TLR3 expression correlates with apoptosis, proliferation and angiogenesis in hepatocellular carcinoma and predicts prognosis
Yuan et al. BMC Cancer
TLR3 expression correlates with apoptosis, proliferation and angiogenesis in hepatocellular carcinoma and predicts prognosis
Ming-Ming Yuan 0 2
Yu-Yin Xu 1
Li Chen 0
Xing-Yu Li 0
Jing Qin 0
Ying Shen 0
0 Department of Pathological Anatomy, Nantong University , Qixiu Road 19, Nantong City, Jiangsu 226001 , China
1 Department of Nephrology, Affiliated Hospital of Nantong University , Nantong, Jiangsu , China
2 Department of Pathology, Nantong Rich Hospital , Jiangsu , China
Background: Toll-like receptor 3 (TLR3) plays a key role in innate immunity. In the present study, we analyzed tissues of patients with human hepatocellular carcinoma (HCC) to determine the significance of the relationship between TLR3 expression and cell proliferation, apoptosis, hepatitis B virus infections, angiogenesis and prognosis. Methods: We collected paraffin-embedded tissues from 85 patients with HCC who had complete histories and were followed for >5 years. The expression and intracellular localization of TLR3 and downstream proteins (TRIF, NF-B, and IRF3) were detected using immunohistochemistry. Further, we determined the expression of proteins that mediate cell proliferation (Ki67, cyclin D1), apoptosis (survivin, bcl-2, caspases 3, 8, and 9), and angiogenesis (CD34, MMP-2) as well as the HBV proteins HBsAg and HBcAg. Apoptosis in HCC tissues was detected using TUNEL. We conducted dual-labeling immunohistochemical analyses of TLR3 expression and TUNEL activity. Results: TLR3 expression was significantly lower in HCC tissues compared with adjacent tissues. TRIF, NF-B, and IRF3 correlated positively with TLR3 expression. Survivin and Bcl-2 expression correlated negatively with TLR3. The frequencies of caspases 3, 8, and 9 expression correlated positively with TLR3 signaling proteins. Cytoplasmic TLR3 and serum levels of HBsAg correlated positively. The apoptotic index determined using the TUNEL method and correlated positively with TLR3 expression. TLR3 expression in the cytoplasm correlated positively with TUNEL-positive cells and HBsAg. Ki67 and cyclin D1 correlated negatively with TLR3 expression. MMP-2 expression, microvessel density (CD34+) and endothelial progenitor cells (EPCs) correlated negatively with TLR3 expression. Kaplan-Meier survival analysis shows that TLR3 expression correlated with longer survival. Conclusions: The expression of TLR3 in HCC tissues may exert a synergistic effect on apoptosis and inhibit the proliferation of HCC cells, MMP-2 expression, generation of EPCs, and angiogenesis. Moreover, TLR3 expression may serve as a prognostic marker of HCC.
TLR3; HCC; Proliferation; Apoptosis; Angiogenesis; Prognostic
Hepatocellular carcinoma (HCC) is the sixth most
prevalent cancer and the third most frequent cause of
cancer-related deaths worldwide . China has a high
frequency of HCC, particularly in Qidong of Jiangsu
Province, the site of this study. The prognosis of patients
with HCC is generally poor, and frequent recurrence or
metastasis after transplantation represents the main
obstacle for long-term survival , which reflects the
complexity and heterogeneity of HCC biology. Therefore,
new approaches to address the mechanisms of HCC
progression are required to develop effective prognostic
techniques and to discover new therapeutic targets.
HCC is a hypervascular carcinoma, and angiogenesis,
in which endothelial cells of pre-existing capillaries
proliferate and migrate to form new vascular tips or
socalled vascular sprouts or endothelial buds , plays
an important role in the progression of HCC and
contributes to its malignant phenotype, invasiveness, and
high rates of recurrence and metastasis . Solid tumors
may not grow beyond 23 mm3 if vascular sprouts are
blocked , suggesting that advances in research on
vascular biology are paramount to the development of
genetic engineering and proteomics technologies that may
provide new and effective therapies for HCC that target
The Toll-like receptor (TLR) family member TLR3
recognizes double-stranded RNA (dsRNA) of viruses,
endogenous dsRNA released from dying cells, or
synthetic dsRNA such as polyriboinosinic:polyribocytidylic
acid (poly I:C). The TLR3 signaling pathway is mediated
exclusively by the TRIF adapter, which is recruited to
TLR3 by interaction between the TIR domains of the
two molecules [5,6]. Various branches of the signaling
pathway emanating from TLR3-TRIF lead to the
activation of IRF3, NF-B, and AP1 [7,8] and to the induction
of apoptosis through pro-caspase-8 activation [9-11].
This pathway activates IRF3 and NF-B, which act
together to induce the production of antiviral IFNs and
other cytokines . Evidence indicates that the
activation of NF-B is mediated by RIP1  and by TRAF6
in some cell types [14,15]. RIP1 and TRAF6
subsequently recruit TAB2  and TAK1. TAK1
phosphorylates IKK and IKK. IKK phosphorylates the NF-B
inhibitor IB, eventually leading to degradation of IB
and the translocation of NF-B to the nucleus .
TLR3 has attracted considerable attention from
investigators in fields such as biochemistry, immunology, and
medicine; however, we know little regarding the
significance of TLR3 in human carcinoma cells. In the present
study, we determined the expression and intracellular
localization of TLR3 in patients HCC tissues and the
relationship between the expression of TLR3 and cell
proliferation, apoptosis, HBV infections, and angiogenesis.
Table 1 Antibodies used for immunohistochemical analyses
Two-step immunohistochemical staining kit
Cell signaling (SER536)
Fuzhou Maixin Biotech (RMA-5542)
Fuzhou Maixin Biotech (RMA-0541)
Fuzhou Maixin Biotech (RAB-0536)
Cell signaling (ASP175)
Cell signaling (ASP391)
Fuzhou Maixin Biotech (MAB-0034)
Fuzhou Maixin Biotech (MAB-0244)
Fuzhou Maixin Biotech (KIT-9801)
Our goal was to investigate whether activation of the
TLR3 signaling pathway inhibits the growth of HCC.
We certify that we have read Nantong Universitys ETHICAL
PRINCIPLES FOR CONDUCTING RESEARCH WITH
HUMAN AND ANIMALS. The protocols for these studies
were approved by the Ethics Committees of the Third
Peoples Hospital of Nantong and the Nantong Rich
The study group included 85 patients who were enrolled at
the Third Peoples Hospital of Nantong and the Nantong
Rich Hospital between 2005 and 2009. We followed 76
patients after their discharge. The follow-up period ranged
from 1 to 96 months (median 26 months). Tissues were
excised from an area 23 cm distant from HCC nodules.
Nontumor tissues were acquired from 15 liver-transplant
donor specimens, 10 liver hemangioma specimens, and five
liver trauma specimens. Patients underwent liver resection
and were subsequently diagnosed histopathologically with
HCC. The patients had clear records of serum AFP level,
tumor size, and lymph node metastasis. The ethics
committee of each institution approved this study, and all patients
granted written informed consent.
Tissue microarrays (TMA) were prepared according to
the method of Qun (Patent Number: ZL 2008 1 0022
170.4). Briefly, all HCC tissues were stained using
hematoxylin and eosin and were reviewed by two
histopathologists. Representative areas free from necrotic and
Figure 1 Immunohistochemical analysis of TLR3 expression and localization in HCC tissues. TLR3 was detected in the cytoplasm (a) and
membrane (b). Magnification 200.
hemorrhagic materials were marked in the paraffin
blocks. Two cylindrical tissue cores (1.6-mm diameter)
were removed from the donor blocks and transferred to
the recipient paraffin blocks, and their array positions
were recorded. Each TMA block contained over 100
cylinders, and the final TMAs comprised samples from 85
cases of HCC and 85 cases of adjacent nontumor tissues
(ANT). Consecutive sections (4 m thick) were cut from
the array blocks and placed on adhesive microscope
slides for immunohistochemical analysis.
The Envision+/DAB analysis method was performed using
formalin-fixed, paraffin-embedded 4-m sections from all
patients. Antibodies against 13 proteins were used to
analyze the sections (Table 1). The sections were dewaxed
in xylene and heated in a microwave oven. For antigen
retrieval, slides were heated at 95C for 10 min in sodium
citrate buffer (10 mM sodium citrate monohydrate,
pH 6.0). The slides were cooled for 20 min at room
temperature and then incubated in Envision + peroxidase
blocking solution (Dako Cytomation, Glostrup, Denmark)
for 5 min and rinsed with 0.05% Tris-buffered saline
(TBS)/Tween 20 buffer, pH 7.4. The slides were then
incubated with primary antibodies for 30 min at room
temperature. The slides were washed with 0.05% Tween 20
in TBS (pH 7.4). Detection was achieved with the DAKO
Envision+/HRP system (DAKO, Carpinteria, CA, USA).
The color was developed using a 15-min incubation with
diaminobenzidine (DAB) solution (DAB kit IL1-9032)
(Fuzhou Maixin Biotech. Co., Ltd., China), and sections
were lightly counterstained with hematoxylin. Positive and
negative controls (TBS was substituted for primary
antibody at concentration of 1:200) were performed for each
Staining patterns and data evaluation
The staining patterns were defined as P (cytoplasm),
M (membrane), and N (nucleus). The average optical
density (OD) of the immunostained HCC cells of each
sample was measured using a morphological analysis
system (Image J). Staining was defined according to
average OD as follows: strongly positive (++), 4060;
weakly positive (+), 2039; and negative (), 019.
CD34 was mainly expressed in a scattered pattern in
endothelial cells of microvessels. The most densely stained
zones were selected at the invading tumor front. We
analyzed blood vessels with a clearly defined lumen or linear
vessel shape, but not single endothelial cells. The mean
vessel count obtained from five fields at 200
magnification was used to define the mean vesicle density (MVD).
Table 2 TLR3 expression in HCC, adjacent tissues, and
other liver tissues
(aP > 0.05, HCC vs Adjacent; bP < 0.05, HCC vs Nontumor; cP > 0.05, Adjacent
Table 3 Association of TLR3 expression and intracellular
localization with the stages of HCC
TLR3 expression pattern
Terminal deoxynucleotidyl transferase-mediated dUTP
nick end labeling (TUNEL)
A TUNEL kit (Promega, USA) was employed to detect
apoptosis. Briefly, paraffin-embedded sections were
deparaffinized and dehydrated. After washing in
phosphatebuffered saline (PBS), sections were treated with 20 g/mL
of Proteinase K for 20 min. After washing in PBS three
times (3 min each), sections were rinsed with 0.3% Triton
X-100 for 10 min followed by washing in PBS. These
sections were incubated with the TUNEL reaction mixture at
37C for 1 h. After washing in PBS three times (3 min
each), sections were treated with horseradish
peroxidaseconjugated streptavidin (1:200; Beijing Zhongshan Biotech
Co., Ltd) at 37C for 30 min. After washing in PBS three
times (3 min each), reactions were visualized by treating
sections with 0.04% DAB and 0.03% H2O2 at room
temperature for 812 min. After washing in water,
sections were counterstained using hematoxylin and
mounted with resin. Control reaction mixtures contained
PBS instead of the TUNEL reagents. Positive-control
sections were pretreated with DNase I for 10 min followed by
TUNEL staining. Cells with blue granules in the nucleus
were regarded as TUNEL-positive.
Dual immunohistochemical staining
Dual immunohistochemical staining to detect
TUNELpositive and TLR3-positive cells was performed as follows:
First, TUNEL staining was performed using the blue-black
BCIP/NBT color reagent (Zymed Histostain-Dskit) at 37C
for 20 min. After the primary staining sequence, slides were
rinsed in TBS and incubated in a double-stain blocking
solution for 3 min (Dako cytomation) and then rinsed in
TBS. The anti-TLR3 antibody (diluted 1:100) was applied
to the tissue sections at 37C for 60 min and incubated at
4C overnight. A secondary antibody against goat
antirabbit IgG conjugated to horseradish peroxidase (PV-6002)
was added for 30 min at 37C followed by rinsing in TBS.
Sections were developed using liquid DAB-plus for 10 min,
rinsed in TBS, stained with Mayers hematoxylin for 10 s,
and mounted in aqueous mounting medium. The nuclei of
apoptotic cells were stained blue-black (BCIP/NBT), and
TLR3 in the cytoplasm was stained brown (DAB).
Statistical analysis was performed using SPSS 19.0 for
Windows. The statistical significance of the differences
in data between cancer tissues and adjacent nontumor
tissues was assessed using Wilcoxon signed-rank tests.
The Spearman rank correlation test was performed to
determine the association of HCC-related markers.
TUNEL, MVD, and EPCs were analyzed using
histograms and linear regression analysis, and P < 0.05 was
considered significant. Univariate survival analyses were
performed to evaluate the prognostic significance of
TLR3 expression. Curves for overall survival (OS) were
generated according to the KaplanMeier method, and
differences were analyzed by applying the log-rank test
to univariate survival analysis.
TLR3 expression and localization in HCC and adjacent
Immunohistochemical analyses of TLR3 expression were
conducted using tissues collected from 85 patients with
HCC along with the respective adjacent uninvolved
tissue as well as 30 non-HCC tissue specimens (Figure 1
and Table 2). TLR3 expression in the cytoplasm or
membrane was detected in 58.8% (50/85), 67.1% (57/85),
and 80.0% (24/30) of the tumor, normal, and non-HCC
sections, respectively. There was no significant difference
Table 4 The association of TLR3 expression with HBV infection of patients with HCC
aTLR3 M expression compared with HBsAg or HBcAg serum levels. bTLR3 P expression compared with HBsAg serum levels. cTLR3 M/P expression compared with
HBsAg serum levels. Abbreviations: M, membrane; P, cytoplasm; MP membrane and cytoplasm.
in TLR3 expression between HCC and adjacent tissues
(2 = 1.236, P > 0.05) or between adjacent and nontumor
tissues (2 = 1.783, P > 0.05). The frequency of TLR3
expression in HCC samples was significantly lower
compared with that of nontumor tissues (2 = 4.334, P < 0.05).
Correlation of TLR3 expression and localization with HCC
The frequencies of TLR3 expression in HCC tissues
according to histological grade were as follows: G1 77.78%
(7/9), G2 62.86% (22/35), and G3 51.22% (21/41). Thus,
TLR3 expression was more frequent in well-differentiated
HCC tissues. There were no significant associations
between expresson of TLR3 in the membrane and in both
the membrane and cytoplasm and HCC histological grade
(2 = 1.057, P = 0.590; 2 = 2.017, P = 0.365; respectively).
Conversely, there was a significant positive relationship
between the expression of TLR3 in the cytoplasm and
HCC histological grade (2 = 8.354, P = 0.015) (Table 3).
The association of TLR3 expression with HBV infection of
patients with HCC
Patients serum levels of HBsAg and HBcAg were
determined using an ELISA. There was a significant positive
correlation between the expression of TLR3 in the cytoplasm
of HCC cells and serum levels of HBsAg (2 = 24.299,
P < 0.001; r = 0.551, P < 0.001) (Table 4). Conversely, there
was a significant negative correlation between the
expression of TLR3 in the membrane or both the membrane and
cytoplasm of cells in HCC tissues with the levels of serum
HBsAg (P > 0.05). However, there was no significant
Figure 2 Immunohistochemical analysis of TRIF, NF-B, and IRF3 expression and localization in HCC tissues. TRIF was detected in the cytoplasm
(a), NF-B was detected in the cytoplasm or nucleus (b, c), and IRF3 was detected in the nucleus (d). Magnification 200.
correlation between TLR3 expression and levels of serum
HBcAg (P > 0.05).
Immunohistochemical analysis of TLR3, TRIF, NF-B, and
IRF3 expression in HCC tissues
TRIF was detected in the cytoplasm of cells in 69.4% (59/
85) of HCC tumor sections. NF-B was detected in the
cytoplasm or nucleus in 63.5% (54/85) of HCC tumor
sections. IRF3 was expressed in the nucleus in 52.9% (45/85)
of HCC tumor sections (Figure 2). The frequency of TLR3
expression in HCC tumor sections correlated positively
with those of TRIF ( = 0.322, P < 0.01), NF-B ( = 0.264,
P < 0.05), and IRF3 ( = 0.317, P < 0.01) (Table 5).
The association of TLR3 signaling-pathway protein
expression and apoptosis in HCC tissues
Using immunohistochemistry, we detected the
cytoplasmic expression of the apoptosis-related proteins
survivin, Bcl-2, and caspases 3, 8, and 9 (Figure 3), which
were expressed in 48.2% (41/85), 38.8% (33/85), 71.8%
(61/85), 68.2% (58/85) and 57.6% (49/85), respectively, of
HCC tissue samples. Survivin expression correlated
negatively with those of TLR3 ( = 0.360, P < 0.01), TRIF
( = 0.234, P < 0.05), IRF3 ( = 0.286, P < 0.01), and NF-B
( = 0.246, P < 0.05). Bcl-2 expression correlated negatively
with that of TLR3 ( = 0.369, P < 0.01), TRIF ( = 0.288,
P < 0.01), and IRF3 ( = 0.236, P < 0.05) in HCC tissue
samples (Table 4). The frequencies of caspases 3, 8, and 9
expression correlated positively with those of TLR3 signaling
proteins (P < 0.01 or P < 0.05) (Table 6).
The results of TUNEL assays (AI) in HCC tissues were
243% (mean = 13.98%). The 85 HCC tissues were divided
according to AI values into the groups as follows: 10%,
11%20%, and >20%. Spearman correlation analysis shows
that AI correlated positively with the frequencies of
expression of TLR3 ( = 0.483, P < 0.01), TRIF ( = 0.566,
P < 0.01), IRF3 ( = 0.548, P < 0.01), and NF-B ( = 0.438,
P < 0.01) (Figure 4, Table 6).
For more precise localization, dual
immunohistochemical analyses were performed to detect TUNEL activity and
TLR3 expression. TLR3-positive and TUNEL-positive
signals were localized to the tumor cell cytoplasm or
membrane and to the nucleus, respectively (Figure 5). The two
markers correlated positively in situ (Figure 5).
Correlation of TLR3 expression with apoptosis (TUNEL
assay) and serum levels of HBV antigens
When TLR3 was expressed in the cytoplasm, the
TUNEL data correlated positively with HBsAg levels in
serum (2 = 9.420, P = 0.003; r = 0.614, P = 0.001) and
negatively with HBcAg levels in serum (P > 0.05). When
TLR3 was expressed in the membrane or in the
membrane and cytoplasm, TUNEL positivity correlated
negatively with HBsAg and HBcAg levels (P > 0.05).
The association of the expression of TLR3-pathway-signaling
proteins with those of proliferation-related proteins Ki67 and
cyclinD1 in HCC tissues
The expressions of Ki67 and cyclin D1 were detected using
immunohistochemistry (Figure 6). Expression was localized
to the nucleus with the frequencies as follows: Ki67 61.2%
(52/85) and cyclin D1 57.6% (49/85). The frequency of Ki67
expression correlated negatively with those of TLR3
( = 0.276, P <0.05), TRIF ( = 0.215, P <0.05), IRF3
( = 0.281, P <0.01), and NF-B ( = 0.265, P <0.05).
Cyclin D1 expression correlated negatively with those of TLR3
( = 0.269, P <0.05), TRIF ( = 0.219, P <0.05), IRF3
( = 0.292, P < 0.01), and NF-B ( = 0.262, P <0.05)
Figure 3 Immunohistochemical analysis of survivin, Bcl-2, and caspase expression and TUNEL analysis of apoptosis in HCC tissues. Survivin (a), Bcl-2
(b), and caspase 3, 8 and 9 were detected in the cytoplasm (c, d and e). Apoptotic nuclei detected using the TUNEL assay are brownish-yellow
(f). Magnification 200.
The association of the expression of TLR3 signaling-pathway
proteins with those of CD34 and MMP-2 in HCC tissues
Immunohistochemical analysis detected MMP-2
expression in the cytoplasm (Figure 6) of cells in 56.5% (48/85) of
samples. MMP-2 expression levels correlated negatively
with those of TLR3 ( = 0.378, P <0.01), TRIF ( = 0.294,
P <0.01), and NF-B ( = 0.248, P < 0.05) (Table 7). CD34
was detected in the membranes or cytoplasm of vascular
endothelial cells and EPCs (Figure 7). The average MVD
of the 85 HCC tissue samples was 183.89 mm2. The
samples were divided into grades as follows: 120, 121240,
and >240. Spearman correlation analysis shows that MVD
Table 6 Correlation between the expression of TLR3 signaling-pathway proteins and apoptosis-related proteins with
apoptosis in HCC tissues
correlated negatively with the frequency of expression of
TLR3 ( = 0.583, P < 0.01), TRIF ( = 0.560, P < 0.01),
IRF3 ( = 0.527, P < 0.01), and NF-B ( = 0.484, P < 0.01)
(Figure 8, Table 8). Moreover, the average EPC count of the
85 HCC samples was 016 (mean = 4.52). The samples were
divided into grades as follows: 5, 610, and >10.
Spearman correlation analysis shows that the EPC count
correlated negatively with the frequencies of expression of TLR3
( = 0.544, P < 0.01), TRIF ( = 0.458, P < 0.01), IRF3
( = 0.277, P < 0.05), and NF-B ( = 0.345, P < 0.01)
(Figure 9, Table 8).
The association of the expression of TLR3
signalingpathway proteins with survival of patients with HCC
The follow-up and loss rates of patients were 89.4% (76/85)
and 10.6% (9/85), respectively. The follow-up period ranged
from 196 months (median = 26 months). Among the 76
patients followed after their discharge, 71 died of HCC and
one died of other causes. The 1-, 3-, and 5-year media
overall survival (OS) rates for these 76 patients were 56.6%
(43/76), 27.6% (21/76), and 10.5% (8/76), respectively. The
survival rates of patients increased significantly as a
function of increased expression levels of TLR3, TRIF, IRF3, and
NF-B as follows (expression levels followed by survival
rates): 1. TLR3 (), (+), and (++): 0% (0/35), 4.5% (1/22),
and 25.0 (7/28), respectively (log-rank = 43.187, P < 0.01)
(Figure 10a). 2. TRIF (), (+), and (++): 0% (0/26), 4.0%
(1/25), and 20.6% (7/34), respectively (log-rank = 21.867,
P < 0.01) (Figure 10b). 3. IRF3 (), (+), and (++): 0% (0/40),
12.5% (3/24), and 23.8 (5/21), respectively (log-rank =
19.818, P < 0.01) (Figure 10c). 4. NF-B (), (+), and (++):
0% (0/31), 9.7% (3/31), and 21.7 (5/23), respectively
(logrank = 23.375, P < 0.01) (Figure 10d).
HCC is the sixth most common malignancy of humans,
accounting for approximately 90% of primary liver cancers
. Hepatocarcinogenesis is complex and characterized
Figure 5 Dual immunohistochemical analysis of apoptosis and TLR3 expression in HCC tissue. (a) In well-differentiated HCC tissue, the nuclei were
TUNEL-positive and TLR3 was overexpressed at equal levels in the cytoplasm and membrane. (b) The nuclei of poorly differentiated HCC cells were
TUNELpositive, and these cells expressed relatively lower levels of cytoplasmic TLR3. Magnification 400.
Figure 6 Analysis of Ki67 and Cyclin D1 expression and localization in HCC cells. (a) Ki67 was detected in the nucleus. CyclinD1 exhibited nucleus
staining (b). Magnification 200.
by myriad molecular abnormalities . Over the past 10
decades, the molecular mechanism of HCC has been
extensively investigated. However, our knowledge of the
pathogenesis of this disease is insufficient for the purposes
of prevention, early diagnosis, and treatment. Increasing
evidence indicates that TLR3 is an important modulator
of HCC progression and a potential target for novel
TLR3 is an endosomal receptor for double-stranded
RNA and is expressed by several subsets of immune
cells, including dendritic cells  and natural killer
(NK) cells . TLR3 is expressed by fibroblasts ,
lung epithelial cells , hepatocytes , and several
types of tumor cells. For example, Yoneda  found
that TLR3 mRNA was expressed in HCC tissues as well
as in nontumor tissues. Using immunocytochemistry,
TLR3 was detected in 52.7% of HCC tissues. Although
cell-surface stimulation of TLR3 with poly (I:C) does not
affect cell viability, it does increase NF-B levels. In
contrast, cytoplasmic stimulation with transfected poly (I:C)
induced apoptosis, which was accompanied by
downregulation of antiapoptotic proteins. In the present
study, we used immunohistochemistry and found that
TLR3 was expressed in 58.8% (50/85) of HCC tissues,
which is slightly higher than previously reported, and that
TLR3 was localized to the membrane and cytoplasmic.
TLR3 was expressed more frequently in tumor tissues
compared with adjacent tissues (67.1%) and tissues from
subjects without HCC (80.0%). Further, TLR3 expression
was stronger in well-differentiated HCC tissues compared
with poorly differentiated HCC tissues, and there was a
significant positive relationship between the cytoplasmic
expression of TLR3 and HCC histological grade, which
suggests that down-regulation of TLR3 may disrupt the
regulation of cell proliferation and homeostasis to
promote malignant transformation.
TLR3 signaling depends solely on the TLR TIR
domain, which recruits the adaptor-inducing IFN- (TRIF)
adapter protein. This leads to activation of the
transcription factors NF-B and IRF3 and induces the antiviral
interferon response . Further, TRIF exhibits
proapoptotic activity, suggesting that TLR3 signaling triggers the
cell death pathway . In the present study, we
detected the TLR3 signaling-pathway proteins TRIF,
NFB, and IRF3 in 69.4% (59/85), 63.5% (54/85), and 52.9%
(45/85) of the HCC samples, respectively. Moreover,
their expression correlated positively with that of TLR3.
Therefore, the increased frequency of TLR3 expression
aTLR3 P expression, positive TUNEL reactions compared with HBsAg or HBcAg levels. bTLR3 M expression, positive TUNEL reactions compared with HBsAg or
HBcAg levels. cTLR3 M/P expression, positive TUNEL reactions compared with HBsAg or HBcAg levels.
may affect the proliferation and apoptosis of HCC cells
through multiple signaling pathways.
TLR3 is unique among TLRs, because it signals through
TRIF (TIR domain-containing adaptor-inducing
interferon), not through MyD88, and may activate the inflammatory
or apoptotic pathways. The inflammatory pathway leads to
NF-B activation, whereas the apoptotic pathway, believed
to be mediated by Rip3, leads to caspase-8 activation .
Our results suggest that TLR3-TRIF-IRF3 or the
TRIF-NFB signaling pathway is activated in HCC cells in the
majority of tissue samples analyzed here.
Apoptosis is a complex process that is mainly
mediated through the Fas ligand/Fas pathway as well as a
mitochondrial pathway . To examine the
mechanisms of TLR3-induced apoptosis in HCC cells, we
studied the association of TLR3 expression by HCC cells
with the expression of apoptosis-related proteins Bcl-2,
survivin, and caspases 3, 8, and 9. Bcl-2 localizes to the
inner mitochondrial membrane  and is important for
cell survival and its antiapoptotic effects. Survivin is
critically required for inhibiting apoptosis and ensuring normal
cell division in the G2/M phase of the cell cycle and is
abundantly expressed in every human tumor compared
with normal tissues. Survivin inhibits apoptosis by
inhibiting activated caspases . The caspases, particularly
caspase-3, act downstream of the Bax/Bcl-2 control and
play a key role in the execution of apoptosis . In the
present study, the expression level of TLR3 correlated
negatively with those of Bcl-2 and survivin, and correlated
positively with those of caspases 3, 8, and 9, indicating that
activation of TLR3 is related to the stimulation of apoptosis.
Detection here of TLR3 in the cytoplasm and
membranes of HCC cells was accompanied by activation of the
components of the Fas ligand/Fas and mitochondrial
apoptotic pathways, suggesting that TLR3 promotes
apoptosis of HCC cells through these pathways. Moreover, we
demonstrate a significant positive relationship between
the expression of the TLR3 signaling-pathway
components TRIF, IRF3, and NF-B as well as those of caspases
3, 8, and 9. TUNEL staining shows that the AI correlated
positively with the expression of TRIF, IRF3, and NF-B.
Our data identify an association of TLR3 with
overexpression of caspases 3, 8, and 9 and suggest that activation of
TLR3 plays an important role in apoptosis in HCC
through the Fas ligand/Fas and mitochondrial pathways.
We demonstrate here a significant positive
relationship between the cytoplasmic expression of TLR3 and
the presence of HBsAg in serum. Moreover, when TLR3
was expressed in the cytoplasm, TUNEL positivity
correlated with the detection of HBsAg in serum, indicating
that the synthesis of viral dsRNA was upregulated and
activated in TLR3, which in turn increased the
population of interstitial immunoreactive cells and induced the
production of inflammatory cytokines.
The cytokine interleukin (IL)-1 is a key mediator of the
inflammatory response and is implicated in the
pathophysiology of acute and chronic inflammation. Poly (I:C)
stimulation of macrophages induces pro-IL-1 processing
via a Toll/IL-1R domain-containing adaptor-inducing
interferon--dependent signaling pathway initiated by
TLR3 . Boulton et al. found that IL-1 administered
parenterally to rats 0 and 12 h after partial hepatectomy
significantly reduced the incorporation of bromodeoxyuridine
into hepatocytes at 18 h, indicating that nonparenchymal
cells isolated from regenerating rat liver express IL-1. These
findings support the hypothesis that IL-1 plays a role in
suppressing hepatocyte proliferation and terminating the
surge of DNA synthesis induced after partial hepatectomy
. In contrast, decreased induction of IL-1 mRNA
synthesis in TLR3-deficient mice leads to the proliferation of
hepatocytes . These findings suggest that TLR3
activation inhibits cell proliferation, which supports the
conclusion that IL-1 mediates antitumor activity. Consistent with
these findings, we show here a negative correlation between
the expression of TLR3 and Ki67 as well as with cyclin D1.
Moreover, we demonstrate a significant negative
relationship between the expression of TLR3 pathway-signaling
proteins and that of Ki67 or cyclin D1.
Angiogenesis plays an important role in the malignant
transformation, growth, and metastasis of parenchymal
tumors. Tumor angiogenesis is regulated by angiogenic
factors generated and secreted by tumor cells. HCC is a
highly vascularized tumor that requires the formation of
numerous blood vessels to receive a sufficient blood
supply required for growth and proliferation. Thus,
angiogenesis is a crucial process in the development of HCC.
MMP-2 is associated with tumor malignancy . EPCs
are considered the primary resource for postnatal
vasculogenesis and are detected in peripheral blood, cord
blood, spleen, vessel walls, and heart and skeletal
muscles . CD34+ EPCs express a range of diverse surface
markers and contain progenitor cells that are capable of
differentiating into endothelial and osteogenic lineages
under the appropriate conditions .
Preclinical studies show that despite their heterogeneity,
human CD34+ EPCs stimulate neovascularization in
ischemic myocardium by increasing capillary density and
improving function in models of acute and chronic
myocardial ischemia . Yang et al.  found that treatment
of EPCs with the TLR3 agonist poly (I:C) upregulates the
expression of the cytokines IL-1, IL-6, IL-8, TNF-,
IFN, and IFN-, indicating that EPCs express functional
TLR3. Poly (I:C) impairs cell proliferation by inducing cell
cycle progress inhibition and cell apoptosis via TLR3 in
EPCs. For example, Guo et al.  found that rat aortic
ring outgrowth and endothelial cell tube formation are
suppressed after treatment with dsRNA and that dsRNA
triggers apoptosis of the MHCC97H, SMMC-7721, and
HUVEC cell lines and inhibits cell migration. Our findings
are consistent with studies showing that MVD and EPCs
correlate negatively with the expression of TLR3
signalingpathway proteins in HCC. Moreover, MMP-2 expression
correlated inversely with the expression of TLR3
signalingpathway proteins in HCC. These findings suggest that
TLR3 activation not only affects the tumor
microenvironment by suppressing angiogenesis but also directly inhibits
tumor cell invasion.
The activation of TLR3 was associated with malignant
progression and prognosis of patients. HCC is the
second most frequent cause of cancer death. Because of the
development of improved treatment strategies and
monitoring methods, the 5-year overall survival rate of HCC
increased by 11.8% in the United States  and by
17.0% in Germany . Our data for 1-, 3-, and 5-year
survival rates were 56.6%, 27.6%, and 10.5%, respectively,
and are still lower compared with those of more
developed countries. Here we show that prognosis was better
for patients with HCC tissues positive for TLR3 and its
downstream signaling molecules TRIF, IRF3, and NF-B,
confirming that activation of TLR3 is an important
factor in improving overall survival rates of patients with
HCC. This may be attributed to the activation of the
TLR3 pathway, which inhibited HCC cell proliferation,
angiogenesis, and induced apoptosis.
Our study shows that the expression of TLR3 was
suppressed in more than 50% of HCC tissues of our patient
population. Decreased TLR3 expression was related to
tumor cell proliferation, upregulated angiogenesis, and
inhibition of apoptosis, which may be associated with
tumor progression and poor prognosis of patients with
HCC. Therefore, the expression of TLR3 may serve as a
valuable marker to estimate HCC progression, and
TLR3 function may play an important role in apoptosis
by inhibiting the growth and invasion of HCC cells, the
production of EPCs, and angiogenesis. However, further
studies are required to confirm these findings and to
provide a better understanding of the mechanisms of
TLR3 signaling in the development of HCC.
All authors declare that they have no competing interests.
LC conceived and designed the research; MMY collected and organized the
patients information and YYX drafted the manuscript. MMY, YYX, XYL, JQ
and YS analyzed the data; MMY interpreted the results of experiments; MMY
and YYX prepared figures; LC and YYX edited and revised the manuscript;
YYX approved the final version of the manuscript; MMY, YYX, XYL, JQ and YS
performed the experiments. All authors read and approved the final
Ming-Ming Yuan and Yu-Yin Xu are joint first authors.
This study was supported by the foundation of the production-study-research
prospective joint research programs of Jiangsu Province, China (BY 201304206), a
project funded by the Priority Academic Program Development of Jiangsu Higher
Education Institutions, and the Science Foundation of Nantong City, Jiangsu
Province, China (No. BK2014001).
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