Quantitative PCR and western blotting were performed on fresh HCC samples that had been collected from patients who underwent surgical resection at Lishui Hospital of Zhejiang University (Zhejiang, China). The Ethics Committee of Lishui Hospital of Zhejiang University approved this study. The protocols were in accordance with the Declaration of Helsinki. Informed consent was obtained from all patients.
HCC tissue microarrays (TMA) were purchased from Servicebio Technology Co. (Wuhan, China), and a standard protocol was used for immunostaining of the TMAs. Immunohistochemistry was performed to determine the PTPN14 (1:150, Santa Cruz Biotechnology, Santa Cruz, CA, USA) levels. Briefly, paraffin was removed from sections, after which they were rehydrated and placed in a microwave oven for heat-induced epitope retrieval. Endogenous peroxide activity was quenched with 3% H2O2. Sections were then blocked in 5% fetal bovine serum (FBS) and incubated overnight at 4 °C with primary antibodies. The following day, horseradish peroxidase secondary antibodies (Invitrogen) were applied and sections were exposed to 3 3′-diaminobenzidine (Zhongshan Golden Bridge Biotechnology, Beijing, China) and counterstained with hematoxylin.
The histochemistry score (H-score) was calculated as previously described . H-Scores were calculated as follows: H-score = (percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + (percentage of cells of strong intensity × 3). Samples with an H-score of more than the median were considered to be high; those less than the median were considered low.
We obtained human HCC cells (Huh7, HepG2 and SK-HEP1) and LO2 cells from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). We cultured HepG2 and LO2 cells in RPMI 1640 medium (Gibco BRL, Rockville, MD, USA) with 10% FBS (Gibco). Huh7 cells were cultured in DMEM medium (Gibco) with 10% FBS. SK-HEP1 cells were grown in MEM medium (Gibco) with 10% FBS. All cell lines were maintained in medium supplemented with 1% penicillin/streptomycin (Gibco) at 37 °C, in a humidified atmosphere of 5% CO2. Hypoxia was induced by incubating cells at 37 °C in an atmosphere containing 93% N2, 5% CO2 and 2% O2 for 24 h. Atmospheric conditions were maintained using a triple-gas incubator(Huaxi Electronic Tec., China). Triple-gas incubator by controlling the input of O2 or N2, with zirconia (ZrO2) sensor to achieve control over O2 content, to perform three gas control of O2, N2 and CO2. When O2 gas concentration was less than 19%, the advanced N2 gas was used, after reaching the O2 concentration set value, and then the way CO2 gas was re fed, to ensure the accuracy of CO2 gas concentration and O2 concentration. When O2 gas concentration was more than 23%, the method of using advanced O2 gas, after reaching the O2 concentration set value, and then the way CO2 gas was re fed, to ensure the accuracy of CO2 gas concentration and O2 concentration.
Cell transfection and stable cell line generation
For PTPN14 knockdown, shRNAs for human PTPN14 were designed and constructed in a GV112 lentiviral expression plasmid (Shanghai Genechem Co., Ltd., Shanghai, China). shRNA plasmids were constructed using PTPN14 target (5′-GGTCTACAGCAACAAACTTGT-3′´) and a scrambled sequence (5′-TTTAGACTTTATGAGCTAA-3′) was used as the negative control.
For PTPN14 overexpression, using cDNA from the human PTPN14 gene, we amplified a fragment encoding the full-length PTPN14 open reading frame sequence using PCR and then cloned it into GV341 lentiviral expression plasmids (Shanghai Genechem Co., Ltd.). Lentivirus with an empty vector served as the negative control.
For production of recombinant lentiviruses, we co-transfected HEK293T cells with their respective recombinant expression lentivectors together with enveloped, packaged plasmids using the Lipofectamine™ 2000 transfection reagent (Life Technologies, Gaithersberg, MD, USA). The viral supernatants were harvested 48 h after transfection, and viral titers were measured. The recombinant lentivirus was infected into HCC cells for 48 h in medium containing 6 μg/ml polybrene (Sigma-Aldrich Co., St. Louis, MO, USA). Fresh culture medium containing 2 μg/ml puromycin was added to select stable transfected cell lines.
siRNAs targeting NPM1 (siNPM1) and YAP (siYAP) were synthesized by Genepharm Technologies (Shanghai, China). Full-length NPM1 was amplified by PCR and subcloned into the pcDNA3-based expression vector (Invitrogen, Carlsbad, CA, USA). Plasmids and all siRNA transfections were performed using Lipofectamine 2000. For transient transfection, we transfected cells with plasmids or siRNAs at various concentrations as indicated for 48 h before performing functional assays.
Total RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using TRIzol (Life Technologies). We performed reverse transcription into cDNA using the SuperScript III cDNA synthesis kit (Life Technologies). We employed cDNA samples (2 μl) for qRT-PCR using SYBR Green PCR Master Mix (Takara, Dalian, China) for 40 cycles on an ABI Prism 7500 detection system (Life Technologies). The following primers were used for qRT-PCR: PTPN14, forward, 5ʹ-TCCCTGTAAAGGACAATCAT-3ʹ and reverse, 5ʹ-GTGGCAAACAACCGAGAA-3ʹ; GAPDH, forward, 5ʹ-TCAAGAAGGTGGTGAAGCAGG-3ʹ and reverse, 5ʹ-TCAAAGGTGGAGGAGTGGGT-3ʹ. We used the 2−ΔΔCt method for relative quantification of gene expression and normalized the data to β-actin expression.
Proteins was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. For nuclear proteins, we used NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. We determined protein concentrations using the bicinchoninic acid method (BCA, Pierce, Rockford, IL, USA). Then, 30 µg of total protein was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrotransfer onto PVDF membranes (Millipore, Bedford, MA, USA). Following blocking, we incubated membranes with antibodies specific for PTPN14 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), YAP or phosphorylated-YAP (p-YAP) (Ser127) (Cell Signaling Technology, Danvers, MA, USA), GAPDH or lamin B (Abcam, Cambridge, MA, USA). Finally, we incubated blots with goat anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology) and visualized signals with enhanced chemiluminescence (Pierce).
Cell viability assay
We seeded cells in 100 µl growth medium at 8 × 103 cells per well in 96-well plates. Following overnight incubation, we treated cells with or without sorafenib (Sigma-Aldrich), then incubated the cells with 10 μl of MTT (5 mg/ml, Cat.11465007001, Sigma-Aldrich) for 4 h at 37 °C. We measured cell viability using a microplate reader (BioTek, Winooski, VT, USA) at 570 nm. IC50 was calculated using GraphPad Prism 5. Each experiment was performed at least three times using separate cultures.
Colony formation assay
We seeded cells into 6-well plates at about 500 cells/well and cultured them in RPMI-1640 containing 10% fetal bovine serum. At day 14, the plates were fixed in 4% paraformaldehyde and stained with 1% crystal violet sequentially. We counted colonies with ≥ 50 cells manually under a dissection microscope.
Invasion and migration assays
After treatment, cells were placed in Transwell chambers (8 μm pore; BD Biosciences, San Jose, CA, USA) as previously described. For the invasion assay, 1 × 105 cells were seeded into the upper chamber coated with Matrigel in a cell invasion system (BD Biosciences). The lower chamber was filled with medium. After incubation, invading cells were fixed with methanol, visualized by staining with 0.1% crystal violet and counted. For the migration assay, 5 × 104 cells were seeded into the upper chamber and the lower chamber was filled with medium. After incubation, the cells that had migrated through the membrane were fixed, stained and counted.
Immunofluorescent staining and confocal microscopy
Confocal microscopic imaging of immunofluorescent staining. Briefly, cells were washed and applied to slides by cytospinning. Then, the cells were fixed for 20 min in PBS containing 4% paraformaldehyde, permeabilized in 1% Triton X-100 for 20 min, then incubated in blocking buffer (5% FBS in PBS) for 30 min. The cells were rinsed in PBS and incubated overnight at 4 °C in dilution buffer containing primary antibodies against PTPN14 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or YAP (1:200, Cell Signaling Technology, Danvers, MA, USA). The cells were washed three times with PBS before being incubated with an appropriate fluorochrome-conjugated secondary antibody (Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibody, Invitrogen) for 1 h at 37 °C in the dark. After nuclear counterstaining with 4, 6-diamidino-2-phenylindole (DAPI, Beyotime, China), coverslips were fixed with mounting medium and cells were visualized using a light microscope (Nikon, Tokyo, Japan).
For co-immunoprecipitation (Co-IP), cells subjected to hypoxia for various durations were harvested. Total lysates were incubated with the human PTPN14 antibody (Santa Cruz Biotechnology) overnight. After overnight incubation, we added protein-G or protein-A sepharose CL-4B (Invitrogen) and incubated the mixture for another 4 h. We then subjected the suspension to several pull-down assays, washed the mixture five times in PBS, and PTPN14-associated protein complexes were separated using SDS-PAGE. Finally, we immunoblotted the membranes using antibodies against human NPM1 or PTPN14.
In vivo tumorigenicity and pulmonary metastasis
Animal studies were approved by the Lishui Hospital of Zhejiang University, Zhejiang, China. Male athymic BALB/c nude mice (4–5 weeks old) were used for this study (Shanghai Slac Laboratory Animal Co., China). For in vivo tumorigenicity experiments, we subcutaneously injected equal numbers (1 × 107) of transduced HCC cells into each mouse flank. We measured tumor volumes using the formula: volume (mm3) = (width)2 × length/2, and mice were euthanized using CO2 inhalation (CO2 flow rate, 20% of chamber volume per minute) 5 weeks after injection.
To generate in vivo models of pulmonary metastasis, we injected luciferase transduced HCC cells into nude mice through the tail vein. After about 8 weeks, mice were injected with D-luciferin (150 mg/kg), anesthetized with 5% isofluorane, and tumor metastases were visualized and imaged using a whole-body fluorescent imaging system. At the end of the experiments, mice were euthanized using CO2 inhalation (CO2 flow rate, 20% of chamber volume per minute), and the whole lungs were excised.
Data were expressed as mean ± SD. The differences in mean values between two groups were analyzed using the Student’s t test, and differences in mean values between several groups were analyzed using one-way ANOVA. All statistical analyses were performed using GraphPad Prism 5.0 software. p < 0.05 was considered statistically significant.