MiR-361-5p inhibits glycolytic metabolism, proliferation and invasion of breast cancer by targeting FGFR1 and MMP-1
Ma et al. Journal of Experimental & Clinical Cancer Research
MiR-361-5p inhibits glycolytic metabolism, proliferation and invasion of breast cancer by targeting FGFR1 and MMP-1
Fei Ma 0 1
Lei Zhang 0 3
Li Ma 2
Yiyun Zhang 4
Jianguo Zhang 1
Baoliang Guo 1
0 Equal contributors
1 Department of General Surgery, the Second Affiliated Hospital of Harbin Medical University , 246 Xuefu Street, Nangang District, Harbin , China
2 Computer Center, the Fifth Hospital of Harbin , Harbin , China
3 Department of Ultrasound, the Second Affiliated Hospital of Harbin Medical University , Harbin , China
4 Department of Endoscopy, Harbin Medical University Cancer Hospital , Harbin , China
Background: MicroRNAs function as key regulators in various human cancers, including breast cancer (BC). MiR-361-5p has been proved to be a tumor suppressor in colorectal cancer and gastric cancer in our previous study. In this study, we aim to find out the function of miR-361-5p in breast cancer progression and elaborate the mechanism that miR-361-5p acts its function in breast cancer. Methods and results: Here we reported that miR-361-5p was down-regulated in breast cancer tissue compared with normal breast tissue and the expression of miR-361-5p was positively associated with prognosis in BC patients. Functional studies showed that overexpression of miR-361-5p suppressed the proliferation, invasion and metastasis of breast cancer cells both in vivo and in vitro. Mechanistically, we found that miR-361-5p inhibited the proliferation of BC cells by suppressing glycolysis. FGFR1, a promoter of glycolysis-related enzyme, was identified as the target of miR-361-5p that promoted glycolysis and repressed oxidative phosphorylation. Furthermore, we demonstrated that miR-361-5p inhibited breast cancer cells invasion and metastasis by targeting MMP-1. An inverse expression pattern was also found between miR-361-5p and FGFR1 or MMP-1 in a cohort of 60 BC tissues. Conclusion: Our results indicate that miR-361-5p inhibits breast cancer cells glycolysis and invasion by respectively repressing FGFR1 and MMP-1, suggesting that miR-361-5p and its targets may serve as therapeutic targets in breast cancer treatment.
miR-361-5p; Glycolysis; FGFR1; MMP-1
Warburg effect was first described as a common metabolic
feature of cancer cells almost 90 years ago, which has also
been known as aerobic glycolysis nowadays [
phenomenon indicates that cancer cells tend to consume
more glucose to produce lactate by glycolysis rather than
oxidative phosphorylation even in oxygen-rich conditions
]. This metabolic shift is thought to provide diverse
glycolytic intermediates for anabolic biosynthesis instead
of energy production in rapidly proliferating cancer cells
]. Thus, the understanding of controlling the shift from
oxidative phosphorylation to aerobic glycolysis is crucial
for cancer treatment.
At present, breast cancer (BC) is the most prevalent
cancer among women in China and the incidence of BC
is still increasing rapidly [
]. Despite numerous evidence
have shown that accumulation of genetic and epigenetic
changes cause tumorigenesis and progression [
mechanisms underlying the pathogenesis of BC remain
to be clearly defined. Given that recrudescence and
metastasis occur frequently and associate closely with BC
], understanding the fundamental mechanism that
facilites cancer progression and finding new sights in
breast cancer treatment are of great importance.
MicroRNAs (miRNAs) are a class of small non-coding
RNAs that can play central regulatory roles in the
development of breast cancer [
]. They can imperfectly pair with
the 3′-untranslated region (UTR) of their target mRNAs
and trigger mRNAs degradation or translation inhibition
]. It has been evidenced that miRNA expression is closely
associated with tumor proliferation and metastasis [
example, miR-146a and miR-301a promotes breast cancer
progression by targeting EMT markers and PTEN,
]. Positive expression of miR-361-5p has been
proved to indicate better prognosis for BC patients .
However, the specific function and regulatory mechanism
of miR-361-5p in BC progression is rarely investigated. In
this study, we sought to reveal how miR-361-5p exerts
influence on BC progression, identify and characterize its
Cell lines and cell culture
Human spontaneously immortal cell line and breast cancer
cell lines, including MCF-10A, MCF-7, MDA-MB-231,
MDA-MB-468, T47D, MDA-MB-549 and HEK-293 T were
cultured under conditions recommended by ATCC. The
cells were maintained in DMEM (Hyclone) supplemented
with 10% FBS (Hyclone) at 37 °C under an air atmosphere
containing 5% carbon dioxide.
RNA extraction and RT-PCR
Total RNA were extracted and reverse transcribed by
using TRIZOL reagent (Invitrogen) and M-MLV RT kit
(Promega). For detecting miR-361-5p, the Mir-VanaTM
MiRNA Isolation Kit (Ambion, USA) was used to isolate
total RNA from cell lines and patient samples following
the manufacturer’s instructions. MiR-361-5p was detected
using Platinum Taq DNA Polymerase (Invitrogen) with
specific primers: miR-361-5p forward: ATAAAGRGCRGA
CAGTGCAGATAGTG, miR-361-5p reverse: TCAAGTA
CCCACAGTGCGGT, and U6 forward: CTCGCTTCGGC
AGCACA, U6 reverse: AACGCTTCACGAATTTGCGT.
Results were expressed as fold change using the 2-△△CT
Plasmid construction and transfection
For the stable transfection of anti-miR-361-5p,
anti-miR361-5p sequence were amplified from miRZip-361-5p
construct (System Biosciences) and cloned into pSilencer4.1
system. BC cells were then transfected with the pSliencer
vector containing the antisense sequence of miR-361-5p.
The cells were selected by puromycin after 48 h
transfection and then diluted. MiR-361-5p mimics, miR-control,
FGFR1 siRNA, MMP-1 siRNA or siRNA negative control
were purchased from Genepharma (China). FGFR1 and
MMP-1 cDNA ORF Clone were purchased from Origene
(Origene Technology). Transient transfections were
performed by using Lipofectamine 2000 (Invitrogen) following
the manufacturer’s protocol. Cells were kept in medium
containing 2% FBS for 48 h and then harvested and used.
Luciferase reporter assay
HEK293T cells were used to perform the luciferase
reporter assay. FGFR1 3′-UTR, mutated FGFR1 3′-UTR,
MMP-1 3′-UTR, mutated MMP-1 3′-UTR, or control
luciferase reporter plasmid was cotransfected with
miR361-5p mimics or negative control (Ambion) using
Lipofectamine 3000 (Thermofisher, USA). Luciferase
activity was measured by SecrePair Dual-Luciferase
Reporter System (GeneCopoeia).
Indicated cells were lysed with RIPA buffer. Protein lysates
were electrophoresed through SDS polyacrylamide gel,
followed by transferring to PVDF membranes (Millipore,
USA). The membranes were blocked with non-fat dry milk
at room temperature and then incubated with primary
antibodies at 4°Covernight. The primary antibodies included:
FGFR1, PDHK1, P-PDHK1, LDHA, P-LDHA, MMP-1 and
β-actin (Cell Signaling Technology). Membranes were then
washed and incubated with secondary antibodies. Proteins
were visualized by the electrochemiluminescence (ECL)
western blot substrate detection (Pierce).
Cell proliferation and colony formation assays
The xCELLigence RTCA DP (Roche) instrument was
used to perform the real-time cell proliferation assays
according to the manufacturer’s instructions. The
concomitant changes in Cell Index reflected the changes in
cell numbers. For colony formation assays, 1 × 103 cells
were seeded in a well of a 6-well plate and cultured for
1–2 weeks. The cells were then fixed and stained. The
cell colonies were imaged and analyzed.
Cell invasion assay
For transwell invasion assays, 2 × 105 cells were plated
into the upper chamber of the insert (Corning) coated
with Matrigel (BD Bioscience). Cells were seeded in
medium without serum in the upper chamber and the
medium in lower chamber was supplemented with 10%
FBS. Cells were cultured for 48 h and cells invaded to the
underside of the membrane were fixed, stained, imaged
Cellular glucose-6-phosphate assay
The levels of glucose-6-phosphate in indicated cells were
measured using Glucose-6-phosphate Fluorometric Assay
kit (Cayman, USA). All results were normalized to total
protein expression levels.
Measurement of the extracellular acidification rate (ECAR)
The extracellular acidification rate (ECAR) was detected
using a Seahorse Bioscience XF24 extracellular flux analyzer
(Seahorse Bioscience). The cartridge sensor was hydrated
overnight with buffer at 37 °C without CO2. Indicated cells
were plated in an XF24 Islet Capture Microplate and the
medium was replaced with serum-free DMEM/F12 without
sodium bicarbonate. ECAR values were observed under
basal conditions and measured after the input of oligomycin
(1 μM), FCCP (1 μM), and antimycin A (1 μM) into the
well. ECAR values were analyzed by using the Seahorse
XF-24 software. Every point represents an average of
five different wells.
Glucose consumption and lactate production
Glucose consumption and lactate production were
measured by using the supernatant of the indicated cells.
The glucose assay kit (Sigma-Aldrich, St. Louis, MO,
USA) was used to measure glucose levels following the
manufacturer’s instructions. The lactate production was
determined by the colorimetric lactate kit (Bio Vision,
Milpitas, CA, USA). The concentrations of metabolite
were examined on deproteinized samples by performing
specific enzymatic assays by a CMA600 analyzer (CMA
Microdialysis AB, Sweden). The results were normalized
to protein content using the Pierce BCA Protein assay
Indicated cells were fixed with formaldehyde and
permeabilized with 0.2% Triton X-100. After blocked with 10%
goat serum, the indicated cells were incubated with primary
antibodies and then corresponding secondary antibodies
(Cell Signaling Technology). Images were taken using Zeiss
Balb/c nude mice and SCID mice were purchased from
Vital Rivers (Beijing, China) and maintained under SPF
conditions. All experiments involving animals were
performed in accordance with the Guide for the
Administration of Affairs Concerning Experimental Animals, the
national guideline for animal experiments. Cells were
suspended in culture medium. A 160 μl sample of medium
containing 1 × 107 cells was injected into the dorsal flank
of nude mice subcutaneously. The growth of tumor was
monitored every week and the tumor xenografts were
collected and weighed after 5 weeks. For lung metastasis
model, 2 × 107 cells were injected through the tail vein of
SCID mice. After 5 weeks, the mice were injected with
luciferin through tail vein 10 min before imaging. Imaging
was performed by the Xenogen IVIS Spectrum Imaging
System (Caliper Life Sciences, USA). Then the mice were
sacrificed and the lungs were collected for detection. The
number of metastatic nodules and tumor volume were
evaluated. For each tissue, HE staining was performed for
histological examination. All the animal experiments were
approved by the Animal Experimental Ethics Committee
of Harbin Medical University.
Sixty pairs of primary breast cancer and corresponding
normal breast tissues were collected and conserved in
−80 °C condition after breast resection and pathological
confirmation between November 2005 and March 2006
in the Second Affiliated Hospital of Harbin Medical
University. The patients should not receive chemotherapy
or radiation therapy before BC resection in this study.
This study was performed according to the ethical
standards of Declaration of Helsinki and all the patients
provided written informed consent for the use of
tissues. The TNM stage was determined in accordance
to the classification proposed by the AJCC Cancer
In situ hybridization and immunohistochemistry
In situ hybridization and immunohistochemistry staining
were performed as previously described [
Results were presented as mean ± standard deviation
from at least three replicates. The Student’s t-test was
used to compare differences between groups. Statistical
analysis was performed by GraphPad Prism 5 software.
Significant data were indicated by asterisks P < 0.05 (*),
P < 0.01 (**).
The expression of miR-361-5p is decreased in breast cancer which is associated with poor prognosis
To decide the role of miR-361-5p in breast cancer, we
first examined the expression levels of miR-361-5p in
normal breast and BC tissues in a group of 60 BC
patients by qRT-PCR and ISH. We found that miR-361-5p
expression was significantly decreased in 49 of 60 BC
tissues compared with corresponding normal breast tissues
(Fig. 1a and b). To explore the clinical significance of
miR-361-5p down-regulation in BC, we further detected
the relationship between clinicopathologic data and
miR361-5p expression levels. It was observed that decreased
miR-361-5p expression was correlated with larger
tumor size and lymph node metastasis (Table 1). As
shown in Fig. 1c, we also found that miR-361-5p
expression was closely associated with TNM stage.
Importantly, Kaplan-Meier analysis indicated that patients
with higher miR-361-5p expression showed significantly
better disease-free survival (Fig. 1d). We next examined
the expression of miR-361-5p in different BC cell lines.
The expression levels of miR-361-5p were found to be
markedly lower in BC cells lines, especially highly
metastatic cells (MDA-MB-231, MDA-MB-468), compared
with spontaneously immortal MCF-10A cells (Fig. 1e).
Altogether, these data suggest that the expression of
miR361-5p is decreased in breast cancer and its downregulation
is associated with poor prognosis.
cancer cells proliferation, invasion and metastasis both in
vitro and in vivo.
MiR-361-5p suppresses breast cancer cells proliferation, invasion and metastasis both in vitro and in vivo
Given the correlation of miR-361-5p expression and
prognosis in BC patients, we further detected the biology
effect of miR-361-5p deregulation on breast cancer cells.
As shown in Fig. 2a, real-time cell proliferation assays
indicated that miR-361-5p overexpression significantly
inhibited growth of MDA-MB-231 cells. Conversely,
miR361-5p inhibitors promoted the growth of MCF-7 cells.
Colony formation assays showed similar results (Fig. 2b).
Matrigel-coated transwell assays indicated that MCF-7
cells transfected with anti-miR-361-5p showed
significantly enhanced invasion ability, whereas overexpressing
miR-361-5p in MDA-MB-231 cells decreased the invasion
ability of cancer cells (Fig. 2c). Consistent with the results
in vitro, MCF-7 cells transfected with anti-miR-361-5p
showed enhanced tumorigenic ability in vivo. In
comparison with the control group, tumorigenic ability dramatically
decreased in the miR-361-5p group (Fig. 2d). Meanwhile,
we observed that mice injected with MDA-MB-231 cells
overexpressing miR-361-5p exhibited less lung metastatic
nodes compared with the control group. Conversely,
transfection with anti-miR-361-5p in MCF-7 cells
markedly increased the number of lung metastatic nodes
in node mice. To conclude, miR-361-5p suppresses breast
MiR-361-5p inhibits glycolysis and increases mitochondrial metabolism in BC cells
To text whether aerobic glycolysis was involved in the
process of breast cancer progression, we cultured BC
cells using galactose instead of glucose, which could
inhibit glycolytic flux and force the cells to rely on
mitochondrial oxidative phosphorylation [
]. It was
observed that BC cells transfected with anti-NC and
anti-miR-361-5p showed similar rate of proliferation
under, indicating that glycolysis might be required by
the increased BC proliferation. Consistent with these
results, there were no difference at proliferation rate
between BC cells transfected with NC and miR-361-5p
under glucose deprivation (Fig. 3a). However, numbers
of invading cells of the anti-miR-361-5p group were
higher than that of the NC group, suggesting that glycolysis
was not necessary for the enhanced BC invasion (data not
shown). Thus, it is possible that miR-361-5p suppressed
BC cells growth by regulating the balance between
glycolytic metabolism and mitochondrial oxidative
phosphorylation. With this idea, we measured the
metabolites of miR-361-5p transfected MDA-MB-231 cells. We
found that metabolites of the TCA cycle were increased
and the intermediates of glycolysis were decreased (Fig. 3b
and c). Morever, levels of glucose from miR-361-5p cells
were significantly higher compared with control group,
whereas levels of the product of glycogenolysis,
glucose-6phosphate, were decreased (Fig. 3d and e). Meanwhile, we
found that miR-361-5p cells consumed less glucose and
released less lactate into the substrate (Fig. 3f ). To
conclude, these results suggest that miR-361-5p controls the
redirection of BC cellular glucose metabolism and inhibits
To explore whether miR-361-5p suppressed glycolysis
due to the increased oxidative capacity, we measured the
rate of glucose uptake of miR-361-5p BC cells treated
with rotenone (an inhibitor of mitochondrial respiration)
or etomoxir (an inhibitor of fatty acid oxidation). We
observed that glucose uptake was declined in the presence
of rotenone and etomoxir (Fig. 3g and h). These data
demonstrated that declined glycolysis in miR-361-5p cells
does not attribute solely to increased mitochondrial
FGFR1 mediated the anti-glycolytic function of miR-361-5p by regulating the activity of PDHK1 and LDHA
MiRNAs were well known to play crucial roles in various
cancers by inhibiting their target genes. Using
bioinformatic methods, we happened to find that FGFR1 might be
a potential target of miR-361-5p among the numerous
candidates, for it was reported to promote aerobic glycolysis
by phosphorylating several glycolytic enzymes [
results showed that Dicer knockdown markedly increased
the expression level of FGFR1 mRNA in MEFs, indicating
that FGFR1 might be targeted by miRNAs (Fig. 4a).
Consistent with these findings, miR-361-5p significantly
decreased the expression levels of both FGFR1 mRNA and
protein (Fig. 4a). To decide whether FGFR1 was the direct
target of miR-361-5p, we constructed luciferase reporters
containing FGFR1–3’UTR with a conserved miR-361-5p
binding sequence or mutated miR-361-5p binding sequence.
It was observed that miR-361-5p inhibited the luciferase
activity of WT 3’UTR-reporter, but not the MUT
3’UTR-reporter (Fig. 4b). These results demonstrated
that FGFR1 was a direct target of miR-361-5p. To test
whether FGFR1 was the functional target of miR-361-5p
in inhibiting glycolysis, we measured the extracellular
acidification rate (ECAR) which was the surrogate of
glycolysis. We found that MDA-MB-231 cells transfected
with miR-361-5p or siFGFR1 showed decreased level of
extracellular acidification rate compared with control cells
(Fig. 4c). Meanwhile, overexpression of FGFR1 in BC cells
reverted decreased glucose uptake and lactate production
of miR-361-5p-transfected cells (Fig. 4d and e). Conversely,
siFGFR1 markedly reverted the enhanced rate of glucose
uptake and lactate production in
anti-miR-361-5p-transfected BC cells (Fig. 4f and g).
It was reported that GLUT1 could translocate from
intracellular membrane compartments to the plasma
membrane in epithelial cells to promote glucose uptake
]. Thus, we used immunofluorescence staining to
evaluate the localization of GLUT1 protein. We found
that in contrast to control MDA-MB-231 cells, where
GULT1 was mostly observed in plasma membrane,
GLUT1 was found in the cytoplasm of
miR-361-5ptransfected cells (Fig. 4h). However, FGFR1 reversed the
effect of miR-361-5p on subcellular translocation of GLUT1
(Fig. 4h). Next, we measured the activation of proteins
correlated with glycolytic pathway in MDA-MB-231 cells.
Using western blot analysis, we found that miR-361-5p
transfection downregulated the phosphorylation of PDHK1
and LDHA in BC cells. However, overexpression of FGFR1
significantly increased the phosphorylation of these proteins
in miR-361-5p-overexpressed or control cells (Fig. 4i).
Studies have shown that phosphorylation of PDHK1
and LDHA is associated with enhanced enzyme activity
and leads to glycolysis increase [
]. Therefore, the
results indicated that FGFR1 reverted the anti-glycolytic
function of miR-361-5p by upregulating the activity of
PDHK1 and LDHA (Fig. 4i). Consistently, colony
formation assays indicated that FGFR1 restored the proliferation
of miR-361-5p-transfected cells (Fig. 4j). However, siFGFR1
transfection failed to convert the increased invasion ability
of anti-miR-361-5p-transfected cells (Fig. 4k), indicating
that FGFR1 could not affected the anti-metastatic function
of miR-361-5p in BC cells. So we argued that there might
be other targets which mediated the anti-metastatic
phenotype of miR-361-5p in BC cells.
MiR-361-5p suppresses the invasion and metastasis of BC by targeting MMP-1
To further elucidate the anti-metastatic function of
miR361-5p in BC, bioinformatic methods were used to
predict other targets of miR-361-5p. MMP-1 was noted
because it contains a putative target sequence of
miR-3615p in the 3’UTR (Fig. 5a) and it was closely correlated
with tumor cell invasion [
]. To examine whether
MMP-1 was a direct target of miR-361-5p, luciferase
reporters containing MMP-1-3’UTR with a conserved
miR-361-5p binding sequence or mutated miR-361-5p
binding sequence were constructed. We found that
miR361-5p was able to decrease the luciferase activity of
WT 3’UTR-reporter, but not the MUT 3’UTR-reporter
(Fig. 5b). Consistently, we detected that overexpression
of miR-361-5p in BC cells decreased the expression level
of MMP-1 mRNA and protein, whereas anti-miR-361-5p
increased the expression level of MMP-1 mRNA and
protein (Fig. 5c). These results suggested that
miR-3615p directly inhibited MMP-1 expression by binding to
its 3′-UTR. Next, we performed restoration assays in BC
cells to decide if MMP-1 mediated the anti-metastatic
function of miR-361-5p. As shown in Fig. 5d, on one
hand, MCF-7 cells transfected with siMMP-1 showed
decreased invasion ability compared with control cells,
on the other hand, siMMP-1 restored the enhanced
invasion ability of miR-361-5p-inhibited MCF-7 cells. In
contrast, MMP-1 overexpression in MDA-MB-231 cells
raised the number of invading cells and restored the
decreased the ability of invasion in
miR-361-5p-transfected cells. Similarly, the results of in vivo assays showed
that MMP-1 reverted the anti-metastasis phenotype of
miR-361-5p in BC cells (Fig. 5e). Altogether, these results
indicated that miR-361-5p suppressed the invasion and
metastasis of BC by targeting MMP-1.
Clinical relevance of miR-361-5p, FGFR1 and MMP-1 expression in BC patients
Given that miR-361-5p downregulated FGFR1 and MMP-1
in BC cells to suppress tumor progression, we next
explored whether this relationship existed in clinical
samples. We found that BC tissues with high
miR-3615p expression showed low IHC score of FGFR1 and
MMP-1. Reversely, BC tissues with low miR-361-5p
expression tended to show high expression of FGFR1 and
MMP-1 (Fig. 6a). A reverse relationship of expression
between miR-361-5p and FGFR1 or MMP-1 was observed
(Fig. 6b). These data support the mechanism that
miR361-5p targets FGFR1 and MMP-1 respectively, inhibits
BC cell glycolysis and proliferation, invasion and metastasis,
and finally suppresses BC progression (Fig. 6c).
In this study, we found that the expression of miR-361-5p
was downregulated in breast cancer and was associated
with poor prognosis. As a tumor suppressor, miR-361-5p
inhibited BC cells aerobic glycolysis and proliferation by
directly targeting FGFR1, a promoter of glycolytic pathway.
Meanwhile, miR-361-5p also targeted MMP-1 to suppress
BC cells invasion and metastasis. Thus, our results provide
evidence that miR-361-5p inhibits glycolytic metabolism,
proliferation and invasion of BC cells and reveal the
specific regulatory mechanism of miR-361-5p in BC,
suggesting that miR-361-5p and its target genes may
serve as potential therapeutic targets for BC patients.
Emerging evidence has demonstrated that microRNAs
play crucial roles in multiple biological and pathological
processes of cancer, including tumor cell proliferation,
invasion and metastasis [
]. It has been reported that
some miRNAs acted as tumor regulators and could reduce
the expression of many target oncogenes [
known as a tumor suppressor, was reported to play
functional roles in several cancers [
]. However, the role of
miR-361-5p and its regulatory mechanism in BC have
rarely been discussed. By coincidence, our previous study
has demonstrated that miR-361-5p inhibits the malignant
phenotype of colorectal and gastric cancer by targeting
SND1 . Morever, another recent study also showed that
increased expression of miR-361-5p predicted improved
BC survival [
]. Similarly, Cao et al. reported that the
clinical outcomes of patients with positive miR-361-5p
expression was better than that of patients with negative
miR-361-5p expression [
]. Consistent with those studies,
we observed that miR-361-5p was down-regulated in breast
cancer compared with normal breast tissue, and decreased
miR-361-5p expression was correlated with poor DFS.
Nevertheless, we found that decreased miR-361-5p
expression was also correlated with larger tumor size, lymph node
metastasis and higher TNM stage, which was not
significantly evidenced in the previous study. This difference in
results might be caused by the distinct grouping modes
between the two studies. In addition, we were the first to
analyze the functional roles of miR-361-5p in BC cells and
found that miR-361-5p suppressed breast cancer cells
proliferation, invasion and metastasis both in vitro and in
vivo. Based on these results, miR-361-5p could be
recognized as a tumor suppressive miRNA in BC.
The type of glucose metabolism in tumors can shift
widely between glycolysis and OXPHOS [
the fact that increased glucose uptake is associated with
enhanced biosynthetic metabolism, the specific molecular
mechanisms that upregulate glycolysis and anabolic
biosynthesis are of great importance. Accumulating evidence
showed that dysregulation of multiple metabolic enzymes
might contribute to the aerobic glycolysis process,
including glucose transporter 1 (GLUT1), pyruvate
dehydrogenase kinase 1 (PDHK1) and lactate dehydrogenase (LDHA)
]. Previous studies found that phosphorylation of
LDHA and PDHK1 by FGFR1 was common in diverse
cancers which could activate LDHA and PDHK1 and
promote glycolysis [
]. Consistent with these studies,
we found that FGFR1 reverted the anti-glycolytic function
of miR-361-5p by upregulating the activity of PDHK1 and
LDHA, which enrich the mechanism that miR-361-5p
inhibited BC cells glycolysis and proliferation. However,
there might be many other questions remain to be solved,
such as possible involvement of other targets and the
detailed post-translational modifications of the metabolic
enzymes. The investigation of these issues will no doubt
enrich the mechanism that miR-361-5p and FGFR1
regulate BC cells glycolysis and proliferation.
Due to the fact that FGFR1 overexpression only partly
converted the invasion ability of miR-361-5p-transfected
cells, we questioned the possibility that another target of
miR-361-5p might mediate its anti-metastatic phenotype.
MMP-1, which belongs to a large family of peptidases,
was identified to cleave the components of extracelluar
matrix and play crucial roles in the movement of epithelial
]. Consistent with that, we demonstrated that
MMP-1 was a direct functional target of miR-361-5p and
mediated the anti-metastatic phenotype of miR-361-5p in
BC cells. Interestingly, a previous study showed that the
activation of FGFR1 might be induced in response to
]. Thus, the regulatory relationship between
FGFR1 and MMP-1 downstream to miR-361-5p in breast
cancer remains to be investigated in the future.
Meanwhile, considering that several studies reported
miR-3615p to act its function by targeting some other factors, such
as Twist1, VEGFA and FOXM1 [
], it remains to be
explored whether other targets may contribute to the
anti-tumor effect of miR-361-5p.
Our study found that miR-361-5p was down-regulated
in breast cancer tissues and the expression of miR-361-5p
was positively associated with prognosis in BC patients.
Functional studies showed that overexpression of
miR361-5p suppressed the glycolysis and proliferation of breast
cancer cells by targeting FGFR1, the invasion and
metastasis by targeting MMP-1. An inverse expression pattern was
also found between miR-361-5p and FGFR1 or MMP-1 in
clinical samples. Our results suggest that miR-361-5p and
its target genes may serve as therapeutic targets in breast
BC: Breast cancer; DFS: Disease-free survival; EACR: Extracellular acidification
rate; EMT: Epithelial-mesenchymal transition; FGFR1: Fibroblast growth factor
receptor 1; GLUT1: Glucose transporter 1; LDHA: Lactate dehydrogenase A;
microRNA: miRNA; MMP-1: Matrix metalloproteinase-1; ORF: Opening reading
frame; OXPHOS: Oxidative phosphorylation; PDHK1: Pyruvate dehydrogenase
kinase 1; PTEN: Phosphatase and tension homolog deleted on chromosome
ten; SND1: Staphylococcal nuclease domain-containing protein 1;
UTR: Untranslated region
This work was supported by the National Natural Science Foundation of China
(No. 81372838), the National Natural Science Foundation of China (No. 81701705),
the Found of Distinguished Young Scholars of the Second Affiliated Hospital of
Harbin Medical University.
FM and LZ carried out most of the experimental work; LM conducted the
analysis of data; YZ and JZ performed the molecular cloning and animal
experiments; FM and BG designed the project and wrote the manuscirpt. All
authors have read and approved the manuscript.
Ethics approval and consent to participate
This study was approved by the Ethical Committee of the Second Affiliated
Hospital of Harbin Medical University. The study was performed according to
the ethical standards of Declaration of Helsinki and all the patients provided
written informed consent for the use of tissues. Additionally, the animal
experiments were performed in accordance with the Guide for the
Administration of Affairs Concerning Experimental Animals.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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