MiR-124 inhibits the migration and invasion of human hepatocellular carcinoma cells by suppressing integrin αV expression
MiR-124 inhibits the migration and invasion of human hepatocellular carcinoma cells by suppressing integrin αV expression
Qian Qian Cai
Yi Wei Dong
Jun Xia Guo
Chun Yi Zhang
Xing Zhong Wu
OPEN Tumor metastasis is the major cause of cancer-related death especially in human hepatocellular carcinoma (HCC). Although microRNAs have been implicated in tumor development, the roles of miR124 in HCC metastasis are still not well understood. We conducted functional analysis in this study to investigate miR-124. We observed that miR-124 significantly retarded the wound healing and migration of HCC SMMC-7721 and BEL-7404 cells. Further analysis indicated miR-124 directly targeting the transcriptional factor Sp1 which is an important transcription factor for the integrin αV subunit gene transcription. Co-transfection of miR-124 with the luciferase reporter containing Sp1 3′ untranslated region (UTR) significantly suppressed the luciferase activities. While mutation of the binding site of miR124 in Sp1 mRNA 3′UTR completely abrogated the suppression of miR-124. Overexpression of miR-124 resulted in robust downregulation of Sp1 and integrin αV expression at either mRNA or protein level. Ectopic expression of miR-124 in HCC dramatically repressed the wound healing and migration in vitro and tumor metastasis in mouse experiments. Our findings demonstrated that miR-124 played as an important role in regulation of integrin αV expression in HCC, and reintroduction of miR-124 might be an alternative therapeutic strategy for controlling integrin αV expression in HCC.
MicroRNA (miR) is a single-stranded, non-coding RNA molecule of 22–25 nucleotides, which are a family of
regulatory molecules involved in controlling gene expression, translation and cellular biological behaviors, even
in tumorigenesis. MiR-124 was first identified by cloning studies in mice1 and is most abundant, well-conserved
and specific microRNA in the brain2. In recent years, some studies indicated that the CPG island (or CG site)
methylation of miR-124 gene is associated with the advanced tumors and the recurrence in patients with renal
carcinoma3. MiR-124 is considered as one of the expression-silenced miRNAs in tumors including gastric cancer
cells4, breast cancer cells5 and nasopharyngeal carcinoma6. MiR-124 was also one of the down-expressed
microRNAs in human hepatocellular carcinoma (HCC)7.
Hepatocellular carcinoma (HCC) is one of the most malignant tumors with poor prognosis largely due to
remote metastasis and postsurgical recurrence. There are more than half a million newly diagnosed patients each
year. The tumor progression and metastasis are the major cause of cancer-related deaths in patients with HCC.
During the process of tumor invasion and metastasis, integrins act as crucial transducers of bidirectional cell
signaling, regulating cell adhesion, migration, and tissue remodeling8,9. Integrins are a family of transmembrane
adhesion receptors composed of 18α and 8β subunits that interact non-covalently to form 24 different
heterodimeric receptors10. Because integrins are the primary receptors to extracellular matrix (ECM) molecules for
cellular adhesion, the heterodimer integrin on the cell surface allows cells to recognize and responds to a variety of
signals from ECM environments. Activation and elevated expression of integrin-coupled signaling effectors have
been implicated in the induction of angiogenesis and metastasis of human cancers11,12. Overexpressed integrin
αVβ3 forms the oncogenic unit with Src to promote tumor cell proliferation and drives the malignance and
stemness of tumor cells13,14. Integrin αVβ3 antagonists have shown encouraging anti-tumor effect on glioma in
the initiatory trials15. Overexpressed integrin αVβ3 helps glioblastoma cells to escape senescence by activation
of the cytoskeletal regulatory kinase PAK416 and promotes the migration of HCC17. However the dysregulation
of integrin αVβ3 in cancer cells especially in HCC is not well understood. The subunit of integrin αV is encoded
in the gene of ITGAV. Specificity protein 1 (Sp1) belongs to the specificity protein/Krüppel-like factor family of
transcription factors that recognizes and binds, via three Cys2-His2 zinc finger motifs localized at its carboxyl
terminus, GC-rich sites, thus regulating the transcription of target genes18. The promoter region of ITGAV gene
encoding integrin αV subunit, contains four GC-rich motifs. In our previous study19 we observed that Sp1 bound
to integrin αV subunit gene promoter and activated its transcription in the pathway of integrin αV transcription
regulated by sulfatide. In this study, we tried to explore further the mechanism of sulfatide inducing integrin αV
subunit gene expression on the base of our previous results. In analysis of integrin αV regulation of cell migration
in HCC we identified miR-124 as a novel regulator of the pathway, targeting Sp1 directly in HCC. Moreover,
overexpression of miR-124 inhibited invasion and metastasis of HCC through integrin αV subunit implicated.
MiR-124 suppresses the wound healing and migration capability of HCC. In order to investigate
the roles of miR-124 in cell migration and metastasis-related behaviors, we performed the wound healing assay
and transmembrane migration assay. The miR-124 expressional construct was transfected into human
hepatocellular carcinoma cells of either SMMC-7721 or BEL-7404 cells, to achieve over-expression of miR-124. 72 hours
after wound healing in SMMC-7721 cells, the scratched wound in miR-124 group was not closed as fast as the
control group. The relative closure in miR-124 group was significantly slower than control (Fig. 1A). Moreover,
the migrated cells through the polyporous membrane were significantly fewer in miR-124 group than control
cells (Fig. 1B). The invasion capability of SMMC-7721 cells transfected with miR-124 through Matrigel in
miR124 group was lower than control (data not shown). To confirm the role of miR-124 in migration further, both
SMMC-7721 and BEL-7404 cells were transfected with specific miR-124 inhibitor. As a result, the group of
antagomirs showed significantly faster wound healing compared to control group in both cell lines (Fig. 1C). These
suggested that miR-124 suppressed cell migration in HCC.
Since cell migration is usually controlled by integrins, we then analyzed the relationship between miR-124 and
integrin αV expression. The miR-124 and integrin αV expression levels were measured in 11 different non-tumor
and tumor cell lines with qPCR (Fig. 2A). We interestingly observed that the expression level of integrin αV
subunit was often low when miR-124 level was high. A strong correlation was noted between miR-124 and integrin αV
subunit expression levels with the correlation coefficient −0.605 (P < 0.01) (Fig. 2B), indicating that miR-124 was
associated with the down regulation of integrin αV subunit expression. Further in Hep3B, HepG2 and SK-Hep-1
cells which expressed high levels of integrin αV, the inhibitory effect on the cell migration by miR-124 was
investigated (Fig. 2C). The inhibitory rate in Hep3B, HepG2 and SK-Hep-1 cells was 86%, 93% and 95%, more robust
than those (40%) in SMMC-771 cells (Fig. 2C bottom right). However the inhibition in LO2 cells was slight since
the basal levels of migration and integrin αV expression were quite low.
MiR-124 directly regulates the transcription factor Sp1. MiR-124 is encoded in three genomic loci
[miR-124a-1 (8p23.1), miR-124a-2 (8q12.3), and miR-124a-3 (20q13.33)], but these three miR-124a loci give rise
to only one mature miRNA, miR-124 (Fig. 3A top). Having found that miR-124 inhibited wound healing and
migration which were related to down-regulating integrin αV subunit, we next investigated the possible
regulation of integrin αV subunit expressions by miR-124. As miRNA functions in the post-transcriptional regulation
of gene expression by targeting 3′ untranslated region (UTR) of mRNAs, we analyzed and screened the
possible target by bioinformatics. The predicated mRNA target for mature miR-124 was analyzed using four online
algorithms including miRanda, TargetScan, PicTar and microRNA.org (Fig. 3A bottom). The analysis results
indicated that there were 53 transcription factors that either matched miR-124 target or were expressed in HCC.
Among them Sp1, Sp3 and ETS1 were associated with integrin αV subunit gene transcription (Fig. 3B). Sp1 as
an important transcription factor for integrin αV gene transcription was demonstrated in our previous study19.
Sp1 3′UTR sequence possesses two conserved binding motifs for miR-124, which are well conserved from worm
to human being (Fig. 3C). In order to confirm that miR-124 directly targets Sp1 3′UTR, we performed luciferase
reporter assay. The wild-type full length of Sp1 mRNA 3′UTR was cloned into the downstream of the Renilla
luciferase gene in the psiCHECTM-2 Vector with a firefly luciferase coding gene as internal control. Besides, two
mutant vectors contained 3 mutated bases in the predicted binding sites were constructed (Fig. 3D). HEK-293T
and HeLa cells were co-transfected with these reporter constructs and miR-124 or control vectors. The group
transfected with miR-124 rather than control vector significantly suppressed the luciferase activity of reporter
genes containing 3′UTR of Sp1 in both cells. However, the suppression was completely abrogated in both
HEK293T and HeLa cells when the binding site 1 was mutated (Fig. 3E). The mutation of the binding site 2 resulted
in the recovery of the reporter activity in HeLa cells. These results indicated that miR-124 directly targeted Sp1
mRNA via the putative binding sites in the 3′UTR.
MiR-124 inhibits integrin αV expression. Sp1 is an important transcription factor for integrin αV
subunit gene19, and the down-regulation of Sp1 by miR-124 might lead to the down-regulation of integrin αV
subunit gene transcription. In order to confirm miR-124 regulation of integrin αV expression, we investigated
and observed the mRNA and protein expression levels of Sp1 and integrin αV subunit in SMMC-7721 and
BEL7404 cells with over-expression of miR-124. SMMC-7721 and BEL-7404 cells were infected with miR-124 or
control lentivirus which is an efficient, stable gene delivery tool in mammalian cells to induce stable gain- and
loss-of-function phenotypes for individual miRNAs20 or shRNAs21. The successful infection of miR-124
lentivirus was observed with fluorescence microscope (Fig. 4A) and the over-expression of miR-124 was validated in
the miR-124 group by RT-PCR measurement (Fig. 4A middle & right). In these cells the expression of both Sp1
and integrin αV was then analyzed. The results of RT-PCR and Western-blotting showed that in the cells with
over-expression of miR-124, the expression levels of Sp1 and integrin αV subunit reduced significantly in both
SMMC-7721 and BEL-7404 cells (Fig. 4B and C). The mRNA expression levels of Sp1 and integrin αV reduced
almost by 50% and 14% respectively in SMMC-7721 cells, and 84% and 53% in BEL-7404 cells. The protein
levels of expression of Sp1 and integrin αV also reduced in miR-124 group by 85% and 74% in SMMC-7721,
and 77% and 58% in BEL-7404 cells compared to control group. After transfection of anti-miR-124 inhibitor in
SMMC-7721 and BEL-7404 cells, the expression of miR-124 was inhibited, and the expression levels of Sp1 and
integrin αV subunit were simultaneously increased by 1.4 and 1.1 folds in SMMC-7721 cells, and 4 and 1.6 folds
in BEL-7404 cells compared to negative control group (Fig. 4D). The overexpression of integrin αV significantly
stimulated the cell migration ability, while integrin αV knockdown had a significant inhibitory effect (Fig. 4E).
These suggested that ectopic expression of miR-124 contributed to the down-regulation of integrin αV subunit
MiR-124 inhibits HCC metastasis in vivo. Since miR-124 overexpression resulted in downregulation of
integrin αV subunit expression, then animal experiments were performed to evaluate the influence of miR-124
overexpression on HCC metastasis in vivo. We employed the SMMC-7721 cells stably expressing miR-124 and
green fluorescence protein (GFP) as the cell model for in vivo metastasis studies. Four weeks after the injection
by tail vein of nude mice, the metastasis foci were examined at the liver and lung of both miR-124 and control
groups. The hepatic metastasis and pulmonary metastases were first examined through organ anatomy and then
pathologic examination. Strikingly, the group of mice receiving miR-124 transfection showed significantly fewer
metastases colonies in the liver and lung by 79% and 77% respectively. However, the control group showed
significant more hepatic and pulmonary metastases foci (Fig. 5A). Parallel with the anatomy results, H&E staining
of lungs also showed that the number and size of micro-metastases foci were significantly more and larger in
control group than those in miR-124 group (p < 0.001, Fig. 5B). These results indicated that miR-124 was capable
of suppressing HCC metastasis in vivo.
We also analyzed human hepatocellular carcinoma gene expression data from TCGA (The Cancer Genome
Atlas), in which 58 cases included both mRNA expression and miRNA expression data. Interestingly, all of these
cases showed decreased miR-124 expression except 3 cases. While integrin αV subunit expression levels were
elevated in all these cases (Fig. 5C). This implied negative association of miR-124 with integrin αV expression levels.
integrin αV which is the driver molecule for the anchorage independence and migration of tumor cells14. This
implied that miR-124 regulation of hepatocellular carcinoma cell migration might be related with the expression
of integrin αV that is important in HCC cell migration and metastasis. We further investigated the possible
regulation of integrin αV subunit expression by miR-124 and noted that miR-124 has two conserved binding
sites on the 3′ untranslated region of transcription factor Sp1 mRNA. Through the luciferase reporter assay and
binding site mutation, the results showed that miR-124 directly targeted Sp1 mRNA. MiR-124 may act together
with miR-137 and miR-128 synergistically to regulate neural cells25. In this study we observed that miR-124 alone
down-regulated the expression of Sp1 efficiently. Moreover, down-regulation of Sp1 by miR-124 subsequently
inhibited integrin αV subunit expression since integrin αV subunit gene transcription relied on Sp1 as the major
transcription factor in human hepatocellular carcinoma cells as demonstrated in our previous study19. Our
current results further showed that overexpression of miR-124 reduced the expression levels of Sp1 and integrin α
V subunit significantly. In functional studies, reintroduction of miR-124 dramatically repressed the migration
and invasion of HCC in vitro and tumor metastasis in vivo. These results indicated that miR-124 functioned as a
negative regulator or tumor suppressor for the cell growth and migration in HCC, which might be related to its
repressing integrin αV subunit expression. Herein, we confirmed Sp1 as a direct target of miR-124, which played
an important role in regulating integrin αV gene transcription.
These findings suggest that miR-124 plays an important role in the metastatic and/or invasive potential of
HCC, which could be a potential therapeutic approach for HCC. Tumor metastasis is a combination of
biological events which requires the flow of erroneous but precisely coordinated basic cellular activities such as cell
migration-invasion, cell survival-apoptosis and cell proliferation. All of these processes require efficient
regulation of cell attachment and detachment, which recruit integrin receptors in this flow of events26,27. In our previous
studies, it had been demonstrated that the expression level of integrin αV was elevated in HCC specimens and
hepatoma cells17,19. Through analysis using online algorithm TargetScan, we found that among 53 transcription
factors screened Sp1 was not only predicted to match miR-124 target but also was the major transcription factor
required for integrin αV gene promoter activation in HCC. Furthermore, the transcription expression of the
integrin αV subunit gene is regulated by transcriptional factors Sp119. In current study, we noted that the expression
level of integrin αV significantly decreased in the cells overexpressing miR-124, but increased when the hepatoma
cells were treated with miR-124 antagomir. Sp1, as a member of the Sp/KLF family, can be the target of miRNAs
such as miR-223, miR-145, miR-129-5p and miR-42917,28–30. Meanwhile, post-translation modifications
including phosphorylation, acetylation, glycosylation and proteolytic processing also significantly affect the activity of
this protein, which can be an activator of integrin αV gene31. In our data, we identified Sp1 mRNA as the direct
target of miR-124, and integrin αV gene as subsequent target in HCC. Overexpression of miR-124 significantly
suppressed the luciferase reporter which contained Sp1 3′UTR and this suppression was interestingly abolished
by the mutation of the miR-124 binding site in Sp1 3′UTR, and overexpression of miR-124 led to a significant
reduction in Sp1 mRNA and protein levels, but the effect was reduced by miR-124 inhibitor.
Accumulating evidence has shed light on the critical role of miR-124 in the development and progression of
cancers, including oncogenesis and proliferation4–6,32,33. These reports shows that the expression of miR-124 was
reduced in carcinomas, which may result from multiple regulatory events, such as the methylation of CpG islands.
Our data is consistent with these reports that miR-124 was down-regulated in HCC. The low level of miR-124
expression may be a candidate biomarker for further prospective molecular stratification of cancer patients possibly
for prognosis prediction. Systemic delivery of miR-124 may perturb the hepatocyte growth and prevent
hepatocellular carcinogenesis34. Our data further indicated that high level of miR-124 expression inhibited the wound healing,
migration and invasion in HCC and suppressed integrin αV which is the malignant driver for anchorage
independence. Ectopic overexpression of miR-124 significantly reduced the metastasis foci in either liver or lung tissues in
nude mice experiments. High level of miR-124 expression in HCC directly resulted in low expression of Sp1, which
subsequently suppressed integrin αV subunit gene expression. Therefore, our findings identified a novel pathway
for miR-124 regulation of HCC metastasis, and miR-124 could possibly be an alternative strategy for controlling
integrin αV expression in liver cancer and a viable anticancer therapeutic approach for HCC metastasis.
Materials and Methods
Cell culture and transfection. Human hepatocellular carcinoma cell lines SMMC-7721, BEL-7404 and
non-tumor hepatocyte LO2 were obtained from Shanghai Institute of Biochemistry and Cell biology, Chinese
Academy of Science. SMMC-7721 and BEL-7404 cells were infected with lentivirus containing miR-124
(SMMC7721miR-124, BEL-7404miR-124) or control virus (SMMC-7721control, BEL-7404 control). HCC cells LM3, Hep3B and
HuH-7 were from Liver Cancer Institute of Zhongshan Hospital, Fudan University, Shanghai, China. Colorectal
cancer cells SW480 and SW620 were from Shanghai Tumor Hospital (Fudan University, Shanghai, China).
Hepatoma cells HepG2, SK-Hep-1, Lung cancer cells A549, cervical cancer HeLa and human embryonic
kidney HEK-293T cells were obtained from ATCC. These cells were cultured at 37°C with 5% CO2 in Dulbecco’s
Modified Eagle’s Medium (DMEM, Gibco, CA, USA) supplemented with streptomycin (100 U/ML), penicillin
(100 U/ML), and 10% newborn calf serum (PAA, Australia). The plasmid pLL 3.7-pre-miR-124 which carries
green fluorescence protein (GFP) was constructed as described in our previous study2. Plasmids
pLL-3.7-premiR-124 and psiCHECK-2 containing Sp1 3′UTR, mutated sequence or psiCHECK-2 control vector were
co-transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at the ratio of 1:3 in weight. The
miR124 inhibitor and their scramble control oligonucleotides were synthesized and provided by Gene Pharma Co.,
Ltd (Shanghai, China). 1 OD inhibitor oligonucleotides were diluted in 125 μl water (20 μM) provided by the
Wound healing assays. 5.0 × 105 cells were cultured into each well of 6-well plates after transfection, when cells
grew to almost confluence, the cell monolayers were scratched by a sterile micropipette tip, and three wounds were
made for each group. The scratched wound was photographed and measured every 24hours under a microscope.
Transmembrane migration assay. Human hepatocellular carcinoma SMMC-7721, Hep3B, HepG2,
SK-Hep1and non-tumor hepatocyte LO2 cells were transfected with miR-124 or control plasmids and 48h after transfection,
the cells were transferred into the upper chamber of the Millicell inserts with 8-μm pore size polyporous membrane
(Millipore, Billerica, MA, USA) in a serum-free DMEM with a cell density of 5 × 106/mL. As the chemoattractant,
DMEM containing 10% fetal calf serum was added to the lower chamber. Cell migration was allowed to proceed for
12 hours at 37 °C in culture incubator. Invasion assay was performed in the same process but the insert membrane was
pre-coated with 20 μg Matrigel and pre-incubated 1 hour to reconstitute a basement membrane. Tumor cell invasion
was allowed to proceed for 24 hours at 37 °C in culture incubator. After incubation, non-migrated or uninvaded cells
were removed from the inner part of the insert by using a cotton swab. Cells that had migrated or invaded through the
membrane were fixed with methanol and stained with 0.5% crystal violet, imaged, and counted under a microscope.
Western blotting analysis. SMMC-7721 or BEL-7404 cell, transfected with miR-124 construct or with
miR-124 inhibitor oligonucleotide for 48 h, were harvested and lysed in lysis buffer (1%SDS containing 50 mM
NaF, 1.5 mM Na3VO4, 0.5 mM PMSF). Proteins was fractionated on 10% SDS-polyacrylamide gels and transferred
to a polyvinylidene fluoride membrane, followed by immunoblotting with the primary antibodies against Sp1
or integrin αV (Santa Cruz, CA, USA). Primary antibodies were then detected using horseradish peroxidase
(HRP)-conjugated secondary antibodies and visualized using enhanced chemiluminescence detection kit (Pierce,
Thermo, Rockford, IL, USA). The images were captured by an E/M CCD camera (Tenon, Shanghai, China), and
the density of protein bands was analyzed and summarized.
RNA extraction and polymerase chain reaction (PCR). Total RNA was isolated from SMMC-7721
or BEL-7404 cells using TRIzol reagent (Life Technologies, Garlsbad, CA, USA), and the RNA extract was
subjected to reverse transcription (RT) reaction using the M-MLV reverse transcriptase (TAKARA, Dalian, China)
according to the manufacturer’s instructions. The polymerase chain reaction was performed in a thermocycler
(FXC-connect, Bio-Rad, Singapore). The relative expression levels of the target genes were evaluated on the base
of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a reference gene, with the principle of SYBR Green
technology. The data analyses were performed using the 2−ΔΔCt method.
Luciferase Reporter Assay. HeLa or HEK-293T Cells were co-transfected with the indicated plasmid and
a dual luciferase reporter constructs containing Sp1 mRNA 3′ untranslated region cloned into the 3 prime of
renilla luciferase gene of psiCHECK-2 vector. Fire fly and renilla luciferase activities were determined using the
Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) with an illuminometer (Lumat LB 9507,
Berthold, Germany) according to manufacturer’s instructions. Data were calculated by normalizing luminescence
of Fire fly luciferase to that of Renilla luciferase.
In vivo metastasis assays. After lentivirus infection and selection, SMMC-7721 cells that stably expressed
miR-124 (SMMC-7721MiR-124) and control cells (SMMC-7721Mock) (5 × 106) were injected into 6-week-old female
nude mice through the tail veins (6 mice/group) for the assessment of in vivo metastasis. Four weeks after
injection, the livers and lungs were isolated and fixed in 10% buffered formalin for hematoxylin and eosin (H&E)
staining. The numbers of the intrahepatic metastatic foci (visible and micro) in the liver and lungs were then
counted. All experiments involving animals were performed according to the Animals (Control of Experiments)
Ordinance and approved by the Animal Ethics Committee of Fudan University Shanghai Medical College with
the permit number of 20140226-001. The BALB/C athymic nude mice for this experiments were from SLAC LAB
ANIMAL LTD, CO. (Shanghai, China). All the animals used in this study were approved by Shanghai Municipal
Government with the permit number of 2015000510941.
Bioinformatics analysis. The targets of miR-124 were analyzed using four online algorithms including
miRanda, TargetScan, PicTar and microRNA.org. All the transcription factors screened were the predicted targets
of miR-124 and all of them were important in HCC. The network for targeted transcription factors and integrin
αV gene was made by Cytoscape (version 3.3.0, NRNB, US, http://www.cytoscape.org).
HCC sample analysis. The human hepatocellular carcinoma data were downloaded and analyzed from
TCGA (The Cancer Genome Atlas, https://genome-cancer.ucsc.edu/proj/site/hgHeatmap/). The data included
both gene expression (IlluminaHiSeq) and miRNA expression (IlluminaHiseq). In 58 cases both integrin αV and
miR-124 expression were measured in the same sample.
Statistical analysis. Data are presented as means ± SD. Statistical differences were measured using one-way
ANOVA and Student’s t test. Chi-square test was used for the analysis of ratio difference.
This work was supported by grants from National Natural Science Foundation of China (31570800, 31400689).
Q.C. and Y.D. did the experiments; R.W., Q.C., J.X.G. and J.P. did partial experiments; Y.D., R.W., Q.C. and Y.L
interpreted the data. R.W., Q.C. and Y.D. drafted the manuscript; C.Z. revised the draft and X.Z.W. designed the
study and revised the manuscript. All authors approved the final manuscript.
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Cai, Q. Q. et al. MiR-124 inhibits the migration and invasion of human hepatocellular
carcinoma cells by suppressing integrin αV expression. Sci. Rep. 7, 40733; doi: 10.1038/srep40733 (2017).
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