miR-589-5p inhibits MAP3K8 and suppresses CD90 + cancer stem cells in hepatocellular carcinoma
Zhang et al. Journal of Experimental & Clinical Cancer Research
miR-589-5p inhibits MAP3K8 and + suppresses CD90 cancer stem cells in hepatocellular carcinoma
Xi Zhang 0
Peng Jiang 0
Ling Shuai 0
Kai Chen 0
Zhonghu Li 0
Yujun Zhang 0
Yan Jiang 0
Xiaowu Li 0
0 Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University , Chongqing 400037 , China
Background: Cancer stem cells (CSCs) are important in the tumorigenesis and progression of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play crucial roles regulating CD133+ and EpCAM+ CSCs in HCC, although it is unclear whether miRNAs regulate CD90+ CSCs in HCC. Methods: The miRNA profiles of CD90+ and CD90- HCC cells were analyzed using a miRNA microarray and quantitative real-time PCR (qRT-PCR). CSC characteristics were examined by qRT-PCR and Western blot of pluripotency-associated genes, clone and sphere formation assay, transwell migration assay, and nude mice tumorigenicity assay. miR-589-5p mimic transfection was used to overexpress miR-589-5p in vitro. The CD90 and miR-589-5p expressions of HCC samples were detected by immunohistochemistry and qRT-PCR, respectively. Results: miR-589-5p and miR-33b-5p were down-regulated in CD90+ cells. Overexpression of miR-589-5p suppressed CD90+ CSC characteristics such as Oct4, Sox2 and Nanog expression, a high likelihood of forming cell spheres, high invasiveness and high tumorigenicity. Luciferase reporter assays demonstrated that miR-589-5p directly binds to the 3ˈ-untranslated region of mitogen-activated protein kinase kinase kinase 8 (MAP3K8) mRNA, and exogenous miR-589-5p down-regulated MAP3K8 expression. In addition, siRNA inhibition of MAP3K8 also suppressed CD90+ CSC characteristics, even in the absence of miR-589-5p overexpression. In HCC tissues, miR-5895p expression was inversely correlated with CD90 expression, and high CD90 expression and low miR-589-5p expression were positively correlated with vascular invasion and recurrence and significantly decreased disease-free and overall survival by clinical analysis. Conclusion: In HCC, miR-589-5p down-regulates the stemness characteristics of CD90+ CSCs in part by silencing MAP3K8. CD90 and miR-589-5p expression predict HCC outcomes and might be novel molecular targets for HCC treatment.
Cellular heterogeneity; Tumorigenicity; CD90; microRNA; MAP3K8; Prognosis
Hepatocellular carcinoma (HCC), which includes over
90 % of liver cancers, is the leading cause of cancer
mortality worldwide . Because HCC is resistant to
chemoradiotherapy and has a high recurrence and metastatic rate
after surgery, HCC patients have a very low five-year
survival rate [2, 3]. In recent years, accumulating evidence has
suggested that in HCC, a population of cells with stem
celllike features, known as cancer stem cells (CSCs) or
tumorinitiating cells (TICs), is essential for the recurrence,
metastasis and resistance to chemoradiotherapy seen in HCC .
Previous studies have shown that CD90, EpCAM, CD133,
CD24, OV-6 and CD44 can be used as CSC markers in
HCC, and cells expressing these markers possessed CSC
characteristics such as self-renewal, tumor generation and
aggressive growth [5–10]. However, the optimal marker for
identifying CSC populations in HCC remains disputed.
MicroRNAs (miRNAs) are a large group of small,
noncoding RNAs that are important regulators of
posttranscriptional gene expression. miRNAs bind to the
3'untranslated region (UTR) of the target mRNA by base
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pairing and suppress protein expression through
translational repression or mRNA degradation [11–13]. miRNAs
play key roles in a variety of physiological and pathological
processes, including embryo development, cellular
differentiation, stem cell self-renewal and tumor
progression . In human solid tumors, many miRNAs have
been found to participate in CSC maintenance [15–17].
To date, in HCC, miR-130b has been shown to
promote CD133+ CSC tumorigenicity and self-renewal ,
whereas miR-181 inhibition reduces the number of
EpCAM+ CSCs and tumor-initiating ability . However,
it remains unclear which miRNA regulates the stemness
of CD90+ HCC CSCs.
Mitogen-activated protein kinase kinase kinase 8
(MAP3K8), also known as tumor progression locus 2
(TPL2) or cancer Osaka thyroid (COT), is a member of
the serine/threonine protein kinase family. MAP3K8
activation is critically involved in inflammation and has
variable effects on tumors [20, 21]. In lung and intestinal
cancer, MAP3K8 is a tumor suppressor gene [22–24].
However, MAP3K8 is predominantly considered a
tumorpromoting oncogene in several tumor types, such as
breast cancer, colon cancer, renal cell carcinoma,
endometrial cancer, gastric cancer, nasopharyngeal carcinoma,
thymoma and lymphoma . MAP3K8 is up-regulated
in multiple tumor types and is closely related to
tumorigenesis and/or cancer progression [25–27]. However, the
role of MAP3K8 in HCC initiation and progression
remains unknown. In this study, we found that miR-589-5p
was down-regulated in CD90+ CSCs and examined the
effects of miR-589-5p expression and its target protein
MAP3K8 on CD90+ CSCs and the clinical outcomes of
Patients and tissue specimens
Tumor specimens were obtained from 2006 to 2008.
All of the patients underwent surgical resection of
primary, pathologically confirmed HCC at the Institute of
Hepatobiliary Surgery, Southwest Hospital, Third
Military Medical University. The tumor stage was based on
the Edmondson grade . Sixty-six formalin-fixed,
paraffin-embedded tumor specimens were used for IHC,
and forty frozen tumor specimens were used for RNA
extraction. All of the patients were followed for 5 years.
Cell lines and culture
For routine culture, MHCC97H, MHCC97L and HepG2
HCC cell lines purchased from the Shanghai Cell
Collection (Shanghai, China) were maintained in high-glucose
DMEM (Gibco, 8113035) containing 10 % FBS in a 5 %
CO2 incubator at 37 °C, whereas the SMMC-7721 cell
line was maintained in RPMI medium 1640 (Gibco,
8112340). Both media contained 10 % fetal bovine serum
(FBS, Gibco, 10099141), penicillin (500 U/ml) and
streptomycin (500 μg/ml).
Cells isolated from HCC cell lines, spheres and tumor
xenografts were labeled with anti-human antibodies at
4 °C for 20 minutes according to the manufacturer’s
instructions (antibodies are described in Additional file 1:
Table S1). For unconjugated primary antibodies, the cells
were subsequently incubated with FITC- or PE-conjugated
secondary antibodies at 4 °C for 20 minutes. The labeled
cells were detected using a FACSAria II system (BD
Biosciences). The FcR Blocking Reagent (Miltenyi, 130-059-901)
was used to increase antibody specificity.
The MHCC97H and MHCC97L HCC cells were
dissociated into single cells and labeled with anti-CD90 antibodies
at 4 °C for 20 minutes. Subsequently, the cells were
magnetically labeled with anti-mouse IgG1 MicroBeads
(Miltenyi) according to the manufacturer’s instructions.
In brief, the cell suspension was loaded onto a MACS
column, which was placed in the magnetic field of a
MACS Separator. The unlabeled cells (CD90- cells) passed
through the column, and the magnetically labeled cells
(CD90+ cells) were retained. After removing the column
from the magnetic field, the magnetically retained CD90+
cells were eluted.
The miRNA expression profiles of CD90+ and
CD90cells isolated from the HCC cell lines MHCC97H and
MHCC97L were compared using a 5th generation
miRCURY LNA™ microRNA Array (v.14.0) (Exiqon). RNA
extraction, RNA quality control, RNA labeling, array
hybridization and data analysis were performed at the
Kangchen Biotechnology Corporation (Shanghai, China).
Then, the scanned images were imported using GenePix
Pro 6.0 software (Axon).
Quantitative real–time PCR
For qRT-PCR of mRNA targets, total RNA was extracted
from cancer cells using RNAiso Plus (TaKaRa, 09108B).
cDNA synthesis was performed according to the
manufacturer’s instructions (TaKaRa, DRR047A), and qRT-PCR
was performed with SYBR Premix Ex Taq II (TaKaRa,
DRR081A) using a LightCycler system (Roche). The PCR
reaction conditions for all of the assays were 94 °C for
30 seconds, followed by 40 cycles of amplification (94 °C
for 5 seconds, 60 °C for 30 seconds and 72 °C for
30 seconds). GAPDH mRNA was used to normalize RNA
inputs. The qRT-PCR primers are listed in Additional file
1: Table S2.
For qRT-PCR of miRNAs, small RNAs were extracted
from cancer cells or tumor tissues using RNAiso for small
RNAs (TaKaRa, D340A). miRNAs were converted to
cDNA using a cDNA synthesis kit (TaKaRa, DRR047A),
and qRT-PCR was performed with SYBR Premix Ex Taq II
(TaKaRa, DRR081A) using a LightCycler system (Roche).
The PCR reaction conditions for all of the assays were
95 °C for 20 seconds, followed by 40 cycles of
amplification (95 °C for 10 seconds, 60 °C for 20 seconds and
70 °C for 5 seconds). U6 was used to normalize the RNA
inputs. All of the primers were from the Bulge-Loop™
miRNA qRT-PCR primer set (RiboBio, MQP-0102, China).
Luciferase gene reporter
The in silico predictions of the potential binding regions
in MAP3K8 mRNA for miRNAs were performed using
TargetScan (http://www.targetscan.org). Wild-type and
mutant MAP3K8 3′-UTR luciferase plasmids were
generated using the pmiR-RB-REPORT™ vector (RiboBio,
China). The full-length wild-type MAP3K8 3′-UTR is
1463 bp. The wild type sense sequence was 5′-GATA
TGCACC GGTCTCAAGG TTCTCATTTC-3′, and the
mutant sense sequence was 5′- GATATGCACC GGTC
TCAAGG AAGACATTTC-3′. Exponentially growing
293 T cells were transfected with wild-type or mutant
vectors using Lipofectamine® 2000 reagent (Invitrogen,
11668027, USA) according to the manufacturer's
instructions. The miR-589-5p mimics or non-target control
(RiboBio, NC#22, China) were co-transfected with the
vectors for 48 hours, and then luciferase activity was
Clone and sphere formation assay
For the clone formation assay, 500 cells were sorted by
MACS and seeded per well in 6-well plates. After 10 days
of culture, the clones were fixed using methanol and
dyed with hematoxylin, and the number of clones (>50
cells) was assessed microscopically.
For the sphere formation assay, 1000 cells were sorted
by MACS and seeded per well in ultra-low attachment
6-well plates (Costar, 3741). The cells were cultured in
DMEM/F12 media (Sigma) containing B27 supplement
(Gibco, 17504-044), antibiotics, 20 ng/ml EGF (Peprotech,
AF-100-15) and 20 ng/ml bFGF (Peprotech, 100-18B).
Fresh medium was added every 3-5 days. After 2 weeks of
culture, spheres with a diameter >75 μm were counted.
For FACS analysis, the spheres were collected and
dissociated into single cells using trypsin.
Cell invasion and migration assays
The invasion and migration assays were performed in
24-well Millicell hanging inserts (Millipore) with or
without a Matrigel layer (BD Biosciences) according to the
manufacturer's instructions. Briefly, 1 × 105 cells were
seeded into the top chamber, and DMEM with 10 % FBS
was added to the bottom chamber as a chemoattractant.
After a 48 hour incubation at 37 °C, the numbers of
cells that invaded the Matrigel (invasion) or membrane
(migration) were counted in 10 fields using a 40×
Tumor formation in nude mice
To assess tumor formation in nude mice, CD90+ and
CD90- cells were sorted and injected (amounts ranging
from 1 × 103 to 5 × 105) subcutaneously into different
sides of 6-week-old male nude mice for controlled
visualization and comparison. The mice were maintained
under standard conditions and were examined for tumor
formation for 12 weeks. After the tumors formed, the
mice were sacrificed, and xenografts were harvested for
IHC and primary culture. The fresh tumor xenografts
from the nude mice were cut into small pieces and
plated in a cell culture flask, and tumor cells migrated
out from these pieces. DMEM containing 15 % FBS was
used to initially establish the primary cultures, and DMEM
containing 10 % FBS was used for subsequent maintenance.
To assess the effect of miR-589-5p on HCC
tumorigenesis, 3 days after 1 × 105 CD90+ MHCC97H cells were
subcutaneously injected into nude mice, micrON™
agomir589-5p (25 nmol, 50 μl) or control RNAs (RiboBio, China)
were injected into the same site every 3 days within the
next 2 weeks. The mice were maintained under standard
conditions and were examined for tumor formation for
miR-589-5p mimic/antagomir transfection
The miR-Ribo™ miR-589-5p mimic/antagomir and
negative control miRs are commercially available (RiboBio,
China), and the experiments were performed according
to the manufacturer's instructions. In brief, 5 × 105 cells
were seeded per well in 6-well plates. The miR-589-5p
mimics/antagomir (or control miRs) and Lipofectamine®
2000 were diluted in Opti-MEM® (Gibco, 31985-062,
USA) separately, were mixed gently and were added to
the culture plates. The final concentration of mimic was
50 nM, and the final concentration of antagomir was
100 nM. After a 24 hour incubation at 37 °C, the cells
were used for additional experiments.
The siRNAs and negative control RNAs were
synthesized and purified by Sangon Biotech (Shanghai, China).
Synthesized siRNAs were transfected into sorted CD90+
MHCC97H and MHCC97L cells with Lipofectamine®
2000 according to the manufacturer’s protocol. The
siRNAs for MAP3K8 were sense: 5′-GCGCCTTTGG
AAAGGTATATT-3′ and antisense: 5′-TATACCTTTC
CAAAGGCGCTT-3′. The negative control siRNAs
were sense: 5′-TTCTCCGAACGTGTCACGTTT-3′ and
antisense: 5′-ACGTGACACGTTCGGAGATT-3′. The
final concentration of siRNAs was 25 nM. After a
24 hour incubation at 37 °C, the cells were used for
Western blot analysis
Prepared cells were lysed in radioimmunoprecipitation
assay (RIPA) buffer supplemented with a protease inhibitor
(Roche, Branford, CT). Total proteins (30 μg/well) were
separated by electrophoresis on 12 % sodium dodecyl
sulfate-polyacrylamide gels. Subsequently, protein
samples were transferred onto nitrocellulose membranes
(Pierce, Thermo Fisher Scientific, Waltham, MA) and
incubated with corresponding primary antibodies
(antibodies are described in Additional file 1: Table S1). The
membranes were incubated with horseradish
peroxidaseconjugated secondary antibodies and developed using
SuperSignal™ chemiluminescence reagent (Pierce, Thermo
Fisher Scientific) according to the manufacturer’s
instruction. Protein expression levels were normalized against
HCC, paired non-tumor tissues and tumor xenografts
from nude mice were fixed with formalin and embedded
in paraffin. Then, samples were sectioned (5 μm) and
attached to poly-L-lysine coated slides. The slides were
deparaffinized, treated with 3 % H2O2 at 37 °C for 1 hour
to block endogenous peroxidase activity, and heated in
10 mM citrate buffer at 120 °C for 2 min and 10 sec for
antigen retrieval. After incubation with the primary
antibody (antibodies are described in Additional file 1: Table S1)
at 4 °C overnight, the samples were incubated with a
secondary peroxidase-conjugated antibody for 60 min
at 37 °C and then developed with DAB (Dako, 00080066).
Hematoxylin was used as a counterstain. The staining
intensity of CD90 was described as low or high according to
the percentage of positively stained cells. Because CD90 is
expressed at a basal level in most liver cells, only cells
expressing a significantly higher level of CD90 were
considered positive .
The data were analyzed using SPSS 18 software (SPSS
Corp., Chicago, IL, USA) and are presented as the mean
values ± standard error of mean (SEM) or the median
with the range. When two groups were compared,
Student’s t test or the Mann-Whitney U test was used.
Chi-square or Fisher’s exact methods were used for
clinical statistical analyses. The Kaplan–Meier and Cox
regression methods were used for survival analyses. p < 0.05
was considered statistically significant and is indicated by
*. p < 0.01 was considered highly statistically significant
and is indicated by **. Experiments were performed in
three independent repeats in triplicate.
miR-589-5p is down-regulated in CD90+ HCC cells
In HCC, several molecules, including CD90, CD133, CD24,
OV-6, EpCAM and CD44, have been used as potential
CSC markers for primary tumors or cell lines [5–10]. An
ideal CSC marker should be expressed in all primary
tumors and cell lines and should be stably expressed in
a small cell population. Using flow cytometric analysis,
we observed that in all of the tested HCC cell lines
(MHCC97H, MHCC97L, HepG2 and SMMC-7721),
CD90 was consistently expressed in a small population
(0.9 % to 3.1 %, Additional file 2: Figure S1A), even
under cell sphere formation conditions, which increased
in all four cell lines up to 11.8 % (Additional file 2:
Figure S1B). However, the expressions of other
markers, such as EpCAM and CD133, were verified in
different cell lines (Additional file 2: Figure S1A). We
further confirmed that CD90+ HCC cells exhibited CSC
characteristics, such as pluripotency-associated gene
(Oct4, Sox2 and Nanog) expression, a greater likelihood to
form cell spheres, a high invasiveness and high
tumorigenicity (Additional file 2: Figure S2). Thus, CD90 might be an
ideal marker to identify the CSC population in HCC.
It has been reported that miRNAs play very important
roles maintaining the stemness of CSCs . Therefore,
we used a miRNA microarray to examine the miRNA
expression profiles in MHCC97H and MHCC97L CD90+
and CD90- cells. As shown in Additional file 1: Table S3,
eleven up-regulated and ten down-regulated miRNAs
were found in MHCC97H CD90+ cells compared with
MHCC97H CD90- cells, and thirteen up-regulated and
twenty-two down-regulated miRNAs were found in the
MHCC97L CD90+ cells compared with the MHCC97L
CD90- cells. Combining the two data sets, a total of eleven
miRNAs with altered expression, five of which were
upregulated and six of which were down-regulated, were
observed in both of the CD90+ HCC cell lines (Fig. 1a).
To confirm the miRNA microarray results, we used
quantitative real-time PCR (qRT-PCR) to demonstrate
that these miRNAs were expressed differently in CD90+
and CD90- cells from the MHCC97H and MHCC97L
cell lines. We found that the expression of miR-589-5p
and miR-33b-5p were down-regulated, which were the
only miRNAs that agreed with the miRNA microarray
results (Fig. 1b). Because miR-589-5p showed a more
significant fold change than miR-33b-5p, we focused on
miR-589-5p in further studies.
miR-589-5p helps regulate the stemness of HCC CSCs
Next, we determined whether miR-589-5p participates in
regulating the stemness of HCC CSCs. The sorted CD90+
Fig. 1 miR-589-5p is down-regulated in CD90+ HCC cells. (a) The results of miRNA microarrays were shown as hierarchical clustering of miRNAs
that are differentially expressed in CD90+ HCC cells. The color scale illustrates the relative expression level of a miRNA: red color represents a high
expression level; green color represents a low expression level. (b) Expression of miR-589-5p and miR-33b-5p in both CD90+ HCC cell lines was
determined using qRT-PCR. All data are representative of three independent experiments and are shown as mean ± SEM (n = 3)
MHCC97H and MHCC97L cells were transfected with
miR-589-5p mimics for 24 hours to overexpress
miR-5895p (Fig. 2a), and cells transfected with miR-589-5p mimics
exhibited lower levels of Oct4, Sox2 and Nanog compared
to the negative control RNA (Fig. 2b). Moreover, the ability
of the cells to form cell spheres and the migration of
MHCC97H and MHCC97L were greatly reduced
compared to the controls (Fig. 2c, 2d). However, overexpression
of miR-589-5p had no impact on the regulation of stemness
in CD90- HCC cells (Additional file 2: Figure S3A-3D).
To determine the impact of miR-589-5p on the
tumorigenic capacity of CD90+ cells, sorted CD90+ cells were
subcutaneously injected into nude mice. As shown in
Fig. 2e, after 1 × 105 CD90+ cells were injected into the
mice, micrON™ agomir-589-5p or control RNAs were
subsequently injected to overexpress miR-589-5p or as a
control. The mice injected with control RNAs initiated tumors
(3/3 mice) within 12 weeks, whereas no tumor formation
was observed in mice injected with the miR-589-5p mimic,
suggesting that miR-589-5p suppresses the CD90+ CSC
characteristics both in vitro and in vivo.
miR-589-5p directly targets MAP3K8
To investigate how miR-589-5p affects the stemness of
CSCs, we used in silico predictions to identify
miR-5895p targets. We identified MAP3K8 as a potential
miR589-5p target; MAP3K8 is a known tumor-promoting
gene in various human tumors. The 3 - UTR of the
MAP3K8 mRNA has a binding site for miR-589-5p,
and this binding site is conserved among different species,
including humans, chimps and mice (Fig. 3a). Thus we
examined the expression of MAP3K8 in CD90+ and
CD90cells sorted from MHCC97H and MHCC97L cells.
Figure 3b showed that the level of MAP3K8 expression
was higher in CD90+ cells than in CD90- cells at both the
mRNA and protein levels.
To determine whether MAP3K8 is a direct target of
miR-589-5p, full-length human MAP3K8 mRNA 3′-UTR
luciferase reporter vectors (wild-type and mutant
plasmids, as described in the Materials and Methods, Fig. 3a)
were co-transfected into 293 T cells with miR-589-5p or
miR-control. Cells co-transfected with miR-589-5p and the
wild-type MAP3K8 3′-UTR exhibited a 45.1 % reduction
Fig. 2 miR-589-5p participates in the regulation of stemness in HCC CSCs. (a) Expression of miR-589-5p increased in CD90+ MHCC97H and MHCC97L
cells after transfection with miR-589-5p mimics. (b) Expression of the pluripotency-associated genes Oct4, Sox2 and Nanog in CD90+ MHCC97H and
MHCC97L cells was measured by qRT-PCR (upper two panels) and Western blot (lower panel) after transfection of miR-control or miR-589-5p mimics.
(c) Sphere formation and clone formation by CD90+ MHCC97H and MHCC97L cells transfected with miR-control or miR-589-5p mimics. (d) Migration
and invasion of CD90+ MHCC97H and MHCC97L cells transfected with miR-control or miR-589-5p mimics. (e) Tumor formation by CD90+ MHCC97H
and MHCC97L cells in nude mice. After the tumor cells were injected, the miR-589-5p agomirs or control RNAs were injected subcutaneously every
three days within the next two weeks. All data are representative of three independent experiments and are shown as mean ± SEM (n = 3)
in luciferase activity compared to the miR-control. In
contrast, the mutant MAP3K8 3′-UTR showed a 93.8 %
restoration of luciferase (Fig. 3c), suggesting that miR-589-5p
directly binds the 3'-UTR of the MAP3K8 mRNA.
To investigate the influence of miR-589-5p on MAP3K8
expression, we transfected miR-589-5p mimics into sorted
CD90+ and CD90- MHCC97H and MHCC97L cells. The
expression of MAP3K8 was decreased at both the mRNA
and protein levels in CD90+ and CD90- cells (Fig. 3d and
Additional file 2: Figure S3E), indicating that miR-589-5p
directly targets and decreases the expression of MAP3K8
by binding its 3′-UTR.
miR-589-5p inhibits the stemness of HCC CSCs through
To examine the significance of MAP3K8 to HCC
stemness, an siRNA targeting MAP3K8 was transfected into
CD90+ cells sorted from MHCC97H and MHCC97L.
qRT-PCR and Western blot analyses showed that MAP3K8
expression was markedly repressed after siRNA transfection
at both the mRNA and protein levels, respectively (Fig. 4a).
The pluripotency-associated genes were also repressed
in the siRNA group compared with the control group
To confirm the role of MAP3K8 in the tumorigenic
and metastatic potential of HCC cells, sphere and clone
formation and cell migration were evaluated in sorted
CD90+ MHCC97H and MHCC97L cells after MAP3K8
siRNA transfection. Compared to the negative control,
down-regulation of MAP3K8 by siRNA significantly
reduced the ability of cells to form spheres and clones
and also suppressed cell migration and invasion in
transwell assays (Fig. 4c, 4d). These data indicate that
siRNA inhibition of MAP3K8 also suppressed the
stemness of CD90+ CSCs even in the absence of
miR589-5p overexpression, and miR-589-5p likely functions
through MAP3K8 to suppress CSC stemness and HCC
Fig. 3 miR-589-5p directly targets MAP3K8. (a) The potential binding sites for miR-589-5p in the MAP3K8 mRNA 3ˈ-UTR were determined with in
silico predictions. The underlined bases were mutated to AAGA in the mutant plasmid. (b) The expression of MAP3K8 in CD90+ and CD90- cells
sorted from MHCC97H and MHCC97L cells measured by qRT-PCR and Western blot. (c) Luciferase activities of MAP3K8 wild-type (Wild) and mutant
reporter plasmids in 293 T cells co-transfected with miR-control or miR-589-5p. (d) CD90+ MHCC97H and MHCC97L cells were transfected with
miR-589-5p mimics for 24 hours, and the level of MAP3K8 expression was measured by qRT-PCR and Western blot. All data are representative
of three independent experiments and are shown as mean ± SEM (n = 3)
The expression of CD90 and miR-589-5p is associated
with a poor clinical prognosis in human HCC
To evaluate the relationship between CD90 expression
and clinical prognosis in human HCC, we detected the
expression of CD90 by immunohistochemistry (IHC) in
sixty-six tissue samples from HCC patients. As shown in
Fig. 5a, CD90 was expressed in all of the non-tumor
tissues at a very low basal level. In contrast, only a fraction
of the cells in the tumor tissues showed significant
positive staining for CD90, ranging from 1.5 to 15.1 %.
Samples with over 5 % CD90 staining were considered CD90
high expressers (CD90High), and the rest were classified
as CD90 low expressers (CD90Low). We found that 57.6 %
(38/66) of the tumor tissues exhibited low CD90
expression, and 42.4 % (28/66) of the tumor tissues displayed
high CD90 expression. Moreover, CD90 expression in the
blood vessel thrombi of tumors was higher than in their
paired primary tumors. Interestingly, CD90 expression
was positively correlated with vascular invasion (p < 0.05)
and recurrence (p < 0.01) (Table 1). Kaplan-Meier survival
analysis showed that high expression of CD90 in HCC
was associated with significantly decreased disease-free
and overall survival (Fig. 5b). Cox regression showed that
high expression of CD90 and Edmondson Grade III/IV
were independent risk factors of disease-free and overall
survival (Table 2). These data suggest that high CD90
expression is associated with a poor prognosis for HCC
Next, we investigated whether there is a relationship
between miR-589-5p and CD90 expression in HCC
tumor tissues. Of the samples used for IHC staining,
forty cases had paired frozen tissues from which RNAs
were extracted and assessed for miR-589-5p expression,
and we observed that miR-589-5p expression was
inversely correlated with CD90 expression (Fig. 5c).
We analyzed the relationship between miR-589-5p
expression level and the clinical outcomes of HCC patients
and found that those with low miR-589-5p expression
had a high risk of vascular invasion (p < 0.05) and
recurrence (p < 0.01) (Table 3). Kaplan-Meier survival analysis
showed that low miR-589-5p expression was correlated with
low disease-free and overall survival (Fig. 5d). Cox regression
showed that low miR-589-5p expression was independent
risk factor of disease-free and overall survival (Table 4).
These data suggest that miR-589-5p down-regulation in
HCC is associated with a poor clinical prognosis.
Because CD90 and miR-589-5p are independent
predictors of HCC outcomes, we investigated whether the
Fig. 4 miR-589-5p inhibits the stemness of HCC CSCs through MAP3K8. (a) CD90+ MHCC97H and MHCC97L cells were transfected with MAP3K8
siRNA, and the mRNA and protein levels of MAP3K8 both decreased. (b) Expression of the pluripotency-associated genes Oct4, Sox2 and Nanog
in CD90+ MHCC97H and MHCC97L cells was measured by qRT-PCR (upper two panels) and Western blot (lower panel) after transfection of
MAP3K8 siRNA or negative control RNA. (c) Sphere formation and clone formation by CD90+ MHCC97H and MHCC97L cells transfected with
MAP3K8 siRNA or negative control RNA. (d) Migration and invasion of CD90+ MHCC97H and MHCC97L cells transfected with MAP3K8 siRNA or
negative control RNA. All data are representative of three independent experiments and are shown as mean ± SEM (n = 3)
combination of CD90 and miR-589-5p is a better
predictor of an HCC prognosis. We found that HCC patients
with CD90HighmiR-589-5pLow expression had a shorter
disease-free and overall survival, larger tumor size, and
higher risks of vascular invasion and recurrence (Fig. 5e,
Additional file 1: Table S4). Therefore, the combination of
CD90 and miR-589-5p may be a better predictor of an
According to the cancer stem cell theory, CSCs are only
a small subset of cells within a tumor, and this
population tends to be stable in various environment. An ideal
CSC marker should distinguish this subset and be
expressed in all primary tumors and cell lines. To date,
CD90, CD133 and EpCAM have been used as
distinguishing phenotypic markers for enriching HCC CSCs
from both primary tumors and cell lines [7–9]. In this
study, these potential CSC markers were examined by
flow cytometry, and the size of the CD133+ and EpCAM+
populations varied greatly among the different HCC cell
lines. In contrast, CD90 was much more consistently
expressed in all of the tested HCC cell lines, ranging from
0.9 % to 3.1 %. In addition, we found that in the cell
spheres, the proportion of CD90+ cells increased in all
cell lines but only up to 11.8 %. Moreover, in every
HCC tumor sample examined, only a fraction of the
tumor cells showed significant positive staining for CD90,
ranging from 1.5-15.1 %. Therefore, CD90 is an ideal CSC
marker that is stably expressed in a small cell population.
In HCC, several miRNAs have been shown to regulate
CSCs and to play cancer promoting or suppressing roles.
It has been reported that exogenous miR-181 increased
EpCAM+ HCC cell quantity and tumor-initiating ability
. In CD133+ HCC cells, miR-130b was overexpressed
and enhanced chemoresistance, tumorigenicity and
self-renewal , whereas miR-150 was down-regulated
and significantly inhibited tumor sphere formation and
cell growth . In this study, we found that
miR-5895p expression was down-regulated in CD90+ HCC cells
Fig. 5 The expression of CD90 and miR-589-5p is associated with a poor clinical prognosis in human HCC. a Expression of CD90 in HCC tissues,
paired non-tumor tissues and tumor thrombus specimens. b Disease-free and overall survival of HCC patients with respect to CD90 expression
levels was analyzed by the Kaplan-Meier method. c Expression of miR-589-5p and its relationship with CD90 in forty HCC tissue specimens.
d Disease-free and overall survival of HCC patients with respect to miR-589-5p expression levels was analyzed by the Kaplan-Meier method.
e Disease-free and overall survival of HCC patients with respect to the combination of CD90 and miR-589-5p expression was analyzed by
the Kaplan-Meier method. All assays were performed in triplicate (n = 3)
*Value expressed in the midian with the range in parentheses
Table 1 The relationship between CD90 expression and clinical
parameters in human HCC (n = 66)
by comparing the miRNA expression profiles of CD90+
and CD90- cells, and this result was confirmed by
qRTPCR. Overexpression of miR-589-5p suppressed the
CSC characteristics of CD90+ HCC cells such as stem
cell-associated gene expression (Oct4, Sox2 and Nanog),
cell sphere formation, invasiveness and tumorigenicity
both in vitro and in vivo. However, overexpression of
miR-589-5p had no impact on the regulation of stemness
in CD90- HCC cells, because CD90- HCC cells do not
possess CSC characteristics . Moreover, transfection of
miR-589-5p antagomir in the whole cell population
suppressed the expression of miR-589-5p, but failed to
increase CD90+ population (Additional file 2: Figure S4).
This might due to the low abundance of miR-589-5p in
HCC cell lines, antagonizing miR-589-5p did not
significantly inhibit miR-589-5p functions. Hence, these data
suggest that miR-589-5p is down-regulated in CD90+
HCC cells and suppresses stem cell characteristics.
MAP3K8 has been reported to be overexpressed in
various human tumors and to promote cell
transformation, proliferation, migration, and invasion by activating
extracellular signal–regulated kinase (ERK), Rac1, and
Vascular Invasion (positive)
Tumer Size(≥5 cm)
Tumer Size(≥5 cm)
focal adhesion kinase (FAK) [31, 32]. However, few
studies have focused on the role of MAP3K8 in HCC
development. One recent study determined that MAP3K8
knockout mice exhibited a significantly lower incidence
of liver tumors compared with wild-type mice in
diethylnitrosamine-induced tumor formation model
. In this study, the in silico analysis predicted that
MAP3K8 was a potential downstream target of
miR589-5p. Luciferase reporter assays showed that
miR-5895p directly bound to the 3 -UTR of MAP3K8 mRNA,
Table 3 The relationship between miR-589-5p expression and
clinic parameters in human HCC (n = 40)
miR-589-5pLow miR-589-5pHigh p Value
Edmondson Grade I/II
Negative 4 (21.1)
Negative 12 (63.2 %)
*Value expressed in the midian with the range in parentheses
and exogenous miR-589-5p decreased MAP3K8
expression at both the mRNA and protein levels. Moreover,
inhibition of MAP3K8 by siRNA significantly reduced the
expression of Oct4, Sox2 and Nanog and suppressed
self-renewal, migration and invasion. The above
findings indicate the importance of MAP3K8 in human
HCC tumorigenesis and progression by promoting
CD90+ CSC stemness characteristics. Overall,
miR-5895p appears to decrease the population of CD90+ cells
and impair stem cell characteristics partly by silencing
The status of CSCs might be a key determinant of
cancer behavior [34–37]. Our clinical study indicated that the
expression levels of CD90 and miR-589-5p were
significantly inversely correlated in the HCC clinical specimens,
and CD90+ HCC samples or samples with decreased
miR-589-5p expression showed more vascular invasion
and reduced disease-free and overall survival. Moreover,
the combination of CD90High and miR-589-5pLow
predicted even poorer prognosis. These results might be
explained by the high invasive and metastatic capacities
of CD90+ HCC and the alteration of stemness by
miRNAs. Additionally, our in vivo study demonstrated that
CD90+ HCC cells initiate tumor xenografts in
immunodeficient mice, whereas CD90- cells and
miR-589-5ptransfected CD90+ cells do not. One mouse injected
with 1 × 105 CD90- cells grew a small tumor by the 11th
week, but this tumor xenograft contained CD90+ cells
(Additional file 2: Figure S5), suggesting that CD90+
cells are required to re-establish the cellular hierarchy
and to generate tumors in HCC. Thus, CD90
overexpression and miR-589-5p down-regulation indicate more
aggressive HCC and poor clinical outcomes.
Vascular Invasion (positive)
Tumer Size(≥5 cm)
Edmondson Grade (III/IV)
Tumer Size(≥5 cm)
Edmondson Grade (III/IV)
In summary, the binding of miR-589-5p to the
MAP3K8 3 -UTR inhibits MAP3K8 expression and
suppresses CD90+ CSC characteristics, and the
expression status of CD90 and miR-589-5p determines
the behavior of HCC. Thus, CD90 and miR-589-5p are
useful predictors of HCC progression, and miR-589-5p
and MAP3K8 might be novel molecular targets for
In HCC, miR-589-5p down-regulates the stemness
characteristics of CD90+ CSCs in part by silencing MAP3K8.
CD90 and miR-589-5p expression predict HCC outcomes
and might be novel molecular targets for HCC treatment.
Additional file 1: Table S1. Antibodies used in this study. Table S2.
The primers have been used for quantitative real-time PCR. Table S3. All
differentially expressed microRNAs in MHCC97H and MHCC97L CD90+
cells. Table S4. The relationship of CD90 and miR-589-5p expression to
clinical parameters in human HCC (n=40). (DOCX 21 kb)
Additional file 2: Figure S1. CD90 is predominantly expressed in a small
population in HCC cell lines. Figure S2. CD90+ HCC cells possess CSC
characteristics. Figure S3. Overexpression of miR-589-5p has no impact on
the regulation of MAP3K8 and stemness in CD90- HCC cells. Figure S4.
Suppression of miR-589-5p fails to alter the CD90+ population in HCC cells.
Figure S5. CD90- tumor xenograft contains CD90+ cells (DOCX 16 kb)
bFGF: Basic fibroblast growth factor; COT: Cancer Osaka thyroid; CSCs: Cancer
stem cells; DFS: Disease-free survival; EGF: Epidermal growth factor;
EpCAM: Epithelial cell adhesion molecule; ERK: Extracellular signal–regulated
kinase; FAK: Focal adhesion kinase; HCC: Hepatocellular carcinoma;
MAP3K8: Mitogen-activated protein kinase kinase kinase 8;
miRNAs: microRNAs; OS: Overall survival; qRT-PCR: quantitative real-time PCR;
SEM: Standard error of mean; TICs: Tumor initiating cells; TPL2: Progression
locus 2; UTR: Untranslated region
Authors thank Dr. Jun Li (Chongqing Cancer Institute & Hospital & Cancer
Center) for his contribution to professional writing revision.
This work was supported by the project of National Natural Science Fund
(81272363, 81430063), the National 863 Project of China (No. 2012AA02A201).
Availability of data and material
The authors declare that all data supporting the findings of this study are
available within the article and its supplementary information files.
XZ and PJ participated in the cell lines experiments. LS conducted qRT-PCR.
KC participated in the immunohistochemistry analysis. ZHL and YJ participated
in Western blot. YJZ and PJ participated in the animal experiments. XZ
performed the statistical analysis. XZ and XWL participated in the design
of the study and drafted the manuscript. All authors read and approved
the final manuscript.
Consent for publication
Written informed consent for the use of tissues was obtained from all of the
patients before surgery, and this study was approved by the Institutional
Review Board of the Southwest Hospital of the Third Military Medical
University. Procedures involving animals and their care were conducted in
conformity with NIH guidelines (NIH Pub. No. 85-23, revised 1996) and was
approved by Animal Care and Use Committee of the Third Military Medical
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