The human HCC cell line MHCC97H was kindly provided by the Stem Cell Bank of the Chinese Academy of Sciences. The MHCC97H cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. Recombinant human CCL2 was purchased from PeproTech (Rocky Hill, NJ, USA). Cyclopamine was purchased from Selleck Chemicals (Houston, TX, USA). The MHCC97H cells in log phase growth were cultured in 6-well plates in media containing only 1% FBS for 24 h before the drug treatment. Subsequently, the drugs were administered at indicated concentrations in medium containing 1% FBS and the plates were incubated for another 48 h before a Matrigel invasion assay was performed. The antibodies were purchased from different resources as follows: CCR2-specific rabbit polyclonal antibody (Proteintech, Chicago, IL, USA), Ptch-specific rabbit polyclonal antibody (Proteintech), sonic Hh homolog (SHH)-specific rabbit polyclonal antibody (Proteintech), SMO-specific rabbit polyclonal antibody (Bioworld Technology, Minneapolis, MN, USA), Gli-1-specific mouse monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), E-cadherin-specific rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), Snail-specific rabbit monoclonal antibody (Cell Signaling Technology), vimentin-specific rabbit monoclonal antibody (Cell Signaling Technology) and β-actin specific mouse monoclonal antibody (Santa Cruz Biotechnology). Horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG (Santa Cruz Biotechnology) were used as the secondary antibody.

Loss-of-function analysis was performed using siRNAs targeting Gli-1 and CCR2 which were purchased from GenePharm (Shanghai, China): siRNA against Gli-1 (5′-GGCUCAGCUUGUGUGUAAUTT-3′, 5′-AUUACACACAAGCUGAGCCTT-3′), siRNA against CCR2 (′-5CTGTCCAC-3′, ′-5CCCAAAGACCCACTCAT-3′) and a negative control siRNA (′-5UUCUCCGAA-3′, ′-5ACGUGACACGUUCGGAGA-3′). The cells were seeded in 6-well plates and were transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen; Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. The knockdown of each target gene was confirmed by western blot analysis. The cells were used for subsequent experiments 48 h after transfection.

Total RNA was extracted from the HCC cells using the Fastgen1000 RNA isolation system (Fastgen, Shanghai, China) according to the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara Bio, Dalian, China). Real-time PCR was used to quantitatively examine the expression of E-cadherin, vimentin, SMO and Gli-1 at the mRNA level. The PCR primer sequences were as follows: for Snail forward, ′-5GGCTCCTT-3′ and reverse, ′-5CTGGAGATCCTTG-3′; for E-cadherin forward, ′-5ATTCTGATTCT-3′ and reverse, ′-5AGTCCTGGTCCTCTTC-3′; for vimentin forward, ′-5CGGGAGAAATTGCAG-3′ and reverse, ′-5AAGGTCAAGACGTGCCA-3′; for SMO forward, ′-5ACGAGGACGTGGAGGG-3′ and reverse, 5′-CGCACGGTATCGGTAGTTCT-3′; for Gli-1 forward, 5′-GGGATGATCCCACATCCTCAGTC-3′ and reverse, 5′-CTGGAGCAGCCCCCCCAGT-3′; for β-actin forward, 5′-AGCGAGTATCCCCCAAAGTT-3′ and reverse, 5′-GGGCACGAAGGCTCATCATT-3′. The expression of each target gene was determined using β-actin as the normalization control. The PCR reactions consisted of 30 sec at 95°C, followed by 40 cycles at 95°C for 5 sec, 30 sec at 60°C and 30 sec at 72°C. After each qRT-PCR, a dissociation curve analysis was conducted. Relative gene expression was calculated using the 2^−ΔΔCt method (19).

Total protein was extracted by RIPA lysis buffer (Beyotime Institute of Biotechnology, Guangzhou, China) and the concentration of protein was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. The proteins were subjected to SDS-PAGE using a 10% polyacrylamide gel with a 5% stacking gel, and then the protein was transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were initially blocked with 5% non-fat dry milk in Tris-buffered saline-Tween (TBS-T) for 2 h and then probed with antibodies against E-cadherin (1:1,000 dilution), Snail (1:1,000 dilution), vimentin (1:1,000 dilution), CCR2 (1:500 dilution), SMO (1:500 dilution), Gli-1 (1:1,000 dilution) or β-actin (1:1,000 dilution, loading control). After co-incubation with the primary antibodies at 4°C overnight, the membranes were blotted with the secondary antibody (1:10,000 dilution) for 2 h at 37°C. Chemiluminescence detection of bound antibodies was performed using an enhanced chemiluminescence ECL Plus system and a Molecular Imager ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA).

The MHCC97H cells were plated into 96-well plates at a density of 5,000 cells/well and allowed to adhere for at least 24 h and serum-starved overnight (1% FBS in DMEM) before the beginning of the experiments. After different treatments, the cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Twenty microliters of 5 mg/ml MTT solution per 100 µl of growth medium was added into each well after the removal of the media and incubation followed at 37°C for 4 h. Following, 100 µl DMSO was added to each well and the optical density (OD) was assessed at 490 nm on a multifunction microplate reader (POLARstar OPTIMA; BMG Labtech Ltd., Ortenberg, Germany). The proliferation rate was calculated according to the following equation: proliferation rate = (OD sample/OD control) × 100%.

The MHCC97H cell invasion was assessed by a chamber-based invasion assay. In brief, the upper surface of a filter (pore size, 8.0 µm; Millipore, Billerica, MA, USA) was coated with basement membrane Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The cells were suspended in DMEM containing 1% FBS. Then the cell suspensions (200 µl containing 40,000 cells) were added to the upper chambers. Simultaneously, 500 µl of DMEM containing 10% FBS was placed in the lower chambers. The cells were allowed to migrate for 24 h at 37°C. The non-invading cells were removed from the upper surface by scraping with a wet cotton swab. After rinsing with phosphate buffered saline (PBS), the filter was fixed and stained with crystal violet. The invasion ability was determined by counting the stained cells on the bottom surface. Five random fields were captured at a magnification, ×200 (n=3).

The cells were fixed in 4% formaldehyde diluted in PBS for 15 min, permeabilized with 0.3% Triton X-100, treated with blocking buffer (5% BSA in PBS), and then incubated overnight with the primary antibody at 4°C. The cells were then incubated with Red-conjugated secondary antibody from Jackson Immunoresearch Laboratories (West Grove, PA, USA) for 1 h at room temperature. Slides were mounted and examined using a Zeiss Instruments microscope (Carl Zeiss, Oberkochen, Germany).

Statistical analysis was performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Each experiment was performed at least three times. Data are presented as the means ± standard deviation (SD). Differences between the groups were analyzed by analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference.

The MHCC97H cells were treated with different concentrations of CCL2 (0, 25, 50, 100 and 200 ng/ml) at different time-points (24, 48 and 72 h) and the cell proliferation was assessed by an MTT assay. Fig. 1A indicates that CCL2 stimulated MHCC97H proliferation in a dose-dependent manner. As shown in Fig. 1B, CCL2 time-dependently stimulated MHCC97H proliferation, indicating that CCL2 could effectively stimulate MHCC97H proliferation. These findings were consistent with the results that CCL2 promoted non-small cell lung cancer A549 cell viability (20). The MHCC97H cells were treated with or without CCL2 for 24 h and then the invasion ability was assessed via a Transwell assay. The results demonstrated that 50 or 100 ng/ml of CCL2 significantly increased the invasion ability of MHCC97H cells (P<0.05, Fig. 1C and D). These results reveal that CCL2 promotes cell invasion.

EMT has been confirmed to play an important role in cancer progression, which is characterized by the loss of cell-cell adhesion with diminished expression of epithelial markers such as E-cadherin and increased expression of mesenchymal markers such as vimentin and EMT transcription factors such as Snail (21). To further investigate the possible role of the CCL2/CCR2 axis on EMT process, the expression of Snail, E-cadherin and vimentin was detected via western blot analysis and real-time PCR at both the protein and mRNA levels. As shown in Fig. 2, both 50 and 100 ng/ml of CCL2 significantly decreased the expression of E-cadherin at both the mRNA and protein levels. Simultaneously, the expression levels of Snail and vimentin at both the mRNA and protein levels were significantly increased after the CCL2 treatment.

Although CCL2 influenced the EMT process and cell invasive ability, the underlying mechanisms remain largely unclear. CCR2 knockdown was carried out in the present study to investigate the role of CCR2 in CCL2-induced HCC invasion. Transfection of CCR2 siRNA significantly reduced CCR2 expression (Fig. 3A). Transfection of CCR2 siRNA reversed the effect of CCL2 on MHCC97H cell invasion (P<0.05; Fig. 3B and C). Furthermore, the CCL2-induced downregulation of E-cadherin and the upregulation of Snail and vimentin in HCC cells were abolished upon knockdown of CCR2 (Fig. 3D-G). Thus, these data reveal that CCL2 promotes HCC progression through its receptor CCR2.

Several studies have revealed that the Hh pathway may be a treatment target for HCC (14,22). Our results revealed that CCL2 significantly increased the transcription of the Hh pathway-related genes, including SMO and Gli-1 in MHCC97H cells. However, the levels of SHH and Ptch remained unchanged, compared to the normal controls (Fig. 4A-C). CCR2 knockdown was carried out in the present study, to investigate the role of CCR2 in CCL2-induced activation of the Hh pathway. Transfection of CCR2 siRNA significantly decreased the CCL2-induced expression of SMO and Gli-1 both at the protein and mRNA levels, but there was no effect on Ptch and SHH expression levels (Fig. 4D-F). These results indicated that Hh signaling was activated in MHCC97H cells under the CCL2 treatment. Upon activation, Gli-1 proteins translocate from the cytoplasm to the nucleus and activate the transcription of target genes (23,24). Additionally, as demonstrated by immunofluorescence, the nuclear translocation of Gli-1 was enhanced as an effect of CCL2 treatment, while transfection of CCR2 siRNA obviously decreased the nuclear translocation of Gli-1 (Fig. 4G). Collectively, these findings indicate that CCL2 activates the Hh pathway in a CCR2-dependent manner.

Since the activation of the CCL2/CCR2 axis simultaneously induces HCC cell invasion and Hh pathway activation, we hypothesized that there is a cross-talk between the CCL2/CCR2 axis and the Hh pathway and this cross-talk contributes to HCC cell invasion. To test this hypothesis, we investigated the relationship between the CCL2/CCR2-induced cell invasion and Hh pathway activation using an SMO antagonist, cyclopamine, as an inhibitor of the Hh pathway (25). Notably, pretreatment with cyclopamine significantly decreased HCC cell invasion (Fig. 5A and B), reversed the downregulation of E-cadherin and the upregulation of Snail and vimentin and reduced the expression of SMO and Gli-1 even in the presence of CCL2 (Fig. 5C). The Snail, SMO, Gli-1, E-cadherin and vimentin mRNA levels were consistent with the protein changes (Fig. 5D-H). In addition, immunofluorescence staining of these treated cells also ascertained that CCL2 induced Gli-1 expression in the nucleus of MHCC97H cells, while pretreatment with cyclopamine markedly decreased the nuclear translocation of Gli-1 (Fig. 5I). Therefore, we concluded that the inhibition of the Hh pathway activation via blocking the function of SMO eliminates cell invasion and EMT induced by CCL2. These findings reveal that there is a cross-talk between the CCL2/CCR2 axis and the Hh pathway activation, which is mediated by an increased SMO expression.

Previous studies have demonstrated that Gli-1 induces mobility and invasion of HCC cells and is an important regulator of EMT (18). In the present study, since we found that the activation of CCR2 by CCL2 simultaneously induces tumor cell invasion and Hh pathway activation, we hypothesized that Gli-1 is a critical mediator of the CCL2-induced EMT and cell invasion of HCC cells. In order to further confirm the effect of Gli-1 on CCL2-induced invasion of HCC cells, we investigated the role of Gli-1 in the CCL2/CCR2 axis-induced invasion and Hh pathway activation using siRNA targeting Gli-1. Transfection of Gli-1 siRNA significantly reduced the Gli-1 protein level (Fig. 6A). Furthermore, Transwell assays revealed that transfection with Gli-1 siRNA significantly inhibited the invasion ability of HCC cells induced by CCL2 (Fig. 6B and C). The knockdown of Gli-1 blocked the CCL2-induced increase in Snail and vimentin and reduction of E-cadherin protein levels (Fig. 5D). These data demonstrate that CCL2 promotes HCC invasion and EMT by modulating Gli-1-dependent Snail, vimentin and E-cadherin expression.