Reagents
TRIzol (15596026) was purchased from Sigma (St Louis, Missouri, USA). MMP inhibitor TAPI-2 (HY-100211) was obtained from MedChemExpress (Shanghai, China). Cathepsin B inhibitor CA-074 methyl ester (CA-074, S7420), GSK-3β inhibitor TWS119 (S1590), EGFR inhibitor PD153035 HCl (S1079), TGFR-1 inhibitor SB431542 (S1067), NF-κB inhibitor PDTC (S3633), STAT3 inhibitor WP1066 (S2796) and FAK inhibitor PF573228 (S2013) were purchased from Selleck (Shanghai, China). α-tubulin antibody (ab80779) was purchased from Abcam (Cambridge, MA, USA); Antibodies specific for E-cadherin (20874-1-AP), GSK-3β (22104-1-AP), lamin B (66095-1-Ig) were obtained from Proteintech (WuHan, China); Cathepsin B antibody (sc-6493) was from Santa Cruz Biotechnology (CA, USA); β-catenin antibody (610153) was obtained from BD Biosciences (San Jose, CA, USA); goat anti-mouse IgG antibody (31430), goat anti-rabbit IgG antibody (31460), were purchased from Thermo Fisher Scientifc (Waltham, MA, USA); CD147-specific antibody was produced by our lab (26).
Cell lines
Human hepatocellular carcinoma cell line Huh-7 was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan). HepG2 cells were obtained from Chinese Academy of Medical Sciences (Shanghai, China). HFF-1 cells were obtained from American Type Culture Collection (ATCC, USA). A Huh-7 CD147-KO (Huh-7 CD147−/−) cell line was generated using a CRISPR/Cas9 system as previously reported (27). All cells were cultured at 37℃, 5% CO2, in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). All cells have been authenticated using short tandem repeat profiling.
3D invasion model
3D cultures referred to the method described by M.Horie et al (28). Briefly, collagen gels were prepared by mixing 0.5 ml cell suspension of HFF-1 (2.5×105 cells) in FBS, 2.3 ml of type I collagen (Cell matrix type IA; 354236, Corning), 670 μl of 5×Dulbecco's Modified Eagle's medium (DMEM), and 330 μl of reconstitution buffer. The mixture (3 ml) was cast into each well of a 6-well culture plate. The solution was then allowed to polymerize at 37℃ for 30 min. For co-culture, cancer cells (2×105 cells) were seeded on the surface of each gel and cultured in DMEM with 10% FBS. After incubation overnight, each gel was detached and cultured with growth medium for five additional days. Then exposed the gels to air by placing it on a filter in new plates with growth medium. After 5 days of the air–liquid interface culture, the gel was fixed in formalin solution and embedded in paraffin, and vertical sections (4 μm) were stained with hematoxylin and eosin.
Cathepsin B Activity assay
Cathepsin B activity was measured using the cathepsin B activity assay kit (KA0766, Abnova, Limerick, PA). 5×106 cells were collected by centrifugation and lysed in 50 μl of pre-chilled CB Cell Lysis Buffer on ice for 10 min. Then centrifuge at top speed for 5 min, transfer the supernatant to a new tube. Add 50 μl of cell lysate to a 96-well plate. Add 50 μl of CB Reaction Buffer to each sample. Add 2 μl of the 10 mM CB Substrate Ac-RR-AFC (200 μM final concentration). Incubate at 37°C for 1-2 hour. Read samples in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter (Infinite M200 Pro, TECAN, Austria).
RNA sequencing
RNA sequencing was performed as previously described (26). Briefly, total RNA was assessed for quantity and quality. Sequence libraries were generated and sequenced by CapitalBio Technology (Beijing, China). A NEB Next Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) was used to construct the libraries for sequencing. A NEB Next Poly(A) mRNA Magnetic Isolation Module (NEB, Ipswich, MA, USA) kit was used to enrich poly(A) tailed mRNA molecules from 1 μg total RNA. The mRNA was fragmented into ∼200 base pair pieces. First-strand cDNA was synthesized from the mRNA fragments using reverse transcriptase and random hexamer primers, after which second-strand cDNA was synthesized using DNA polymerase I and RNase H. The end of the cDNA fragments were subjected to an end repair process including the addition of a single “A” base, followed by ligation of the adapters. The resulting products were purified and enriched by PCR to amplify the library DNA. The final libraries were quantified using a KAPA Library Quantification kit (KAPA Biosystems, South Africa) and an Agilent 2100 Bioanalyzer. After qRT-PCR validation, the libraries were subjected to paired-end sequencing with a pair-end 150-base pair reading length on an Illumina HiSeq sequencer (Illumina) (29). The clean reads were subsequently aligned to the reference genome and the processed reads from each sample were aligned using HISAT (Johns Hopkins University, USA). Cuffdiff was used to analyze the differentially expressed genes (DEGs) between samples. The standardization method of Cuffdiff is geometric, with per-condition and pooled as discrete model (30). Thousands independent statistical hypothesis tests were conducted on DEGs, separately, after which a p value was obtained that was corrected using an FDR method.
Tissue specimens and immunohistochemistry
HCC tissue specimens were collected from the Department of Pathology, Eastern Hepatobiliary Surgery Hospital, which is affiliated with the Second Military Medical University, from 2008 to 2012 and were histological confirmed by staining with hematoxylin and eosin (HE). All patients provided written informed consent, and the study was approved by the Hospital Ethics Committee.
Immunohistochemical (IHC) staining was performed on 5 μm tissue sections. Paraffin sections were dewaxed, followed by antigen retrieval with 10 μM citrate buffer at pH 6.0. The deparaffinized sections were treated with methanol containing 3% hydrogen peroxide for 15 min. After washing with PBS, the sections were incubated with blocking serum for 30 min. Then, the sections were incubated with primary antibody at 4ºC overnight. Following incubation, immunoperoxidase staining was conducted using a streptavidin-peroxidase kit (Zhongshan Jinqiao Co., Beijing, China) and the sections were treated with 3,3′-diaminobenzidine (Zhongshan Jinqiao Co., Beijing, China) to detect the target proteins. Hematoxylin was used to counterstain the nuclei. The expression levels were independently evaluated by two senior pathologists according to the proportion and intensity of positive cells. The following criteria were used to score each specimen: 0 (no staining), 1 (any percentage with weak intensity or < 30% with intermediate intensity), 2 (> 30% with intermediate intensity or < 50% with strong intensity) or 3 (> 50% with strong intensity).
Immunofluorescence assays
Immunofluorescence was performed as described previously (31). Briefly, cells were harvested and allowed to attach for 24 h to cell culture dishes with glass bottoms (NEST Biotechnology Co., LTD.). After washing twice with PBS, the cells were fixed in paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and blocked with 1% BSA in PBS for 1 h. The cells were first incubated with the indicated antibodies at 4ºC overnight, washed twice with PBS, and then incubated with the corresponding fluorescein-conjugated secondary antibodies for 1h in the dark. Cell nuclei were stained with DAPI (Vector Labs). After washing, the cells were visualized using an A1R-A1 confocal laser microscope system (Nikon, Japan).
Transfection and generation of stable cell lines
One day prior to transfection, 4×105 cells were seeded per well in a 12-well plate in complete medium. Subsequent transfection was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After transfection, the cells were subjected to selection in 10 μg/ml blasticidin (for shCTSB) or in 8μg/ml puromycin (for shCD147 and CD147OE) for 2 weeks. Antibiotic resistant colonies were subsequently picked, pooled, and expanded for further analysis under selective conditions.
Western blotting
Western blotting was performed as described previously (31). Briefly, equal amounts of protein were separated by denaturing SDS-PAGE and transfered to polyvinylidene fluoride (PVDF) microporous membranes (Millipore, Boston, MA). Next, the resulting blots were blocked with 5% nonfat milk in TBS/0.5‰ Tween (TBS-T). The primary antibodies were diluted in TBS-T, and the blots were incubated with these antibodies at 4 ºC overnight followed by washing in TBS-T and incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Signal detection was conducted using a ChemiDoc™ Touch Imaging System and analyzed using Image Lab™ Software (Bio-Rad, CA, USA).
Scratch wound healing assay
In vitro scratch wound-healing assays were performed as described previously (31). Briefly, 24 h after treatment, the cells were harvested, seeded in 24-well plates and grown until confluence. Next, a pipette was used to scratch (‘wound’) the monolayer after which the remaining cells were washed with serum-free medium. Subsequently, photomicrographs were taken at various time points.
In vivo metastasis assay
Immunodeficient nude mice (strain BALB/c, 6–8 wk) were obtained from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences, and the animal study was approved by the Animal Care and Use Committee of Fourth Military Medical University. The experimental metastatic potential of HCC cells was assessed following intrasplenic injection as described previously (32).
RNA interference
Cells were transfected with siRNAs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. siRNAs targeting CD147 were designed and synthesized by Shanghai GenePharma Co. (Shanghai, China). The siRNA sequence is depicted in Table S1. GSK-3β siRNA was obtained from Santa Cruz Biotechnology (sc-35527).
Quantitative real-time PCR analysis
Total RNA was extracted using TRIzol reagents. Reverse transcription was performed using a PrimeScript RT reagent kit (TaKaRa Biotechnology, Japan). All primers were synthesized by BGI (BGI, Shenzhen, China) and their sequences are listed in Table S1. Quantitative real-time PCR (qRT-PCR) was performed using a SYBR Premix Ex Taq II Kit (TaKaRa Biotechnology, Japan).
Luciferase reporter assay
CTSB promoter (-1100~+200) was cloned to pGL3-basic (pGL3-CTSB). pGL3-CTSB and pRL-TK (50:1) were cotransfected using Lipofectamine 2000. The luminescence was measured using Dual-luciferase reporter assay system and GloMax luminometer (Promega, WI, USA) according to the manufacturer’s instructions at 48 h after transfection.
Statistical analysis
Differences were compared by Student’s t test (two groups) or ANOVA followed by post-hoc tests (three groups) as indicated in figure legends. Spearman R was used to determine correlations between relative expression of genes. Quantitative results are presented as mean values with SD. P<0.05 was considered significant (GraphPad Prism 6, San Diego, CA).