MiR-124 inhibits the migration and invasion of human hepatocellular carcinoma cells by suppressing integrin αV expression

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 miR-124 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 miR-124 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.

Scientific RepoRts | 7:40733 | DOI: 10.1038/srep40733 stemness of tumor cells 13,14 . Integrin α Vβ 3 antagonists have shown encouraging anti-tumor effect on glioma in the initiatory trials 15 . Overexpressed integrin α Vβ 3 helps glioblastoma cells to escape senescence by activation of the cytoskeletal regulatory kinase PAK4 16 and promotes the migration of HCC 17 . However the dysregulation of integrin α Vβ 3 in cancer cells especially in HCC is not well understood. The subunit of integrin α V 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 genes 18 . The promoter region of ITGAV gene encoding integrin α V subunit, contains four GC-rich motifs. In our previous study 19 we observed that Sp1 bound to integrin α V subunit gene promoter and activated its transcription in the pathway of integrin α V transcription regulated by sulfatide. In this study, we tried to explore further the mechanism of sulfatide inducing integrin α V subunit gene expression on the base of our previous results. In analysis of integrin α V 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 α V subunit implicated.

Results
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 miR-124 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 α V expression. The miR-124 and integrin α V 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 α V subunit was often low when miR-124 level was high. A strong correlation was noted between miR-124 and integrin α V 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 α V subunit expression. Further in Hep3B, HepG2 and SK-Hep-1 cells which expressed high levels of integrin α V, 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 α V 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 α V subunit, we next investigated the possible regulation of integrin α V 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 α V subunit gene transcription (Fig. 3B). Sp1 as an important transcription factor for integrin α V gene transcription was demonstrated in our previous study 19 . 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 psiCHEC TM -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 HEK-293T 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 α V subunit gene 19 , and the down-regulation of Sp1 by miR-124 might lead to the down-regulation of integrin α V subunit gene transcription. In order to confirm miR-124 regulation of integrin α V expression, we investigated and observed the mRNA and protein expression levels of Sp1 and integrin α V subunit in SMMC-7721 and BEL-7404 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 miRNAs 20 or shRNAs 21 . 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 α V 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 α V subunit reduced significantly in both SMMC-7721 and BEL-7404 cells ( Fig. 4B and C). The mRNA expression levels of Sp1 and integrin α V 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 α V 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 α V 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 α V significantly stimulated the cell migration ability, while integrin α V knockdown had a significant inhibitory effect (Fig. 4E). These suggested that ectopic expression of miR-124 contributed to the down-regulation of integrin α V subunit gene expression.

MiR-124 inhibits HCC metastasis in vivo.
Since miR-124 overexpression resulted in downregulation of integrin α V 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 α V subunit expression levels were elevated in all these cases (Fig. 5C). This implied negative association of miR-124 with integrin α V expression levels.

Discussion
More and more studies have revealed that miRNAs abnormally expressed in human cancers involve in tumor progression as either oncogenes or tumor suppressors 22,23 . Moreover, the abnormal expression of miRNAs may have molecular functional links with the malignant hallmarks such as aberrant cell growth, angiogenesis, invasion and metastasis 24 . Hence, investigation of miRNA regulation network could help us understand the progression of tumor diseases and stratify their prognostic risks. MiR-124 is abundantly expressed in the brain tissues, but the roles of miR-124 were also noted in gastric cancer 4 and nasopharyngeal carcinoma 6 . In this study, we demonstrated that miR-124 was involved in the regulation of migration and metastasis of human hepatocellular carcinoma cells (SMMC-7721 and BEL-7404). It was noted in our study that miR-124 played an important negative role in regulation of migration and metastasis of HCC cells. MiR-124 significantly slowed the wound healing and transmembrane migration of HCC cells. Through further analysis we noted that the expression levels of miR-124 in 11 HCC cell lines and other cancer cells was strikingly inversely correlated with the expression of integrin α V which is the driver molecule for the anchorage independence and migration of tumor cells 14 . This implied that miR-124 regulation of hepatocellular carcinoma cell migration might be related with the expression of integrin α V that is important in HCC cell migration and metastasis. We further investigated the possible regulation of integrin α V 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 cells 25 . 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 α V subunit expression since integrin α V subunit gene transcription relied on Sp1 as the major transcription factor in human hepatocellular carcinoma cells as demonstrated in our previous study 19 . 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 α V subunit expression. Herein, we confirmed Sp1 as a direct target of miR-124, which played an important role in regulating integrin α V 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 events 26,27 . In our previous studies, it had been demonstrated that the expression level of integrin α V was elevated in HCC specimens and hepatoma cells 17,19 . 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 α V gene promoter activation in HCC. Furthermore, the transcription expression of the integrin α V subunit gene is regulated by transcriptional factors Sp1 19 . In current study, we noted that the expression level of integrin α V 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-429 17,28-30 . 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 α V gene 31 . In our data, we identified Sp1 mRNA as the direct target of miR-124, and integrin α V 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 proliferation [4][5][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 carcinogenesis 34 . Our data further indicated that high level of miR-124 expression inhibited the wound healing, migration and invasion in HCC and suppressed integrin α V 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 α V 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 α V expression in liver cancer and a viable anticancer therapeutic approach for HCC metastasis. Wound healing assays. 5.0 × 10 5 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 24 hours under a microscope.

Materials and Methods
Transmembrane migration assay. Human hepatocellular carcinoma SMMC-7721, Hep3B, HepG2, SK-Hep-1and non-tumor hepatocyte LO2 cells were transfected with miR-124 or control plasmids and 48 h after transfection, the cells were transferred into the upper chamber of the Millicell inserts with 8-μ m pore size polyporous membrane (Millipore, Billerica, MA, USA) in a serum-free DMEM with a cell density of 5 × 10 6 /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 μ g 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 Na 3 VO 4 , 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 α V (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-7721 MiR-124 ) and control cells (SMMC-7721 Mock ) (5 × 10 6 ) 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 α V 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 α V 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.