Human HCC cell lines were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Roswell Park Memorial Institute (RPMI)-1640 medium containing 100 mg/ml streptomycin, 100 U/ml penicillin, and 10% foetal bovine serum (FBS) Gibco, Mulgrave, Australia) was used for cell culture. Cell culture was maintained under standard cell culture conditions in humidified 5% CO2.
Patients and specimens
The present study obtained HCC and adjacent non-tumorous tissues from patients with HCC (n =40) who received radical resection in Affiliated Cancer Hospital & Institute of Guangzhou Medical University (Guangzhou 510515, China). The patients provided signed informed consent for the use of their collected samples, according to the internal review and ethics boards of the Affiliated Cancer Hospital & Institute of Guangzhou Medical University. Histopathology was used to confirm a diagnosis of HCC.
Lentiviral construction and transduction
Genechem (Shanghai, China) constructed the U6-sh-CMTM6-CMV-GFP lentiviral vector, which was used to silence CMTM6 expression. The negative control comprised a lentiviral vector containing a non-silencing short hairpin RNA (shRNA). To silence CMTM6, HCC cells were infected with the lentiviral vector encoding the CMTM6-specific shRNA sequences, control cells were infected with the negative control vector. Puromcyin (Sigma Aldrich, St. Louis, MO, USA) was used to select stable clones for 2 weeks. Expression of the CMTM6 protein was detected using western blotting. Supplementary Table 1 shows the shRNA sequences.
Small interfering RNA (siRNA) transfection
RiBoBio (Guangzhou, China) provided the small interfering RNA (siRNA) targeting VIM (encoding vimentin ). Cells were seeded into six-well culture plates and grown until they reached 50% confluence (usually the second day). Lipofectamine 3000 reagent (Invitrogen, Waltham, MA, USA) was used to perform the transfections, as described in the manufacturer’s instructions. At 48 h after transfection, the cells were subjected to functional assays. Western blotting was used to evaluate the transfection efficiency. Supplementary Table 2 shows the siRNA sequences.
Quantitative real-time reverse transcription PCR (qRT-PCR)
The TRIzol Reagent (Invitrogen) was used to extract total RNA from HCC cells or tissues, following the manufacturer’s guidelines. A SYBR Green PCR kit (Takara Biotechnology, Dalian, China) was used to perform quantitative real-time PCR (qRT-PCR). Data were analysed and normalized to 18S rRNA expression. Supplementary Table 3 shows the primer sequences.
Western blotting analysis
Cell Signaling Technology (Danvers, MA, USA) provided the primary antibodies recognising glyceraldehyde-3-phosphate dehydrogenase (GAPDH (control), CMTM6, N-cadherin, vimentin and E-cadherin. Total protein was extracted from HCC tissues or cells by lysis using Radioimmunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitor cocktails (Sigma Aldrich). The proteins were separated using 8% to 15% SDS-PAGE and then transferred electrophoretically onto a polyvinylidene fluoride membrane (Invitrogen, Carlsbad, CA, USA). The membranes were blocked using 5% bovine serum albumin, washed, incubated with primary antibodies at 4 °C overnight, washed, and incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibody as appropriate. The immunoreactive protein bands were visualised using a chemiluminescence system (Pierce Biotechnology, Rockford, IL, USA).
Tissue multiarray analysis
Shanghai Outdo Biotech (Shanghai, China) produced tissue microarray sections comprising paired HCC and adjacent non-tumorous tissue samples from 90 patients with HCC. The detailed clinical follow-up data related to outcomes were provided by National Engineering Center Biochip at Shanghai. The follow up began at the date of surgery. Survival was calculated as the time from the baseline date to the clinical outcome diagnosis date or the date of last available clinical record. The patient’s history, physical examination, and laboratory tests were evaluated during follow-up, as needed. The time from surgery to death was defined as overall survival (OS).
Cell counting kit-8 (CCK-8) assay
The CCK-8 assay was used to assess cell proliferation ability. Cells transfected with siRNAs targeting CMTM6 were seeded into 96-well plate at of 5 × 103 cells/well. The CCK-8 solution was used to treat the cells at 0, 24, 48, 72, and 96 h. Absorbance at 570 nm was tested using a microplate spectrophotometer from triplicate groups.
Colony formation assay
Cell seeded into 6-well plates were allowed to grow for 12 d. 4% paraformaldehyde was then used to fix the colonies before staining for 30 mins with 0.1% crystal violet. The plates were washed with phosphate-buffered saline (PBS), imaged, and the colonies were counted by visual inspection.
Equal amounts of cells were plated and allowed to grow to 90% confluence. A sterile pipette tip was used to make a wound in the cell monolayer. The cells on the plate were rinsed gently using medium, which was then replaced with fresh medium. The wound areas were marked and photographed at 0, 12, 24, and 48 h under a phase-contrast microscope. Wound healing was determined by comparison with the wound area at 0 h.
Cell invasion assay
The cell-invasive assay was performed using 24-well Matrigel invasion chambers with 8 µm‑diameter pore inserts (BD Biosciences, Madrid, Spain). Cells grown in serum-free medium (1.5× 105 cells/100 μl) were added to the top chamber. 10% FBS was added to the bottom chamber as a chemoattractant. Over 24 h, the cells were allowed to migrate through the porous membrane. Then, methanol was used to fix the lower chambers, followed by staining with 0.3% crystal violet for 5 min. After washing with PBS, images were captured under an inverted microscope (Olympus Microscopes, Tokyo, Japan). Cells were counted in five random high power fields in three independent inserts and then averaged.
Cells were seeded into glass coverslips in a 12-well plate at a density of 3 × 104 per well. The cells were fixed with paraffin and permeabilized using 0.3% Triton X-100. Subsequently, the fixed cells were incubated with primary antibodies against CMTM6 (Abcam, Cambridge, MA, USA) and Vimentin (Proteintech, Rosemont, IL, USA) overnight at 4 °C, and then incubated with secondary antibodies (Alexa 488 goat anti-Rabbit antibodies; Molecular Probes, Eugene, OR, USA). After counterstaining with Hoechst 33258 (Sigma), images were obtained under a confocal microscopy (Leica Microsystems, Milton Keynes, UK).
Myc-tagged Vimentin and Flag-tagged CMTM6 expression plasmids were transfected into HCC cells. To precipitate the target proteins, cells were lysed with lysis buffer and incubated with 5 µg of the primary antibody, followed by incubation with a slurry of precleared protein agarose bead (Roche, Mannheim, Germany) at 4 °C for 2 h. After thorough washing, western blotting of the precipitates was used to analyse the potential interacting proteins.
The animal experiments were performed strictly according to the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Guangzhou Medical University (Guangzhou, China). To assess in vivo tumorigenesis, 5 × 106 CMTM6-silenced MHCC-97H cells in of serum-free RPMI 1640 (0.1 ml ) were injected into the left shoulder of 4-week-old female BALB/cnu/nu nude mice subcutaneously (four mice in each group). The size of the resulting tumour was measured weekly. The tumour volumes were calculated using the following formula:(length × width2)/2.
The mice were sacrificed at 32 days after inoculation, and the tumours were dissected and weighed.
To evaluate the mouse liver metastasis potential of cancer cells, 5×106/0.2 ml CMTM6‑silenced Bel-74029 cells were injected into BALB/c nude mice tail veins(four mice in each group). After 8 weeks, the mice were sacrificed, and the lung metastatic tumours were dissected for further assays. In another group of mice, at 8 weeks after HCC cell injection, the mice were sacrificed and their lungs were removed. An In-Vivo F Imaging System (Kodak, Rochester, NY, USA) was used to assess the metastatic tissues in the lungs after hematoxylin and eosin (H&E) staining.
Tissues were fixed in paraformaldehyde and embedded in paraffin. The sections were deparaffinized and hydrated, and then sodium citrate buffer was used to pretreat the sections by heating in a microwave to retrieve the antigen, followed by blocking with normal goat serum. Rabbit anti-CMTM6，anti-Vimentin or anti-Ki67 antibodies (Cell Signaling Technology) were then incubated with the sections at 4 °C overnight. The next day, biotinylated goat anti-rabbit IgG secondary antibodies were incubated with the sections at room temperature (19–21 °C) for 1 h. Lastly, avidin-biotin peroxidase complex (GeneTex, Irvine, CA, USA) was used to stain the sections. Staining intensity was assessed using a semi-quantitative approach (0, negative; 1, weak; and 2, strong) and the percentage of positively stained malignant cells was scored as: 0, 0–4%; 1, 5–24%; 2, 25–49%; 3,50–74%; and 4, 75–100%. The final IHC scores were determined by multiplying the intensity score by the percentage counts. Positive expression of CMTM6 was defined as having an IHC score ≥ 3.
All statistical data are shown as the mean ± the standard deviation (SD). Student’s t-test or analysis of variance (ANOVA) were used to perform the statistical analyses. To analyse the relation of CMTM6 expression with the clinicopathological characteristics of the patients, the chi-squared test was used. Survival analyses were assessed using the Kaplan-Meier plotter. SPSS 19.0 software (IBM Corp, Armonk, NY, USA) was used for all statistical analyses. P < 0.05 was considered to indicate statistical significance.