findings suggested that DLK1 may be
involved in hepatocarcinogenesis.
Delta-like 1 promotes colony formation and
tumorigenicity of hepatocellular carcinoma cells
Based on the intrinsic DLK1 expression in HCC cell lines,
stable HCC cell lines of SMMC-7721 and Huh7 with
ectopic DLK1 expression were established (Fig. 2A and
B). DLK1 is a putative secreted transmembrane protein,
and the ectodomain can be shed from the membrane (20,
21). Along with this, the secreted ectodomain and the
intracellular domain fused with GFP were detected in
SMMC-7721-DLK1 and Huh7-DLK1 cells, but not in the
empty vector control cell lines (Fig. 2B). The cytoplasmic
membrane localization of exogenous DLK1 was detected
by the anti-GFP antibody, but the most intrinsic DLK1
was found to be distributed in both nuclei and cytoplas-
mic membrane using the anti-DLK1 antibody (Fig. 2C).
In vitro experiments have shown that SMMC-7721 and
Huh7 cells with ectopic DLK1 expression formed sig-
nificantly more amount and larger size of colonies than
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Fig. 1. Dysregulated expression of DLK1 in human liver tumours. (A) Representative immunohistochemical staining for DLK1 in a tissue array.
DLK1 was strongly expressed in 4 HCC specimens (a, b), moderately expressed in 16 liver cancers (c, d), weakly expressed in 21 specimens (e, f)
and not expressed in 16 samples (g, h). i, j: the representative DLK1 stain for NT-liver. The nuclei were counterstained with hematoxylin. Arrows
on the enlarged image (b, d, f) indicated the DLK1 positive cells. a, c, e, g, i  50; b, d, f, h, j  400. (B) RT-PCR for DLK1 expression in HCC
tissue (C) and the adjacent non-cancerous liver (N). b-actin used as an internal control. (C) RT-PCR for DLK1 expression patterns in eight HCC
cell lines. RT (-): Fetal liver mRNA in which RT was omitted as a negative control.

those of the cells transfected with the empty vector (Fig.
2D). Furthermore, SMMC-7721 and Huh7 cells stably
overexpressing DLK1 or GFP were inoculated into athy-
mic mice to assess their tumorigenicity in vivo. As shown
in Fig. 2E, the tumour weight of ectopic DLK1-expressing
cells was two-fold more than that of the vector control in
SMMC-7721 cells, which was 1.78 fold in Huh7 cells.
Consistently, both SMMC-7721-DLK1 and Huh7-DLK1
cells provided with strong tumorigenicity. These data
suggested that the upregulation of DLK1 could indeed
contribute to the growth of HCC cells.
Interferon-inducible protein 16 is a downstream target of
Delta-like 1
Microarray was performed to explore the underlying
mechanism of how DLK1 proliferated the growth of
HCC cells using the RNA prepared from the cells of
SMMC-7721-DLK1 and SMMC-7721-GFP. Data analysis
showed that the expression levels of 463 genes were
altered: 283 genes were upregulated and 180 were down-
regulated. The representative data are listed in support-
ing information Tables S2 and S3, which include only
those genes with more than a 2.5-fold change in expres-
sion levels when compared with the control. To validate
the results obtained in the microarray analysis, the
expression level of seven genes (upregulated: DLK1,
mitogen-activated protein kinase 1 (MAPK1), tumour
suppressor candidate 3 (TUSC3) and IFI16; downregu-
lated: Janus kinase 1 (JAK1), matrix metallopeptidase 16
and caspase-1) selected from those identified in the
microarray were analysed using qRT-PCR with the cDNA
prepared from the RNA of SMMC-7721-DLK1 or
SMMC-7721-GFP cells. The qRT-PCR data of all the
tested genes were consistent with the results of the
microarray (Fig. 3A). IFI16 is a member of the HIN-200
(haemopoietic IFN-inducible nuclear/200-amino acid
repeat) family and contains domains involved in DNA
binding, transcriptional regulation and protein–protein
interactions. In our previous work, we found that some
DLK1 overexpressing primary HCC samples with in-
creased IFI16 expression, and hence the IFI16 gene was
selected for further investigation.
Upregulation of IFI16 by DLK1 was further deter-
mined by Western blot and RT-PCR analysis in SMMC-
7721 and Huh7 cells with ectopic DLK1 expression (Fig.
3B). To determine whether the downregulation of DLK1-
affected the IFI16 expression, the intrinsic DLK1 expres-
sion was silenced in BEL-7402 cells, which endogenously
expressed both DLK1 and IFI16. IFI16 expression was
significantly downregulated in response to DLK1 silen-
cing (Fig. 3C). To explore whether IFI16-affected the
DLK1 expression, intrinsic IFI16 expression was silenced
in Huh7 cells, which expressed a high level of IFI16.
Notably, a decreased IFI16 expression did not alter the
expression of DLK1 (Fig. 3D). In a cotransfection
experiment, IFI16 promoter activity was activated by a
transient overexpression of DLK1 in a dose-dependent
manner in SMMC-7721 and Huh7 cells; it suggested that
the IFI16 promoter is responsive to the DLK1 protein
(Fig. 3E). Moreover, a higher expression of both DLK1
and IFI16 was observed in HepG2 and BEL-7402 cells,

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Fig. 2. The effect of DLK1 on the colony formation and tumorigenicity of HCC cells. (A) RT-PCR for DLK1 expression in SMMC-7721 and Huh7
cells stably transfected with DLK1 or GFP. b-Actin was used as a loading control. RT (-): SMMC-7721-DLK1 mRNA, in which RT was omitted as a
negative control. (B) A Western blot confirmed the results in A. D: cells transfected with DLK1; V: cells transfected with GFP; B: blank cells.
(C) The subcellular localization of DLK1 detected by immunofluorescence microscopy using anti-GFP and anti-DLK1 antibodies. (D). DLK1
contributed to the colony formation of SMMC-7721 and Huh7 cells. Left: Representative plates of three independent experiments
demonstrating increased colony formation of SMMC-7721 and Huh7 cells induced by DLK1. Right: The average numbers of colonies were
calculated from three independent wells. (E) DLK1 increased the tumorigenicity of SMMC-7721 and Huh7 cells in athymic nude mice.
Left: Tumours excised from the experimental mice. Right: The columns represent the average weight of each group. P o 0.05.

compared with SMMC-7721 cells (Fig. 3F). These results
suggested that IFI16 is modulated by DLK1 and might be
a downstream target gene of DLK1 in HCC cells.
Overexpression of delta-like 1 leads to a decreased
p21waf1/cip1 expression and cell cycle acceleration
Interferon-inducible protein 16 can affect p53 and
p21waf1/cip1 transcription (22, 23), and can also de-
crease the p21waf1/cip1 protein stability (23). As shown
in Fig. 4A, a slight decrease of p53 expression and a
significant decrease in p21waf1/cip1 protein expression,
relative to control cells, were detected in SMMC-7721
and Huh7 cells that stably overexpressed DLK1. To assess
the physiological response of the cells to p21waf1/cip1
downregulation, the cell cycle progression was analysed
using flow cytometry. DLK1-overexpressing cells had an
approximately 10% increase in the number of cells in the
S phase (Fig. 4B and C). Rb is an important regulator of
the transition from the G1 to the S phase of the cell cycle.
Therefore, we examined the phosphorylation state of Rb.
Rb phosphorylation was significantly increased in
SMMC-7721 and Huh7 cells that overexpressed DLK1
relative to cells that expressed GFP (Fig. 4A). Then,

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Fig. 3. DLK1 upregulates IFI16 expression in HCC cells. (A) Validation of microarray data by qRT-PCR. The fold changes of selected genes were
assayed and normalized using GAPDH. The columns represent the average value of three independent experiments with standard deviations
(SDs). P o 0.05 except for JAK1 (P o 0.1). (B) RT-PCR (left) and Western bolt (right) analysis of IFI16 in the indicated SMMC-7721 and Huh7
cells. b-Actin was used as a loading control. (C) The effect of DLK1 silence on IFI16 expression in BEL-7402 cells. Left: The efficiency of DLK1
silence in BEL-7402 cells (upper panel) was detected by RT-PCR. IFI16 expression was examined (middle panel). b-actin was used as a loading
control (lower panel). BEL7402-m: mock-transfected BEL-7402 cells; RT (-): total mRNA of BEL7402-m in which RT was omitted as a negative
control. Right: A Western blot confirmed the results in left. (D) DLK1 expression was not affected by decreased IFI16 expression. Left: The
efficiency of RNAi-mediated silencing of IFI16 in Huh7 cells (upper panel) was detected by RT-PCR. DLK1 expression was not affected by IFI16
silencing (middle panel). b-actin was used as an internal control (lower panel). Right: A Western blot confirmed the results in left. (E) The IFI16
promoter-luc construct (50 ng) and Renilla luciferase plasmid (5 ng) were transiently contransfected in SMMC-7721 and Huh7 cells with DLK1-
EGFP or pEGFP expression constructs in 96-well plates. Cells were transfected in triplicate, cultured for 48 h, then lysed for relative luciferase
activity assay. Standard deviations were obtained from triplicate determinations. (F) RT-PCR analysis of intrinsic expression of DLK1 and IFI16 in
HCC cell lines. b-Actin was used as a loading control.

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Fig. 4. Effect of DLK1 overexpression on the cell cycle distribution. (A) Overexpression of DLK1 decreased p53 and p21waf1/cip1 protein
expression and increased Rb phosphorylation. Cell lysates of SMMC-7721 and Huh7 cells were analysed by Western blot using antibodies
against p53, MDM2, p21waf1/cip1, phosphorylated Rb (Phos-Rb), total Rb and b-actin. (B, C) Effect of DLK1 overexpression on the cell cycle
distribution. (D, E) Analysis of the cell cycle distribution of the paired stable cells treated with doxorubicin ( mg/ml).

doxorubicin treatment further enhanced the S-phase
distribution of DLK1-overexpressing cells compared
with empty vector control cells (Fig. 4D and E). These
data suggested that the DLK1-accelerated cell growth
might correlate with an increased IFI16 expression.
Interferon-inducible protein 16 is essential for the delta-
like 1-induced hepatocellular carcinoma cell proliferation
To further confirm the functional relevance of IFI16 in
the DLK1-induced HCC cell proliferation, the expression
of IFI16 was silenced in SMMC-7721 and Huh7 cells that
stably overexpressed DLK1 or GFP (Fig. 5A). Cell pro-
liferation was measured using the BrdU incorporation.
The SMMC-7721 and Huh7 cells that stably overex-
pressed DLK1 had significantly higher levels of BrdU
incorporation than the cells that stably expressed GFP
(1.34-fold increase for SMMC-7721 cells, P o 0.01; 1.40-
fold increase for Huh7 cells, P o 0.01), as did the
SMMC-7721 and Huh7 cells that stably overexpressed
DLK1 or GFP and were transiently transfected with siNC
(1.36-fold increase for SMMC-7721 cells, P o 0.01;

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Fig. 5. The effect of IFI16 silencing on the growth of HCC cells forced expressing DLK1. (A) The efficiency of RNAi in silencing IFI16 expression
in SMMC-7721 and Huh7 cells stably expressing DLK1 or GFP was validated by RT-PCR. b-actin was used as a loading control. (B) DLK1-induced
HCC cell proliferation was associated with IFI16. Left: Paired SMMC-7721-DLK1 and SMMC-7721-GFP cell groups, transfected with and
without siNC or siIFI16-3. Cell proliferation was measured using BrdU incorporation. The average absorbance numbers and SDs from triplicate
wells are reported. The results represent one of three independent experiments. P o 0.05, P o 0.01, DLK1 vs. vector. Right: Similar results
were obtained with Huh7-DLK1 and Huh7-GFP cells.

1.42-fold increase for Huh7 cells, P o 0.01). Conversely, Discussion

the difference in BrdU incorporation in SMMC-7721 and
Huh7 cells that stably overexpressed DLK1 or GFP and
were transiently transfected with siIFI16, was signifi-
cantly decreased (1.10-fold increase for SMMC-7721
cells, P o 0.05; 1.12-fold increase for Huh7 cells,
P o 0.05; Fig. 5B). Overall, IFI16 silencing resulted in
the distinct decrease of DLK1-induced BrdU incorpora-
tion. These data demonstrated that DLK1 accelerates the
growth of HCC cells by increasing the IFI16 expression.
Ectopic delta-like 1-expressing hepatocellular carcinoma
cells had a low susceptibility to ultraviolet-induced
apoptosis
Flow cytometry analysis showed that ectopic DLK1-
expressing HCC cells had a low susceptibility to DNA
damage-induced apoptosis from 12 to 24 h after UV
treatment. The cell percentage of sub-G1 phase reached
8.7% in vector control cells, but in that of ectopic DLK1-
expressing cells only 5.4% in SMMC-7721 cells at 12 h
after UV treatment. Compared with 12 h, they were 29.4
and 8.1% at 24 h respectively. In Huh7 cells, the cell
numbers of the sub-G1 phase (apoptotic cells) reached
42.3% in vector control cells, and that of ectopic DLK1-
expressing cells 24.1% at 12 h after UV treatment. At
24 h, they were 52.4% and 38.9% respectively (Fig. 6A).
The cell cycle distribution was compared, and showed
that ectopic DLK1-overexpressing HCC cells were sig-
nificantly resistant to cell growth arrest induced by UV
(Fig. 6B). Western blotting indicated that DLK1 decreas-
ing the susceptibility of HCC cells to UV-induced cell
apoptosis might be correlated with the increased Bcl-2
and Bcl-xL proteins (Fig. 6C).
Delta-like 1 has been identified as a HSC marker ex-
pressed in liver progenitor cells, but not in healthy adult
liver cells (24, 25). In this study, we have shown that (i)
fetal liver and most HCC samples expressed a high level
of DLK1 compared with their matched adjacent livers;
and (ii) overexpression of DLK1 significantly stimulated
the proliferation of HCC cells in vitro and in vivo. These
results suggest that DLK1-positive tumours may be
derived from primitive hepatoblasts or HSCs.
Delta-like 1 has been found to be expressed in almost
100% of hepatoblastomas (15) and in 73.2% of liver
cancers (16). Our results (71.9% of DLK1-positive in
HCC samples) were consistent with the previous reports,
implying that DLK1 may serve as an independent prog-
nostic factor for liver cancer (14).
In present study, we have shown that DLK1 increased
the growth of SMMC-7721 and Huh7 cells, and en-
hanced the colony formation of these cells. Moreover,
SMMC-7721 and Huh7 cells with an ectopic DLK1
expression subcutaneously implanted in nude mice,
accelerated tumour growth in vivo compared with those
of empty vector controls. In vitro and in vivo promotion
of SMMC-7721 and Huh7 cell growth by DLK1 were
associated with an increased expression of IFI16, and
might be correlated with a decreased apoptosis suscept-
ibility induced by UV irradiation as well as with an
increased expression of Bcl-2 and Bcl-xL (Fig. 6). Con-
sistently, most HCC cell lines with DLK1 expression also
express a high level of IFI16 in vitro (Fig. 3F). It resembles
that of human primary HCC samples compared with
their matched adjacent livers (supporting information
Fig. S1). Conversely, the silence of intrinsic IFI16

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DLK1 upregulates IFI16 in HCC Yu et al.

Fig. 6. Decreased susceptibility to UV-induced cell apoptosis and cell growth inhibition in DLK1 overexpressed cells. (A) Flow cytometry
analysis of cellular DNA contents after UV irradiation. Ectopic DLK1 cells and vector t control cells (SMMC-7721, up panel; Huh7, down panel)
were treated with UV (SMMC-7721, 300 J/m2; Huh7, 30 J/m2 at 254 nm). At 12 h and 24 h after UV irradiation, cells were collected and fixed
with 70% ethanol, and stained with propidium iodide. The representative data presented are one of three independent experiments. The
percentage of apoptosis cells (Sub-G1 cells) were indicated in each panel. (B) The analysis of the cell cycle distribution after UV irradiation
(including Sub-G1). The columns represent the average value of three independent experiments with standard deviations (SDs). P o 0.05,
P o 0.01. (C) Overexpression of DLK1 decreased the susceptibility to UV-induced cell apoptosis. Cell lysates of SMMC-7721 and Huh7 cells
12 h and 24 h after UV irradiation were analysed by Western blot using antibodies against GFP, IFI16, p53, phosphorylated p53 (p-p53), Bcl-2,
Bcl-xL, Bax, p21waf1/cif1, and b-actin.

expression significantly the decreased DLK1-induced cell
proliferation. These results suggest that IFI16 may be an
essential downstream target of DLK1 in HCC cells
and that IFI16 may be required for DLK1-induced cell
proliferation.
Recently, Kawakami and colleagues found that DLK1
expression was significantly decreased in human renal
cell carcinoma, and the expression of exogenous DLK1
could inhibit renal cell carcinoma cell growth. They
concluded that DLK1 was a putative tumour suppressor

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Yu et al.
in renal cell carcinoma (26). Conversely, the overexpres-
sion of DLK1 could promote glioblastoma multiforme
cell proliferation (13), and the downregulation of intrin-
sic DLK1 significantly inhibited HCC cell growth (16).
Our data would rather support the role of DLK1 as an
oncogenic gene in HCC because the ectopic expression of
DLK1 significantly accelerated the growth of HCC cells
in vitro and in vivo.
Our findings that IFI16 was required for DLK1-
induced cell growth conflicted with prior studies that
IFI16 acted as a tumour suppressor and growth inhibitor
in breast cancer cell lines and in human osteosarcoma
and chondrosarcoma cell lines (27, 28). However, it also
demonstrated that the inhibition of the intrinsic IFI16
expression by siRNA could induce the p21waf1/cip1
mRNA and protein expression through an increased
expression of p53, thus arresting the U2OS cell growth
(23). Furthermore, Kim et al. (19) found that the Wilms’
tumour cell lines CCG99-11 and SK-NEP-1 cells toler-
ated extremely high levels of IFI16 protein and that the
depletion of the intrinsic IFI16 protein expression in-
duced a growth arrest in these cells. In accordance with a
previous report demonstrating that IFI16 was expressed
in highly proliferative tissues (29), we also found that
HCC cell lines (HepG2, Huh7 and BEL-7402) expressed
high levels of IFI16. Therefore, it appears that the effect of
IFI16 on cell growth may be dependent on different cell
types and cellular contexts. In this study, we found that
DLK1-induced HCC cell proliferation mainly through an
increase in IFI16 expression and a decrease in p21waf1/
cip1 protein expression, which accelerated the G1–S
phase transition of the cell cycle.
Ruiz-Hidalgo et al. (30) found that DLK1 could
activate the Erk/MAPK signalling in response to IGF-1
in adipocyte. Our gene chip data indicated that DLK1
can upregulate the MAPK1 mRNA expression. There-
fore, we inhibited the MAPK signalling pathway by
U0126 to investigate whether DLK1 regulates the IFI16
expression via a MAPK signalling. The results showed
that DLK1 upregulated IFI16 independent of the MAPK
pathway (data not show). DLK1 also appears to mod-
ulate pro-inflammatory cytokines and immune re-
sponse-related factors expression through the NF-kB
activation in human bone marrow mesenchymal stem
cells (hMSC) (31). As IFI16 is also one kind of immune
response-related factors, therefore we speculated that
DLK1 increasing IFI16 expression might correlate with
the NF-kB signalling pathway.
Although most HCC cell lines and HCC tissues
displayed both high levels of DLK1 and IFI16 expression,
our current results do not imply that DLK1 is a IFI16
unique upstream regulator as some human HCC samples
shown a high IFI16 expression and are without a
significant upregulation of DLK1 (supporting informa-
tion Fig. S1). One of mechanisms of DLK1 upregulating
IFI16 is that DLK1 increases the promoter activation of
IFI16 gene (Fig. 3E). Taken together, our data suggest
that IFI16 is modulated by DLK1 and that DLK1 induces
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c 2010 John Wiley & Sons A/S
DLK1 upregulates IFI16 in HCC
the growth of HCC cells mainly through the upregula-
tion of IFI16. The significance of the signalling between
DLK1 and IFI16 and the functional role of DLK1 in
hepatocarcinogenesis need to be further elucidated.
Acknowledgements
This investigation was supported in part by grants from
the National Key Sci-Tech Special Project of China
(Grant 2008ZX10002-022), Program Of Shanghai Sub-
ject Chief Scientist (A type) (09XD1403600), National
Key Program for Basic Research of China (973)
(2009CB521803) and National Natural Science Founda-
tion of China/Research Grants Council Joint Fund
(NSFC/RGC) (30731160004).
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Sequences of the primers used for real-
time PCR analysis.
Table S2. Genes differentially up-regulated by DLK1
overexpression in SMMC-7721 cells.
Table S3. Genes differentially down-regulated by
DLK1 overexpression in SMMC-7721 cells.
Fig. S1. RT-PCR analysis of DLK1 and IFI16 expres-
sion in HCC tissues.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
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