Cell culture
The HCC cell lines were kept in the Institute of Liver and Gastrointestinal Diseases (Tongji Hospital, Huazhong University of Science and Technology). HepG2, Huh7, SMMC-7721, MHCC97L, MHCC97H, HCCLM3, and SK-hep-1 were cultured in DMEM medium, HL7702 were cultured in RPMI 1640 medium, and Hep3B were cultured in MEM medium. These cell lines were incubated in at 37 °C, 5% CO2. All of the medium were added 10% fetal bovine serum (Invitrogen Gibco, USA).
Human samples
The human HCC and adjacent nontumor tissues were obtained from patients who underwent excisional surgery of HCC in Tongji hospital. The patients received no preoperative adjuvant therapy. The diagnosis of HCC was confirmed pathologically. This study was approved by the Ethics Committee of Tongji Hospital and complied with the World Medical Association Declaration of Helsinki.
In vitro silicone chamber model
A silicone membrane stretching model for detecting live cell extrusion was invented by Professor Jody Rosenblatt [6]. Briefly, silicone chambers (2 cm × 2 cm) were pre-stretched by 28% through a metal retractor plate (a layer of extracellular matrix adhesive was placed on the silicone membrane to facilitate cell adhesion), and then treated at 4 °C for 24 h. Approximately 750,000 cells were seeded on the stretched silicone membrane. When the cells were fully fused, the silicone chambers were slowly removed to instantly reduce the cell growth area and provide a state of cell overcrowding. Two hours later, the extruded cells were collected from the culture medium, centrifuged and resuspended, and the number of extruded cells was counted (Fig. 1A).
In vitro petri dish inversion model
We have established an in vitro petri dish inversion model to detect crowding-induced cell extrusion [15]. As shown in Fig. 1B, cells grown to approximately 100% confluence in a small culture dish (60 × 15 mm) were inverted in a large culture dish (150 mm × 25 mm) with enough culture medium to ensure that the medium in the large dish could cover the bottom of the small dish. The gas in the small dish was drawn out using an aseptic bending needle. Live cells extracted from the small culture dish could live in the large culture dish below. Twenty-four hours later, the small culture dish was gently removed. The cells shed from the small dish were able to grow for 7 days. For BVES expression analysis, HCC cells in the large culture dish were collected, and the expression level of BVES in the extruded HCC cells was detected by RT-qPCR as described below. For cell counting analysis, HCC cells in the large culture dish were then stained with crystal violet to detect the formation of cell clones. All steps were aseptic, and touching the bottom of the small dish was also avoided.
Immunofluorescence (IF) staining
For IF assays, cells were fixed with 4% paraformaldehyde at room temperature for 15 min and, then, permeabilized with phosphate-buffered saline (PBS) containing 0.3% Triton X-100 for 10 min, blocked with 10% goat serum for 40 min, and incubated with primary antibodies at 4 °C overnight. Primary antibodies include GM130 (Abcam, cat#ab52649), BVES (Santa cruz, cat#sc-374081), ZO-1 (GenTex, cat#GTX108592), F-actin (Keygene Biotech, cat#KGMP001 and cat#KGMP0012), GEFT (Proteintech, cat#14839-1-AP). The cells were washed with cold PBS and then incubated with the appropriate secondary antibodies (Promoter, Wuhan, China). Fluorescence was detected using an Olympus fluorescence microscope. As for confocal microscope analysis, images were obtained under a laser confocal microscope or reconstructed by continuous multilayer scanning with a layer spacing of 1.0 mm by the FV10-ASW 2.1 Viewer software.
Three-dimensional (3D) cell culture
The procedure of 3D cell culture was performed as described previously [16]. In short, 1 × 105 cells were resuspended in 120 μl of 10% sucrose solution, and were quickly mixed with an equal volume of 0.5% hydrogel solution. Then, the cells were immediately seeded in a glass-bottomed cell culture dish. The medium was changed every other day. The cells were allowed to grow for several days and then imaged by microscopy or immunofluorescently stained.
Immunohistochemistry (IHC) assays
As we previously described [17], the tissues were paraformaldehyde-fixed and paraffin-embedded, and the SP Detection System Kits were used for immunohistochemistry staining (ZSGB-BIO, cat# SP-9000) according to the instructions. The intensity of staining was divided into 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The percentage of positive cells was scored from 0 to 4 (0%, 1–25%, 26–50%, 51–75%, 76–100%, respectively). Total score ranged from 0 to 12.
Western blot analyses
The protein of HCC cell lines was digested in RIPA buffer containing phosphatase inhibitor cocktail, and PMSF (Wuhan Sevier Biotechnology Co., Ltd.). All samples were centrifuged at 12,000 × g for 15 min after grinding and sonication. The protein concentration was determined with BCA Protein Assay Kit (Wuhan Sevier Biotechnology Co., Ltd.). Then, 40 μg protein of each sample was fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked with 5% milk in TBST (120 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20) for 1 h at room temperature. Blots were incubated with the specific antibody overnight at 4 °C. Primary antibodies include BVES (Santa cruz, cat#sc-374081), ZO-1 (GenTex, cat#GTX108592), GEFT (Proteintech, cat#14839-1-AP). Anti-GAPDH (Proteintech, cat#60004-1-Ig) was used as an internal control. The membranes were then washed with PBS three times and incubated with the appropriate HRP-conjugated secondary antibodies. Immune complexes were visualized using Super ECL Detection Reagent (Yeasen, 36208ES60).
Quantitative real-time-PCR (RT-qPCR)
The reverse transcription was performed using HiScript II Q RT SuperMix for qPCR Kit (Vazyme, cat#R223-01), and quantitative PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, cat#Q321-02) according to the manufacturer’s protocol. The conditions for the reactions were as follows: 95 °C for 30 s, 95 °C for 10 s and 60 °C for 30 s for 40 cycles. Gene expression was normalized to GAPDH mRNA content, and the relative expression of target gene was determined from replicate samples using the 2−ΔΔCt (Ct, cycle threshold). Primer sequences were as follows: GAPDH: 5′-TCATTGACCTCAACTACATGGTTT-3′ (sense) and 5′-GAAGATGGTGATGGGATTTC-3′ (antisense); BVES: 5′-ACCAGCGAGCCTCTGCCAAGA-3′ (sense) and 5′-CCTCACTTCCTCCCTCCGACTCT-3′ (antisense).
Plasmids
The stable cell lines Huh7-shBVES, SK-hep-1-BVES, and SMMC-7721-BVES were constructed using plasmids transfection. Plasmid vectors encoding the human BVES short hairpin RNAs (shRNA) (shBVES) and BVES expression plasmids (BVES) were obtained from Genechem (Shanghai, China). The sequences were listed as follow: shBVES: 5′-CCTCCAGATTTGTTCAGAA-3′. For BVES expression plasmid construction, the open reading frame of BVES (NM_147147) was cloned into the plasmid vector GV141. The method of BVES plasmid lentiviral transfection was performed as described previously [18].
Co-immunoprecipitation (Co-IP) analysis
Cells were lysed on the ice with NP-40 solution for 40 min. After centrifugation, supernatants were mixed with Protein A/G Agarose Beads (Abmart, cat#HY-K0202) and appropriate antibodies BVES (Santa cruz, cat#sc-374081), Normal Mouse IgG (Abmart, cat#2729) overnight at 4 °C. The agarose beads were separated from the mixture, and incubated with the supernatants. Precipitated proteins were washed with lysis buffer four times. Samples were collected for subsequent western blotting analyses.
In vivo metastasis analysis
All animal experimental procedures were conducted following National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Committee of Ethics of Animal Experiments of Tongji Hospital, Huazhong University of Science and Technology. Six-week-old BALB/C male nude mice were raised in specific pathogen-free (SPF) conditions. Firstly, 5 × 106 luciferase labeled HCC cells were injected subcutaneously into the mice to establish a subcutaneous xenograft tumor model. When the subcutaneous tumor of the nude mice grew to 1 cm3 in size, the tumor was resected and cut into small tumors of 1 mm3 in size. Eight-week-old nude mice were randomly divided into SMMC-7721-BVES-luc group and SMMC-7721-Control-luc group, with 10 mice in each group. After anesthesia, the isolated small tumor tissues were placed into the liver lobes of the mice. Tumor metastasis was monitored using the bioluminescence every 2 weeks. For in vivo signals detection, d-luciferin at 15 mg/kg was injected intraperitonially into the nude mice. After 8 weeks, the mice were sacrificed, liver and lung tissues were resected and collected for further histological examination.
RhoA activity analysis
The RhoA activity was analyzed by a RhoA activation assay kit (Wuhan Newstead Biotechnology, cat# 80601), and the operating procedure was performed according to the instructions. In brief, a murine monoclonal antibody that specifically recognizes the RhoA-activated conformation could specifically bind to RhoA-GTP active protein in cell lysates. Then, Protein A/G was used to adsorb the antigen–antibody conjugate, following by detection through western blotting analyses using a rabbit polyclonal antibody that specifically recognized RhoA. Moreover, before conjugation by Protein A/G, a fraction of cell lysates of each sample was used to detect total RhoA, ZO-1, GEFT, BVES and GAPDH protein expression level as described in section of Western blot analyses.
Statistical analysis
Student’s t-test was used for quantitative data analysis, and Fisher’s exact test was utilized to analyze categorical data. Two-sided P < 0.05 was considered statistically significant. Statistical analyses were calculated using SPSS software (version 21.0) and Prism 6.0 (GraphPad Software).