Cell lines culture and regents
The human hepatocarcinoma cell (HCC) lines SMMC-7721 and HepG2 were purchased from the American Type Culture Collection (ATCC, Rockville, MD). All cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 complete medium supplemented with 10% fetal bovine serum (Gibco, MA, USA) at 37 °C in a 95% humidified atmosphere containing 5% CO2. The inhibitors, such as SCH772984, PF-573228, JSH-23 were purchased from Selleck Chemicals (MA, USA). GLPG0187 was purchased from MCM (MA, USA). The chemotherapeutic agents Adriamycin (ADM) and cis-diamine dichloro platinum (DDP) were purchased from Sangon (Shanghai, China).
Cell proliferation
The cell proliferation was examined by MTT assay. Briefly, a density of 2 × 103 per/well SMMC-7721 and HepG2 tumor cells were seeded in 96-well plates and cultured with RPMI 1640 complete culture medium supplemented with 10% fetal bovine serum. Cell proliferation was examined at 0, 24, 48, and 72 h. Samples were added with 10 μl MTT (5 mg/ml) and incubated for 4 h at 37 °C. The formazan crystals in the cells were solubilized with stop solution (100 μl/well). Subsequently, the samples were analyzed at 570 nm using a Microplate Reader Model 550 (BIO-RAD, Shanghai, China).
Colony formation
SMMC-7721 and HepG2 cells were seeded in 6-well plates at 200 cells per well and cultured in RMPI complete medium in a humidified incubator. After 14 days, colonies were fixed with paraformaldehyde and stained with crystal violet (Beyotime, Beijing, China). Visible colonies were counted. Each experiment was performed three times independently.
Cell invasion ability detection by transwell assay
The cell suspension (1 × 106 cells /ml, 200 μl) was added into the upper insert of a transwell chamber (8 μm, Corning, USA), and RMPI 1640 complete culture medium containing 15% fetal calf serum was added to the lower chamber. After 24 h, the migrating cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Then the migrating cells numbers were counted. Each experiment was performed three times independently.
Preparation of different stiffness PA gel
PA gels used in cell culture with different stiffness were prepared as indicated in previous studies [17]. Briefly, the coverslips were coated with a thin layer of gel containing a mixture of 3 to 10% acrylamide and 0.01-0.3% bis-acrylamide, producing gels of 2, 8 and 20 kPa stiffness. Addition of 10% APS (1/100 volume) and TEMED (3/1000 volume) promoted the PA gel polymerization. Then the coverslips were washed with PBS twice for 20 min, followed by sterilizing in PBS solution for 1 h with ultraviolet light. Next, 50 μl heterobifunctional sulphosuccinimidyl 6-(4′-azido-2′-nitrophe-nylamino) hexanoate were added and photo-activated for 5 min with ultraviolet light. Then, the coverslips were coated with 10 μl/ml fibronectin for 1.5 h and rinsed before cell seeding. Gels were soaked in serum-free culture media for 24 h for usage. All regents of PA gels were purchased from Solarbio (Beijing, China).
Patients’ tumor tissues samples
Formalin-fixed, paraffin-embedded human hepatocarcinoma tumor tissue sections were obtained from the Second People’ s Hospital of Yibin (Sichuan, China), and were divided into low degree malignant group (LD, stage A ~ B) and high degree malignant group (HD, stage C ~ D) according to the Barcelona Clinic Liver Cancer criterion. The protocols were approved by Regional Scientific Ethics Committee of the Second People’ s Hospital of Yibin (Sichuan, China). Written informed consent was attained from all subjects, and all methods were performed in accordance with the Declaration of Helsinki.
AFM analysis
Atomic force microscopy and analysis were performed as previously indicated [18]. Frozen tissue blocks were cut into 20 μm thick sections. All the sample sections were immersed in PBS at room temperature before AFM measurement. The samples were maintained in proteinase inhibitor in PBS (protease inhibitor cocktail, Roche 14 Diagnostics, 11,836,170,001) and supplemented with Propidium Iodide (SIGMA P4170, 20 μg/ml) during the AFM session. MFP3D-BIO inverted optical AFM (Asylum Research) mounted on a Nikon TE2000-U inverted fluorescent microscope was used for AFM analyze as previously described. Ten frozen sections were analyzed in each tumor tissue, and 10 tumor tissues were collected and examined in each group.
Western blot analysis
The total proteins were extracted by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) with the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Shanghai, China). Cell lysates were separated on 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), then blocked and incubated with the corresponding primary antibodies: anti-integrin β1 (1:1000, Abcam, Cambridge, UK), anti-phosphorylated FAK (1:1000, Abcam, Cambridge, UK), anti-FAK (1:1000, Abcam, Cambridge, UK), anti- phosphorylated ERK1/2 (1:1000, Abcam, Cambridge, UK), anti-ERK1/2 (1:1000, Abcam, Cambridge, UK), anti-NF-κB (1:1000, Abcam, Cambridge, UK). Subsequently, samples were incubated with an HRP-conjugated secondary antibody (1:1000, Abcam, Cambridge, UK). β-actin served as an internal control.
Immunofluorescence staining
The sections of tumor tissues were dewaxed, rehydrated, quenched of endogenous peroxidase, blocked by 5% BSA, and incubated with the primary antibodies: anti-p-FAK, anti-p-ERK, anti-NF-κB (1:200, Abcam, Cambridge, UK) overnight at 4 °C, and followed by signal amplification using the ABC HRP Kit (Thermo, MA, USA) for 2 h at room temperature, and the nucleus was stained with DAPI. All immunofluorescence images were captured by FV1000 confocal microscope (Leica, Barnack, Germany) and the intensity of protein expression was calculated by image J software.
Real-time PCR
Total RNA was extracted from tumor cells using TRIzol (Thermo, MA, USA) according to the manufacturer’s protocol. The quantification of mRNA levels was conducted by real-time PCR using SYBR green dye (Thermo, MA, USA). And 1μg cDNA was used as template for amplification. GAPDH was used as the internal control and normalized the target gene level to the GAPDH by the ΔΔCt method to quantify the relative expression. The primer pairs in our study were used as follow: integrin β1: Forward, 5′- CCTACTTCTGCACGATGTGATG-3′, and reverse, 5′- CCTTTGCTACGGTTGGTTACATT-3′; integrin β2: Forward, 5′- TGCGTCCTCTCTCAGGAGTG-3′, and reverse, 5′-GGTCCATGATGTCGTCAGCC-3′; integrin β3: Forward, 5′-GTGACCTGAAGGAGAATCTGC-3′, and reverse, 5′-CCGGAGTGCAATCCTCTGG-3′; integrin β4: Forward, 5′-GCAGCTTCCAAATCACAGAGG-3′, and reverse, 5′-CCAGATCATCGGACATGGAGTT-3′; integrin β5: Forward, 5′-TCTCGGTGTGATCTGAGGG-3′, and reverse, 5′-TGGCGAACCTGTAGCTGGA-3′; integrin β6: Forward, 5′-TCCATCTGGAGTTGGCGAAAG-3′, and reverse, 5′-TCTGTCTGCCTACACTGAGAG-3′; integrin β7: Forward, 5′-AGAATGGCGGAATCCTCACCT-3′, and reverse, 5′-TGAAGTTCAGTTGCTTGCACC-3′; integrin β8: Forward, 5′-ACCAGGAGAAGTGTCTATCCAG-3′, and reverse, 5′-CCAAGACGAAAGTCACGGGA-3′. Each experiment was performed three times independently.
Flow cytometry
The SMMC-7721 and HepG2 tumor cells were collected and fixed with 4% paraformaldehyde, washed by PBS for three times. Subsequently, the cells were stained with anti-human CD133 (Biolegend, MA, USA) at a dilution of 1:500 at 4 °C. Human IgG Isotype was stained as control at a dilution of 1:500 in flow cytometry analysis. After 30 min, samples were washed by PBS and were examined using an AccuriC6 (BD, MA, USA). FlowJo software 2.0 (Biolegend, MA, USA) was used for analysis. Each experiment was performed in three independent times.
Si RNA interference
For integrin β1 or NF-κB silence, SMMC-7721 and HepG2 cells were infected with Lipofectamine 8000 (Beyotime, Beijing, China) according to the manufacturer’s protocol. The relevant NF-κB siRNA sequence as followed: siRNA#1: 5′-GAAGGGTTGCCAACCAAGT-3′ and siRNA#2: 5′-GGTGGCTTTGATGCAATCA-3′;
Animal experiments
Four to six weeks female nude mice were purchased from Huafukang company (Beijing, China), and raised in SPF level. The animal protocols were approved by the Animal Care and Use Committee of the Second People’ s Hospital of Yibin Committee (#2018-02-13), according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal studies were conducted in accordance with the Public Health Service Policy and complied with the ARRIVE guidelines for the humane use and care of animals. For establishing subcutaneous hepatocarcinoma model, 2 × 106 SMMC-7721 tumor cells in 100 ul PBS were subcutaneously inoculated on their armpits of right anterior limbs. All the mice were randomly divided into 4 group (6 mice per group). After 2 weeks, tumor bearing mice were treated with ADM (4 mg/kg), DDP (0.5 mg/kg), or DDP (0.5 mg/kg) combining GLPG0187 (i.p 100 mg/kg; dissolved in 2% DMSO in PBS), and 2% DMSO in PBS for control. Mice were treated every 3 days and the treatment lasted for 2 weeks. Tumor volume was measured using the formula: volume = length × width2/2. The survival time of mice was recorded. For tumorigenesis analysis, SMMC-7721 cells (2 × 105) were subcutaneously injected into the right side of mice, and tumor numbers on each mouse was measured after 2 weeks. Mice were sacrificed by cervical dislocation.
Statistical analysis
Results are presented as the mean ± standard deviation (SD), and the data in bar graphs are represented as the mean fold change relative to the untreated or control groups with SD of three independent experiments. Statistical significance between groups was calculated by Student’s t test for two groups or by one-way ANOVA for more than two groups using Graphpad 6.0. The log-rank (Mantel–Cox) test was used to analyze the long-term survival curve. The ARRIVE reporting guidelines were used to complete the analysis [19]. Statistical significance was set at P < 0.05. All error bars are expressed as the mean ± SD of three independent experiments.