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ORIGINAL ARTICLE
MicroRNA-125b promotes apoptosis by regulating the expression
of Mcl-1, Bcl-w and IL-6R
J Gong
1,3
, J-P Zhang
1,3
,BLi
2
, C Zeng
1
,KYou
1
, M-X Chen
2
, Y Yuan
2
and S-M Zhuang
1
The microRNA miR-125b is multi-faceted, with the ability to function as a tumor suppressor or an oncogene, depending on the
cellular context. To date, the pro-apoptotic role of miR-125b and its underlying mechanisms are unexplored. In this study, both
gain- and loss-of-function experiments revealed that miR-125b expression not only induced spontaneous apoptosis in various cell
lines derived from the liver, lung and colorectal cancers, but also sensitized cancer cells to diverse apoptotic stimuli, including
nutrient starvation and chemotherapeutic treatment. Furthermore, downregulation of miR-125b was a frequent event in
hepatocellular carcinoma (HCC) tissues, and the miR-125b level was positively associated with the rate of apoptosis in HCC tissues.
Subsequent investigations identified Mcl-1, Bcl-w and interleukin (IL)-6R as direct targets of miR-125b. Restoration of miR-125b
expression not only diminished the expression of Mcl-1 and Bcl-w directly but also indirectly reduced the Mcl-1 and Bcl-xL levels
by attenuating IL-6/signal transducer and activator of transcription 3 signaling. Consistent with these findings, introduction of
miR-125b reduced the mitochondrial membrane potential and promoted the cleavage of pro-caspase-3. These data indicate that
miR-125b may promote apoptosis by suppressing the anti-apoptotic molecules of the Bcl-2 family and miR-125b downregulation
may facilitate tumor development by conferring upon cells the capability to survive under conditions of nutrient deprivation and
chemotherapeutic treatment. Our findings highlight the importance of miR-125b in the regulation of apoptosis and suggest
miR-125b as an attractive target for anti-cancer therapy.
Oncogene (2013) 32, 3071–3079; doi:10.1038/onc.2012.318; published online 23 July 2012
Keywords: miR-125b; apoptosis; Mcl-1; Bcl-w; IL-6R; hepatocellular carcinoma.
INTRODUCTION
MicroRNAs (miRNAs) belong to a novel class of small noncoding
RNAs that repress gene expression by binding to the
30untranslated regions (30UTRs) of their target messenger RNAs
(mRNAs). miRNAs have important roles in various biological
processes, such as development, cell proliferation, differentiation
and apoptosis.
1,2
Accumulating evidence suggests that
deregulation of miRNAs contributes to the development of
diseases, including cancer.
3
The miRNA miR-125b is multi-faceted, with the ability to
function as a tumor suppressor or an oncogene, depending on the
cellular context. It is downregulated in malignancies originating in
the ovary, bladder and breast
4–6
but is upregulated in leukemia,
prostate cancer and glioma.
7–9
Interestingly, studies from mouse
models reveal that ectopic expression of miR-125b in
hematopoietic stem cells can induce leukemia 12–29 weeks post
transplantation. Furthermore, miR-125b overexpression also
accelerates the development of BCR-ABL-induced leukemia.
10
Most published studies have focused on analyzing the effect of
miR-125b on cell proliferation. Overexpression of miR-125b
promotes the proliferation of glial cells
7
but suppresses the
proliferation of ovarian, bladder, breast and liver cancer cells.
6,11–13
Regulators of proliferation, including BCL3, CDKN2A, E2F3, RAF1,
ETS1, LIN28B2, ERBB3, ERBB2 and placenta growth factor
6,7,11–16
have been identified as direct targets of miR-125b. It has been
shown that overexpression of miR-125b inhibits apoptosis in
glioma and prostate cancer cells, and miR-125b regulates the
expression of pro-apoptotic proteins such as p53, Bak1, Puma and
Bmf.
17–20
To date, studies addressing the pro-apoptotic effect of
miR-125b are limited
14,21
and the detailed mechanisms remain
unclear.
Hepatocellular carcinoma (HCC) is one of the most common
cancers worldwide. It is characterized by resistance to anti-cancer
therapy, progressive development, high postsurgical recurrence
and extremely poor prognosis. Emerging evidence indicates that
miR-125b has an important role in the development of HCC. miR-
125b is significantly downregulated in HCC
2,14,15,22
and overex-
pression of miR-125b not only inhibits proliferation, anchorage-
independent growth, migration and invasion in HCC cells but also
represses tube formation of malignant hepatic endothelial cells.
14,15
In a previous study, we observed that miR-125b overexpression
induced spontaneous apoptosis in the HCC cell line PLC/PRF-5 and
in a malignant hepatic endothelial cells.
14
However, the role of
miR-125b in promoting apoptosis and the underlying mechanisms
are still largely unknown. In this study, both gain- and loss-of-
function analyses revealed that miR-125b not only induced
spontaneous apoptosis in cancer cells but also sensitized cancer
cells to various apoptotic stimuli, including nutrient deprivation
and treatment with chemotherapeutic agents. A significant
correlation between decreased miR-125b expression and a
1
Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, Department of Biochemistry, School of Life Sciences, Guangzhou, PR China
and
2
Department of Hepatobiliary Oncology, State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-sen University, Guangzhou, PR China. Correspondence:
Dr Y Yuan, Department of Hepatobiliary Oncology, Cancer Center, Sun Yat-sen University, Dongfengdong Road 651#, Guangzhou 510060, PR China or Dr S-M Zhuang,
Department of Biochemistry, School of Life Sciences, Sun Yat-sen University, Xin Gang Xi Road 135#, Guangzhou 510275, PR China
E-mail: yuanyf@mail.sysu.edu.cn or zhuangshimei@163.com or LSSZSM@mail.sysu.edu.cn
3
These authors contributed equally to this work.
Received 27 December 2011; revised 7 June 2012; accepted 11 June 2012; published online 23 July 2012
Oncogene (2013) 32, 3071– 3079
&
2013 Macmillan Publishers Limited All rights reserved 0950-9232/13
www.nature.com/onc
reduced rate of apoptosis was observed in HCC tissues. Moreover,
we showed that Mcl-1, Bcl-w and interleukin (IL)-6R were direct
targets of miR-125b, and they were involved in miR-125b-
mediated apoptosis. In addition, we demonstrated that the
mitochondrial pathway and caspase-3 were activated in miR-
125b-promoted apoptosis. Our results suggest that miR-125b may
be an attractive target for cancer therapy.
RESULTS
Analysis on the function of miR-125b in apoptosis
To explore the effect of miR-125b in apoptosis, SMMC7721,
HCT116 and 95D cell lines (derived from the liver, colorectal and
lung cancers, respectively) were transfected with a miR-125b
duplex or a negative control (NC). The morphological examination
revealed that compared with the NC, miR-125b significantly
increased spontaneous apoptosis in all three cell lines (Figure 1a).
Rapid growth of malignancies results in insufficient blood
supply, solid cancer cells thus should develop resistance to
nutrient starvation-induced apoptosis. Therefore, we analyzed
whether miR-125b could affect the apoptosis of cancer cells that
were grown in nutrient-deprived medium. Twenty-four hours after
transfection with NC or miR-125b, the cells were deprived of
serum for different lengths of time. We observed that miR-125b
expression significantly increased the sensitivity of different types
of cancer cells to nutrient deprivation-stimulated apoptosis
(Figure 1b).
Resistance to chemotherapeutic agents is another important
characteristic of cancer cells. Thus, we determined whether
miR-125b could enhance the chemosensitivity of tumor cells.
Twenty-four hours after transfection with NC or miR-125b, the
cells were treated with doxorubicin for various lengths of time.
Compared with NC transfection, introduction of miR-125b
obviously enhanced the apoptotic rates in all examined cell lines
that were treated with doxorubicin (Figure 1c).
Next, the apoptosis-promoting effect of miR-125b from our
above observation by morphological analysis was further con-
firmed by the use of terminal deoxynucleotidyl transferase-
mediated nick end labeling (TUNEL) staining (Supplementary
Figure S1). Moreover, activation of caspase-3 was also found in
miR-125b-promoting apoptosis (Figures 1d and e).
To verify the findings from the gain-of-function study, loss-of-
function analysis was firstly performed in HCC cell line MHCC97L,
which expressed higher levels of miR-125b among examined cell
lines (Supplementary Figure S2). An inhibitor of miR-125b (anti-
miR-125b), which obviously decreased the level of endogenous
miR-125b (Supplementary Figure S3), was transfected into
MHCC97L. In response to either serum deprivation or doxorubicin
treatment, anti-miR-125b-transfected cells displayed significantly
reduced rates of apoptosis, compared with the control, anti-miR-C
transfectants (Figures 2a and b). Similarly, miR-125b inhibition also
attenuated both nutrient deprivation and chemotherapeutic
agent-induced apoptosis in another two hepatoma cell lines,
SMMC7721 and SK-HEP-1 (Figures 2c and d).
To confirm the association of miR-125b with apoptosis in vivo,
we further evaluated the miR-125b level and the apoptotic rate in
human HCC specimens using quantitative PCR and TUNEL
staining, respectively. Compared with adjacent non-tumor tissues,
miR-125b was significantly downregulated in HCC tissues
(Supplementary Figure S4A). Linear regression analysis disclosed
a significant positive correlation between the level of miR-125b
and the percentage of TUNEL-positive cells in HCC tissues
(Figure 3, Supplementary Figure S4B), suggesting that miR-125b
downregulation may lead to decreased apoptosis in HCC cells.
Taken together, these results indicate that miR-125b promotes
apoptosis in our cell models and deregulation of miR-125b may
confer cellular resistance to nutrient starvation and chemotherapy-
induced apoptosis.
Characterization of direct target genes of miR-125b
We next explored the molecular mechanisms responsible for the
apoptosis-promoting effect of miR-125b. First, the potential target
genes that may mediate the effect of miR-125b were chosen for
experimental validation, based on bioinformatic prediction and
the known function of the genes in apoptosis regulation.
Predicted target genes of miR-125b were retrieved from the
TargetScan database. We then focused our attention on Mcl-1,
Bcl-w and IL-6R, because Mcl-1 and Bcl-w are anti-apoptotic
members of the Bcl-2 family and are frequently overexpressed in
tumor tissues;
23,24
IL-6R is the binding subunit specific to IL-6, and
the binding of IL-6 to IL-6R activates the Janus kinase/signal
transducer and activator of transcription (Stat)3 pathway and in
turn induces the expression of genes involved in apoptosis and
cell cycle regulation.
25
A dual-luciferase reporter system was first used to evaluate
whether Mcl-1,Bcl-w and IL-6R were direct target genes of miR-
125b. The wild-type or mutant 30-UTR fragments of these genes
(Supplementary Figure S5) were cloned downstream of the firefly
luciferase reporter gene. We found that co-transfection of miR-
125b significantly suppressed the activity of firefly luciferase that
carried the wild-type but not mutant 30UTR of Mcl-1,Bcl-w or IL-6R
(Figure 4a), indicating that miR-125b may suppress gene
expression through its binding sequences at the 30UTR of target
genes.
Subsequently, the effect of miR-125b on cellular endogenous
expression of its potential target genes was examined. Ectopic
expression of miR-125b obviously diminished the expression of
Mcl-1, Bcl-w and IL-6R proteins in SMMC7721 cells (Figure 4b),
whereas antagonism of endogenous miR-125b expression
increased the levels of these three proteins in MHCC97L cells
(Figure 4c). Similar suppressive effect was obtained for Mcl-1 and
Bcl-w after miR-125b expression in HCT116 and 95D cells
(Supplementary Figure S6), while IL-6R was undetectable in these
two cell lines (data not shown).
All together, these data imply that miR-125b may repress the
expression of Mcl-1, Bcl-w and IL6-R by directly binding to their
30UTRs.
Elucidation of signaling pathways involved in miR-125b-
promoting apoptosis
It has been demonstrated that once IL-6 binds to IL-6R, gp130 is
recruited to the IL-6–IL-6R complex, followed by sequential
phosphorylation and activation of Janus kinases and Stat3.
Phosphorylated-Stat3 (p-Stat3) dimerizes, translocates to the
nucleus and initiates the transcription of genes that promote
growth and prevent apoptosis, like cyclin D1, Mcl-1 and Bcl-xL.
Therefore, we investigated whether miR-125b could repress IL-6/
Stat3 signaling. Transfection with miR-125b or siIL-6R resulted in
decrease of constitutive p-Stat3 (Figure 5a, lanes 1–4). In response
to IL-6 stimulation, p-Stat3 significantly increased in control cells
(Figure 5a, lanes 1, 2, 5, 6), but remains unchanged in both miR-
125b- and siIL-6R transfectants (Figure 5a, lanes 3, 4, 7, 8).
Immunofluorescent staining also revealed that miR-125b effi-
ciently blocked IL-6-induced phosphorylation and nuclear translo-
cation of Stat3 (Figure 5b). Consistently, IL-6 treatment increased
the Mcl-1 and Bcl-xL level (Supplementary Figure S7A), but
restoration of miR-125b or silencing of IL-6R significantly
attenuated the expression of Mcl-1 and Bcl-xL in IL-6-exposed
cells (Figure 5c). Interestingly, although restoration of miR-125b
decreased the level of both Mcl-1 and Bcl-w, knockdown of either
IL-6 or IL-6R only downregulated Mcl-1, but had no effect on Bcl-w
expression (Figure 5d, Supplementary Figure S7B–D). Accordingly,
the expression of Mcl-1 but not Bcl-w was enhanced in response
to IL-6 treatment (Supplementary Figure S7A). These data indicate
that miR-125b can attenuate IL-6/Stat3 signaling and thereby
indirectly reduces the levels of Mcl-1 and Bcl-xL.
miR-125b promotes apoptosis
J Gong et al
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To explore the role of Mcl-1, Bcl-w and IL-6R in miR-125b-
promoted apoptosis, we examined whether knockdown of these
genes could mimic the effect of miR-125b overexpression. siRNA
duplexes targeting Mcl-1, Bcl-w or IL-6R, which significantly
decreased the expression of their respective target genes
(Figure 4b), were transfected into cells for 24 h, followed by
serum starvation for 72 h. Compared with NC-transfected cells,
siMcl-1, siBcl-w and siIL-6R transfectants displayed higher levels of
apoptosis (Figure 5e), and the apoptotic rates increased with the
concentrations of siRNA (Supplementary Figure S8). Moreover, the
apoptosis-promoting effect of triple-knockdown of Mcl-1, Bcl-w
and siIL-6R was greater than that of any one individual silencing
Figure 1. miR-125b promotes apoptosis in various cancer cell lines. (a) Ectopic expression of miR-125b-induced spontaneous apoptosis.
SMMC7721, HCT116 and 95D cells were transfected with miR-125b or NC for the indicated times. (b,c) miR-125b sensitized cancer cells to
different apoptotic stimuli. Twenty-four hours after transfection, SMMC7721, HCT116 and 95D cells were deprived of serum (b) or treated with
0.05 (for HCT116 or 95D) or 0.1 (for SMMC7721) mg/ml doxorubicin (c) for the indicated times. (d,e) miR-125b expression increased the
cleavage of pro-caspase-3. Twenty-four hours after transfection, SMMC7721 cells were deprived of serum (d) or treated with 0.2mg/ml
doxorubicin (e) for 24 h, followed by detection of pro- and active caspase-3 using western blotting. b-Actin, internal control. * indicates a
nonspecific band. For (a-c), apoptosis was analyzed by 40-60-diamidino-2-phenylindole staining. RNAiMAX, cells were exposed to transfection
mixture that contained Lipofectamine RNAiMAX but not the RNA oligoribonucleotide. **Po0.01; ***Po0.001.
miR-125b promotes apoptosis
J Gong et al
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&2013 Macmillan Publishers Limited Oncogene (2013) 3071 – 3079
(Figure 5e) but similar to that of miR-125b expression (Figure 1b).
These results suggest that miR-125b may promote apoptosis by
targeting Mcl-1, Bcl-w and IL-6R.
As Mcl-1, Bcl-w and Bcl-xL belong to the Bcl-2 family, which has
crucial roles in the mitochondrial apoptosis pathway, we next
evaluated whether overexpression of miR-125b disrupted the
mitochondria membrane potential (DCm) by double staining with
MitoTracker Deep Red FM (MTRed) and MitoTracker Green FM
(MTGreen). The intensity of MTRed staining depends on DCm,
whereas the intensity of MTGreen remains the same regardless of
DCm and was thus used as an internal staining control. As shown,
loss of DCm was distinctly increased in the miR-125b transfec-
tants compared with the control group (15.7% versus 7.8%,
Figures 6a and b). The results suggest that miR-125b may promote
apoptosis by suppressing the anti-apoptotic molecules of Bcl-2
family and in turn activating the mitochondrial apoptotic pathway.
Taken together, our findings suggest that miR-125b not only
directly represses the endogenous expression of Mcl-1 and Bcl-w,
but also indirectly reduces the levels of Mcl-1 and Bcl-xL by
attenuating IL-6/Stat3 signaling, which result in activation of the
mitochondrial pathway and in turn induction of apoptosis
(Figure 6c).
DISCUSSION
Although abnormal miR-125b expression is frequently observed in
various types of cancers, the biological outcome of miR-125b
deregulation is largely cellular context-dependent.
4–7,9,10
To date,
the role of miR-125b in the regulation of apoptosis remains
unclear. Here, we found frequent downregulation of miR-125b in
HCC tissues, as well as a significant correlation between a reduced
miR-125b level and a decreased rate of apoptosis. We also showed
that restoration of miR-125b significantly increases apoptosis in
the liver, colorectal and lung cancer cell lines in the absence of
apoptotic stimuli and upon nutrient starvation or chemo-
therapeutic drug exposure. Our results highlight the importance
of miR-125b deregulation in cancer development and the
possibility of miR-125b as a new target for anti-cancer therapy.
miR-125b is a multi-faceted molecule that may promote or inhibit
apoptosis, depending on cellular context. The opposite effects
mediated by miR-125b in different types of cells may provide a
basis for targeted therapy, if the underlying mechanisms are
disclosed.
Apoptosis serves as a natural barrier to cancer formation and
development. Tumor cells must evolve a variety of strategies to
circumvent apoptosis so that they can escape clearance by
immune surveillance systems or anti-cancer therapy and survive in
the harsh tumor microenvironment, where hypoxia and nutrient
deprivation are common.
26
Multiple mechanisms are used by
tumor cells to evade apoptosis, such as the functional loss of the
p53 tumor suppressor, increased expression of anti-apoptotic
regulators (Bcl-2, Bcl-xL, Mcl-1 and Bcl-w) and survival signals
(insulin-like growth factor 1/2), downregulation of proapoptotic
factors (Bax, Bak1, Bim and Puma) and short-circuiting of the
extrinsic ligand-induced death pathway. The Bcl-2 family, which
consists of both pro- and anti-apoptotic proteins, regulates the
permeability of the mitochondrial membrane. Mcl-1, Bcl-xL and
Bcl-w, the anti-apoptotic members of the Bcl-2 family, suppress
apoptosis largely through binding to pro-apoptotic proteins (Bax
and Bak1) that are embedded in the mitochondrial outer
Figure 2. Antagonism of miR-125b desensitizes cancer cells to
various apoptotic stimuli. (a,b) Antagonism of miR-125b desensi-
tized MHCC97L cells to apoptotic stimuli. Twenty-four hours after
transfection with anti-miR-125b (miR-125b inhibitor) or anti-miR-C
(negative control), the cells were deprived of serum (a) or treated
with 0.2 mg/ml doxorubicin (b) for 72 h. (c,d) Antagonism of miR-
125b desensitized SMMC7721 and SK-HEP-1 cells to apoptotic
stimuli. Twenty-four hours after transfection with anti-miR-125b or
anti-miR-C, SMMC7721 and SK-HEP-1 cells were deprived of serum
(c) for 96 and 72 h, respectively, or treated with 0.1 and 0.3 mg/ml
doxorubicin, respectively (d) for 72 h. Apoptosis was analyzed by
40-60-diamidino-2-phenylindole staining. RNAiMAX, the cells were
exposed to transfection mixture that contained Lipofectamine
RNAiMAX but not the RNA oligoribonucleotide. *Po0.05;
**Po0.01 and ***Po0.001.
Figure 3. miR-125b expression is positively correlated with the level
of apoptosis in HCC tissues. The level of mature miR-125b was
examined by real-time qPCR and normalized to RNU6B expression.
Apoptosis was determined using TUNEL staining. The correlation
between the miR-125b level and the rate of apoptosis in HCC tissues
was analyzed using Spearman’s correlation coefficient. The median
level of miR-125b in all examined samples was set to 1.
miR-125b promotes apoptosis
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membrane and preserve the integrity of mitochondria. Mcl-1,
Bcl-xL and Bcl-w are frequently upregulated in various types of
cancer
23,24,27
and function as essential pro-survival molecules in
tumor progression. We show that miR-125b directly suppresses
Mcl-1 and Bcl-w and indirectly represses Bcl-xL by targeting IL-6R.
Knockdown of Mcl-1, Bcl-w and IL-6R closely mimics the
apoptosis-promoting effect of miR-125b expression, and
activation of the mitochondrial and caspase-3 pathways is
involved in miR-125b-promoted apoptosis. These results clearly
indicate that Mcl-1, Bcl-w and IL-6R are the predominant
mediators of miR-125b-induced apoptosis in liver cancer cells,
although other unidentified targets may also be involved.
The reason why miR-125b can exert both apoptosis-promoting
and inhibiting function is still unclear. It has been reported that
miR-125b suppresses apoptosis by negatively regulating p53,
Bak1, Puma and Bmf in leukemia, glioma and prostate cancer
cells.
17–20
However, neither the level of p53 mRNA nor the Bak1
protein in HCC cell lines is altered by miR-125b expression.
14,15
In
our HCC cell model, no obvious change was found in the protein
level of p53 after either overexpression or suppression of
miR-125b (Supplementary Figure S9). We speculate that the
following mechanisms may be responsible for the opposite role of
miR-125b in different cells. First, the relative concentration of
target mRNAs in different cell types may decide which set of
targets will be regulated by miR-125b. Second, cell-type-specific
competitive mRNAs or UTR-binding cofactors may affect the
binding of miR-125b to its target sites. For example, the
pseudogene PTENP1 could compete with PTEN for miRNA binding,
thereby attenuating the repression of miRNAs on PTEN.
28
Third,
the location of miRNA target genes may affect the repressive
effect of miRNA. For instance, the repression of CAT-1 by miR-122
is relieved by the relocation of CAT-1 mRNA from cytoplasmic
processing bodies to polysomes under the cellular stress.
29
Collectively, the regulatory effect of miRNA may be more
dynamic than previously anticipated. Much more extensive
studies are needed to understand the mechanisms that
contribute to the diverse roles of miR-125b.
IL-6 is a multifunctional cytokine that has pleiotropic roles in
immune regulation, inflammation, liver regeneration and tumor-
igenesis. Serum IL-6 levels are elevated in patients with hepatitis,
Figure 4. Mcl-1, Bcl-w and IL-6R are direct targets of miR-125b. (a) miR-125b suppressed the activity of firefly luciferase that carried the wild-
type but not mutant 30UTR of Mcl-1,Bcl-w or IL-6R. SMMC7721 cells were co-transfected with the indicated RNA duplex, pRL-TK and a firefly
luciferase reporter plasmid containing either wild-type (WT) or mutant (MUT) 30UTR of putative target gene. pRL-TK, which expresses Renilla
luciferase, was co-transfected to calibrate the differences in both transfection and harvest efficiency. Luciferase activity was detected 48 h post
transfection. The activity of firefly luciferase in each sample was normalized to that of Renilla luciferase. The normalized activity of NC
transfectants was set as relative luciferase activity 1; therefore, no error bar is shown for NC transfectants. **Po0.01. (b) Ectopic expression of
miR-125b decreased the endogenous levels of Mcl-1, Bcl-w and IL-6R in SMMC7721 cells. Forty-eight hours after transfection, endogenous
protein levels were examined by western blotting. (c) Antagonism of miR-125b increased the levels of Mcl-1, Bcl-w and IL-6R in MHCC97L cells.
Cellular proteins were analyzed by western blotting 72 h post transfection. b-Actin, internal control.
miR-125b promotes apoptosis
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&2013 Macmillan Publishers Limited Oncogene (2013) 3071– 3079
cirrhosis and HCC.
30
IL-6 signals through IL-6R/gp130 and activates
Janus kinases, which then phosphorylate several substrates to
induce signaling through the Stat3, mitogen-activated protein
kinase and phosphatidylinositol 3-kinase pathways. Among them,
the Stat3 pathway is the main one activated by IL-6 because most
effects of IL-6 can be abolished by disrupting Stat3 signaling.
31
IL-6/Stat3 signaling pathway is found to be over-activated in
various types of human malignancies.
25
It has been reported that
Stat3 is a target of miR-125b in myeloid cells.
32
However, enforced
expression of miR-125b has minimal effects on the expression of
Stat3 in our cell models. Alternatively, we found that miR-125b
could suppress the phosphorylation of Stat3 and in turn the
expression of its target genes, such as Mcl-1 and Bcl-xL.
Furthermore, restoration of miR-125b decreased the level of all
Mcl-1, Bcl-xL and Bcl-w, whereas IL-6 treatment and knockdown of
IL-6 or IL-6R only affected the expression of Mcl-1 and Bcl-xL, but
had no effect on Bcl-w level. Therefore, miR-125b may promote
apoptosis via several different mechanisms, that is, direct
suppression of Mcl-1 and Bcl-w expression by binding to their
30UTRs and/or indirect repression of Mcl-1 and Bcl-xL expression
by attenuating IL-6/Stat3 signaling. Our results highlight an
intriguing facet of miRNA action, that is, miRNAs can regulate
certain cell activities by suppressing the expression of several
cooperating proteins.
Figure 5. miR-125b promotes apoptosis by targeting Mcl-1, Bcl-w and IL-6R. (a) miR-125b suppressed constitutive and IL-6-induced
phosphorylation of Stat3. Forty-eight hours after transfection, SMMC7721 cells were incubated in serum-free Dulbecco’s modified Eagle’s
medium (DMEM) with or without 50 ng/ml IL-6 for 30 min, followed by western blotting. b-Actin, internal control. (b) miR-125b suppressed
IL-6-induced phosphorylation and nuclear translocation of Stat3. SMMC7721 cells transfected with the indicated RNA were treated with
50 ng/ml IL-6 for 30 min and then analyzed by immunofluorescent staining. The nuclei were stained blue with 40-60-diamidino-2-phenylindole
(DAPI). Scale bar, 20 mm. (c) Restoration of miR-125b or silencing of IL-6R diminished the expression of Mcl-1 and Bcl-xL in IL-6-exposed cells.
Twenty-four hours after transfection, SMMC7721 cells were cultured in serum-free DMEM for 20 h and then exposed to 10 ng/ml IL-6 for 4 h
before western blotting. (d) miR-125b overexpression reduced the levels of Mcl-1, Bcl-xL and Bcl-w. SMMC7721 cells were transfected with
miR-125b or siIL-6R for 48 h before western blotting. b-Actin, internal control. (e) Knockdown of Mcl-1, Bcl-w and IL-6R-promoted apoptosis.
Twenty-four hours after transfection with the indicated single siRNA (50 nM) or siRNA mixture (16.7 nMof each siMcl-1, siBcl-w and siIL-6R),
SMMC7721 cells were deprived of serum for 72 h and then subjected to DAPI staining. **Po0.01; ***Po0.001.
miR-125b promotes apoptosis
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Membrane-bound IL-6R is mainly expressed on hepatocytes
and immune cells,
25
which may explain why IL-6R is undetectable
in HCT116 and 95D, cell lines that originate from colorectal and
lung cancers. Except when expressed on the cell membrane, IL-6R
exists as a soluble form (sIL-6R) in serum and other biologic fluids.
sIL-6R is generated by alternative splicing or by proteolytic
cleavage from the membrane-bound IL-6R. IL-6 can also bind to
sIL-6R and form an IL-6/sIL-6R complex, which then associates with
gp130, the ubiquitously expressed IL-6-signal transducer. This
ternary complex can transduce the IL-6 signal into cells in the
absence of membrane-bound IL-6R.
25
Notably, sIL-6R levels are
elevated in sera from HCC patients.
30
Therefore, downregulation
of miR-125b may not only enhance IL-6 signaling in hepatic cells
but may also affect other cells by increasing the level of sIL-6R.
In summary, we investigated the potential role of miR-125b in
apoptosis and the mechanisms underlying its actions. Our data
suggest that downregulation of miR-125b may favor malignant
transformation and tumor progression, and implicate potential
application for miR-125b in anti-cancer therapy.
MATERIALS AND METHODS
Reagents
The reagents used were as follows: rabbit polyclonal antibodies for Mcl-1
(sc-819, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-w (sc-130701),
IL-6R (sc-661, Santa Cruz Biotechnology), caspase-3 (no. 9662, Cell
Signaling Technology, Beverly, MA, USA); mouse monoclonal antibodies
against Tyr705-p-Stat3 (sc-8059), Stat3 (sc-8019), Bcl-xL (sc-8392), p53
(sc-126, Santa Cruz Biotechnology) and b-actin (BM0627, Boster, Wuhan,
China); ECL kit (Pierce, Rockford, IL, USA); 40-60-diamidino-2-phenylindole
(Sigma-Aldrich, St Louis, MO, USA); IL-6 (206-IL, R&D Systems, Oxon, UK);
cell culture plates (Corning Glass Works, Corning, NY, USA). All other
reagents were purchased from Sigma-Aldrich, unless otherwise indicated.
Tissue specimens and cell lines
A total of 32 paired HCC and adjacent nontumor liver tissues were
collected from patients who underwent surgical resection at the
Department of Hepatobiliary Oncology at Cancer Center, Sun Yat-sen
University in Guangzhou, PR China. The non-tumor tissues were collected
at least 2-cm distant from the tumor border. Both tumor and non-tumor
tissues were histologically confirmed. All patients were unrelated ethnic
Han Chinese who lived in Southeast China. None of the patients had
received local or systemic anticancer treatment before the operation. This
study was approved by the Institute Research Ethics Committee at the
Cancer Center. Informed consent was obtained from each patient.
Cell lines derived from the human HCC (MHCC97L, SMMC7721), liver
adenocarcinoma (SK-HEP-1), colorectal (HCT116) and lung (95D) cancers
were maintained in Dulbecco’s modified Eagle’s medium (Hyclone, Logan,
UT, USA) supplemented with 10% fetal bovine serum (Hyclone, Thermo
Fisher Scientific, Victoria, Australia).
RNA oligoribonucleotides and plasmids
All RNA oligoribonucleotides were purchased from Genepharma (Shanghai,
PR China). The miRNA duplexes corresponding to mature miR-125b were
designed as described previously.
33
The siRNAs targeting mRNA of human
Mcl-1 (GenBank accession no. NM_021960), Bcl-w (NM_004050), IL-6R
(NM_000565, NM_181359) or IL-6 (NM_000600.3) were denoted as siMcl-1,
siBcl-w, siIL-6R and siIL-6, respectively. The NC RNA duplex for both the miR-
125b mimic and the siRNA was nonhomologous to any human genome
sequences. The miR-125b inhibitor (anti-miR-125b) with a sequence
complementary to the mature miR-125b and its control (anti-miR-C) were
20-O-methyl-modified RNA oligoribonucleotides.
The bioinformatic analysis predicted one miR-125b-binding site in Mcl-1
and Bcl-w and two binding sites in IL-6R (Supplementary Figure S5). A wild-
type 30UTR segment of human Mcl-1 (492 bp) or IL-6R (484 and 589 bp for
two sites, respectively) mRNA that contained putative binding site for miR-
125b was PCR-amplified and inserted into the EcoRI/XbaI sites downstream
of the stop codon of firefly luciferase in pGL3cm vector, which was created
based upon the pGL3-control vector (Promega, Madison, WI, USA), as
Figure 6. The signaling pathways that are involved in miR-125b-regulated apoptosis. (a,b) miR-125b overexpression activated the
mitochondrial pathways. Seventy-two hours after transfection, SMMC7721 cells were double-stained with MTRed and MTGreen, followed by
flow cytometry analysis of DCm variations. Both dot plot (a) and statistic histograms (b) are shown. **Po0.01. (c) Schematic showing the
network of miR-125b and its target genes in the regulation of apoptosis.
miR-125b promotes apoptosis
J Gong et al
3077
&2013 Macmillan Publishers Limited Oncogene (2013) 3071– 3079
described previously.
34
A wild-type 30UTR segment (668 bp) of human
Bcl-w mRNA was inserted into the EcoRI/ApaI sites of pGL3cm. The mutant
30UTR (Supplementary Figure S5), which contained the mutated sequence
in the complementary site for the seed region of miR-125b, was generated
using fusion PCR based on the construct with wild-type 30UTR.
All RNA oligoribonucleotides and the primers are listed in Supplemen-
tary Table S1.
Cell transfection
RNA oligoribonucleotides were reversely transfected using Lipofectamine
RNAiMAX (Invitrogen, Carlsbad, CA, USA). A final concentration of 50 nM
RNA duplex or 200 n MmiRNA inhibitor was used for each transfection,
unless otherwise indicated. The RNA transfection efficiency using this
method is B70–80%,
34
and the overexpression of miRNA mimics persists
for at least 4 days.
35
Co-transfection of the RNA duplex and plasmid DNA
was conducted using Lipofectamine 2000 (Invitrogen). All transfections
were performed according to the manufacturer’s protocol.
Apoptosis analysis
Apoptosis was detected by morphological examination and TUNEL assay.
For morphological examination, cells were stained with 40-60-diamidino-2-
phenylindole and those with condensed or fragmented nuclei were
considered to be apoptotic cells. At least 500 cells were counted for each
sample. TUNEL staining was conducted using the In situ Cell Death
Detection Kit (Roche, Penzberg, Germany), according to the manufacturer’s
protocol. At least 750 cells were counted for each sample.
Luciferase reporter assay
SMMC7721 cells plated in a 48-well plate were co-transfected with 10 nM
RNA duplex, 2 ng pRL-TK (Promega) and 10 ng firefly luciferase reporter
containing the wild-type or mutant 30UTR of the indicated target gene.
pRL-TK, which expresses Renilla luciferase, was co-transfected as an
internal control to correct for differences in both transfection and harvest
efficiency. Transfections were performed in duplicate and repeated in at
least three independent experiments. Luciferase activity was detected as
described.
34
Analysis of gene expression
miRNA level was evaluated by quantitative real-time PCR, mRNA level by
semiquantitative RT–PCR and protein expression by western blotting.
Among the 32 pairs of HCC and matched adjacent liver tissues, miR-125b
expression has been previously reported in 19 of these samples.
14
Analysis of DCm
Cells were stained with both MitoTracker Deep Red FM and MitoTracker
Green FM (Invitrogen) at 37 1C for 20 min in the dark and washed with
Ca
2þ
-free phosphate-buffered saline, followed by flow cytometry analysis
(Gallios, Beckman Coulter, Fullerton, CA, USA).
Immunofluorescent staining for p-Stat3
Cells were cultured on coverslips, fixed with methanol and stained with
monoclonal antibodies against p-Stat3 (sc-8059, Santa Cruz Biotechnol-
ogy), followed by incubation with Alexa Fluor 488-conjugated goat anti-
mouse immunoglobulin G (Molecular Probes, Carlsbad, CA, USA) and
nuclear counterstaining with 40-60-diamidino-2-phenylindole. Fluorescent
images were examined and photographed with a Zeiss Axio Imager Z1
(Zeiss, Jena, Germany).
Statistical analysis
Data were expressed as the mean±s.e.m. from at least three independent
experiments. The Student’s t-test was applied to compare the differences
between two groups. Correlation between the miR-125b level and the
apoptotic rate in HCC tissues was analyzed using Spearman’s correlation
coefficient. A P-value of o0.05 was used as the criterion of statistical
significance, and all statistical tests were two-sided.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This study was supported by grants from the Ministry of Science and Technology of
China (2010CB912803 and 2011CB811305), the Ministry of Health of China
(2012ZX10002-011) and the National Natural Science Foundation of China
(30925036).
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
miR-125b promotes apoptosis
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