Human tissue samples
Patients who underwent curative hepatectomy as initial treatment for HCC at Peking University Cancer Hospital between 2009 and 2011 were considered for enrollment. Patients with other malignancies, absence of paraffin-embedded clinical tissue specimens and with incomplete clinicopathological information were excluded. In total, 128 cases were eligible for analysis, including 128 paired tumor tissue and non-tumorous samples. Tumor staging was performed using the Barcelona Clinical Liver Cancer (BCLC) staging system . All patients underwent follow-up evaluations until June 2016. This investigation was approved by the Ethics Committee of Peking University Cancer Hospital. Informed consent was obtained from each patient at the time of sample collection.
Cell culture and reagents
SMMC7721, Huh7 and HepG2 cells were used in this study. HepG2 and SMMC7721 cells were cultured in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and Huh7 cells were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). All media was supplemented with 10% fetal bovine serum (FBS). Cells were incubated in a humidified chamber of 5% CO2 at 37 °C. The reagents for this study are listed in Supplementary Table 1.
Cells were transfected with plasmid DNA or siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the instructions provided by the manufacturer. Plasmid DNA was kept consistent with empty vector in transient transfection experiments. To construct stable PPP1R26 knockdown cell lines, pLKO.1-PPP1R26 shRNA or pLKO.1-ctrl shRNA were transfected into HEK293T cells with the packaging vectors pMD2.G and psPAX2 to produce lenti-viral particles. HCC cells were infected with lentiviruses delivering PPP1R26 shRNA or control shRNA to obtain stable PPP1R26 knockdown cell lines and control cell lines. Small interfering RNAs (siRNAs) targeting PPP1R26, PTBP1 or control siRNA were synthesized (GenePharma, Shanghai, China). The sequences of all shRNAs and siRNAs are listed in Supplementary Table 2.
Cell proliferation assay
MTS assay and colony formation were used to determine cell proliferation. For the MTS assay, the CellTiter 96 AQueous One Solution Cell Proliferation Assay system (Promega, Madison, WA, USA) was used according to the manufacturer’s instructions. Briefly, HCC cells were seeded at a density of 1 × 103 cells/well in 96-well plate. At different time points, 10 μl of MTS were added to each well in the plate and incubated for 4 h at 37 °C. Absorbance at 490 nm was measured using a Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA). For colony formation, HCC cells were seeded into 6-well plates at a density of 500 cells/well and cultured under normal conditions for about 14 days. Cell clones were fixed with 4% paraformaldehyde and stained with crystal violet. Visible colonies were counted under the microscope.
Cell wound healing and transwell assays
The wound-healing experiment was performed as previously described . The wound area was measured by ImageJ software. Transwell invasion and migration assays were performed using chambers (Corning, NY, USA) with or without Matrigel coating, respectively. Chambers were placed in 24-well plates. Cells were serum-starved overnight and plated to the upper compartments containing serum-free DMEM. DMEM containing 10% FBS was added to the lower compartments. After a 24 h incubation period at 37 °C, the migratory or invasive cells were stained with crystal violet and counted in five random fields.
Glucose uptake and lactate production assays
After transfections, the culture medium was collected for measurement of glucose and lactate concentration while cells were harvested for preparation of cell lysates. Glucose concentration was measured by the Amplex-Red Glucose assay kit (Invitrogen, Carlsbad, CA, USA). Glucose uptake was calculated by deducting the glucose concentration in the media from the initial glucose concentration. For evaluating lactate production, cells were cultured under DMEM without pyruvate for 24 h after transfection, and the concentration of lactate was measured using the L-Lactate Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instruction. All values were normalized based on the cell number.
Extracellular acidification rate (ECAR)
The glycolysis stress test was performed using the Seahorse XF Glycolysis Stress Test Kit (Agilent Technologies, Palo Alto, CA, USA) according to the instructions provided by the manufacturer. The ECAR reported by this method reflected the main parameters of glycolysis, including glycolysis rate and glycolysis capacity. The ECAR value was normalized by the cell number per well on the website of Agilent (https://seahorseanalytics.agilent.com). The reagents for ECAR are listed in Supplementary Table 1.
Immunoprecipitation was performed as previously described . Briefly, cells were harvested and cell lysates were prepared in Buffer A (25 mM Tris-Cl pH 7.5, 150 mM KCl, 1 mM dithioerythritol, 2 mM ethylenediaminetetraacetic acid, 0.5 mM phenylmethylsulfonyl fluoride, and 0.2% Nonidet P-40) and used directly for immunoprecipitation. Antibody (1 μg) was incubated with 20 μL of Protein A Sepharose beads (GE Healthcare, Madison, WI, USA) in Buffer IPP500 (500 mM NaCl, 10 mM Tris-Cl pH 8.0, 0.2% Nonidet P-40) for 2 h at 4 °C. After centrifugation, the Ab-coupled beads were incubated with cell lysates for 2 h at 4 °C. After washing three times with Buffer B (10 mM Tris-Cl pH 8.0, 150 mM KCl, 5 mM MgCl2, 0.05% NP-40), the precipitates were subjected to Western blot using indicated antibodies.
RNA immunoprecipitation (RIP)
RIP assay was carried out as previously described [28, 29]. In brief, 1 × 107 cells were treated with UV irradiation and lysed in RIP lysis buffer. Then, cell extracts were immunoprecipitated with anti-PPP1R26-coated magnetic beads overnight at 4 °C. After being washed six times, the PPP1R26-immunoprecipitated complexes were incubated with proteinase K digestion buffer. RNA was extracted from the precipitates using phenol: chloroform: isoamyl alcohol (125:24:1) RNA extraction methods. The relative expression of RNA was determined by RT-qPCR and normalized to the input. Normal mouse IgG was used as a negative control.
Chromatin Immunoprecipitation (ChIP)
ChIP was performed as described previously . Briefly, 1% formaldehyde was added to the cells to cross-link nuclear proteins with genomic DNA, and 0.125 M glycine was added to stop the cross-linking. The cells were collected, centrifugated and resuspended in FA lysis buffer (1% SDS, 10 mM EDTA, protease inhibitors and 50 mM Tris-HCl pH 8.0) and genomic DNA was sonicated to get a length of approximately 300 to 1000 bp. Cell lysates were clarified by centrifugation, diluted by 1:10 in ChIP dilution buffer (0.01% SDS, 1.0% Triton X-100, 1.2 mM EDTA, 16.7 mM NaCl, protease inhibitors and 16.7 mM Tris-HCl pH 8.0) and incubated with anti-TGIF2 or control IgG overnight at 4 °C with rotation. ChIP-Grade Protein G Magnetic Beads (Cell Signaling Technology) were added to each reaction and incubated for 2 h. Protein G magnetic beads were pelleted by centrifugation and washed with the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris-HCl pH 8.0), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl and 20 mM Tris-HCl pH 8.0) and LiCl wash buffer (0.25 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl pH 8.0). Finally, beads were washed twice with 1 ml of TE buffer (1 mM EDTA and 10 mM Tris-HCl pH 8.0). The protein-DNA cross-linking was reversed by incubation with 200 mM NaCl and proteinase K for 2 h at 65 °C. DNA was extracted and subjected to real-time qPCR reaction.
Cellular fractionation was prepared as described previously . Briefly, HCC cells were collected and resuspended gently in Buffer A (1 mM HEPES-KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5% NP-40), incubated on ice for 10 min and then centrifugated to collect supernatant as cytoplasmic fraction. The pellet were washed with Buffer A and suspended in Buffer T (1% NP-40, 450 mM NaCl, 50 mM Tris-Cl pH 7.4, 1 mM PMSF, 0.2 mM Na3VO4, 5 mM β-glycerophase, 20% glycerol, 2 mM DTT). The supernatant was collected as nuclear extracts after centrifugation at 12000 rpm for 30 min.
Protein extraction from human HCC frozen tissues
Proteins for Western blot were extracted from human HCC frozen tissue samples using cold lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 150 mM EDTA, 10% SDS, 10% Sodium deoxycholate, 1 × cocktail, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF). Protein concentration was measured by Coomassie brilliant blue G250 assay (Beyotime Biotechnology, Shanghai, China).
Western blotting and antibodies
Western blot was performed as described previously . Primary antibodies specific to PKM1 (1:1000), PKM2 (1:1000), PTBP1 (1:1000), phospho-Ser37 PKM2 (1:1000), E-cadherin (1:2000), Vimentin (1:1000), TOP1 (1:2000), Tubulin (1:3000), Flag (1:3000), GFP (1:5000) and β-actin (1:10000) were used. Anti-PPP1R26 was generated in our laboratory (1:1000) [13, 16]. The detailed information on antibodies for this study is listed in Supplementary Table 3.
Real-time qPCR was done as previously described . Briefly, total RNA was extracted from cells by TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reversely transcribed into cDNA. Reverse transcription was performed using PrimeScript™ RT Master Mix (Roche, Nutley, NJ, USA). Real-time PCR was performed with SYBR® Premix Ex Taq™ GC (Roche, Nutley, NJ, USA) to determine the relative mRNA expression levels. The human β-actin was included as an internal control. Each sample was assessed in triplicate and each real-time qPCR experiment was independently repeated at least three times. Gene expression was presented as the ratio of target gene mRNA levels to the β-actin mRNA level. The primers were described in Supplementary Table 4.
IHC was performed as previously described . The tissues were fixed in formalin and embedded in paraffin. Four-micrometer-thick sections of paraffin-embedded tissues were mounted on poly-L-lysine-coated slides. Then, the slides were deparaffinized in xylene and rehydrated with a gradient of ethanol and distilled water. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min at room temperature. After antigen retrieval, the sections were incubated with primary antibody overnight at 4 °C. The primary antibody was probed with a two-step Poly-HRP Anti-Mouse/Rabbit IgG Detection System (Zsbio, Beijing, China). The expression of proteins was classified according to a semi-quantitative score. The percentage of positive tumor cells were scored as following: 0% (0), 1–25% (1), 26–50% (2), 51–75% (3), and > 75% (4). The intensity was assessed as follows: negative staining (0), weak staining (1), moderate staining (2), and strong staining (3). The scores of staining intensity and proportion were multiplied for comprehensive evaluation. The expression of proteins in HCC tissues was scored independently by two senior pathologists.
Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 20 min at room temperature. Cells were incubated with primary antibodies overnight at 4 °C after blocking with 10% goat serum. After washing with 1 × PBS, the cells were incubated with secondary antibodies conjugated with DyLight 488 or DyLight 594 (1:100) (Earthox, Millbrae, CA, USA) for 1 h at 37 °C. The cells were stained with DAPI (Beyotime, Nantong, China) to visualize the nuclei.
Xenograft mouse model
Female BALB/C nude mice aged 5-week (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly separated into three groups (n = 6, per group). SMMC7721-control shRNA, SMMC7721-PPP1R26 shRNA, Huh7-control-shRNA, Huh7-PPP1R26-shRNA, Huh7-PPP1R26-shRNA1 + GFP-PTBP1 or Huh7-PPP1R26-shRNA2 + GFP-PTBP1 cells (3 × 106) were injected subcutaneously into the right and left flanks in each mouse, respectively. The tumor volume was measured every four days and calculated using the formula length×width2 × 0.5 (mm3). The tumor size, volume and weight were measured when the mice were sacrificed at day 28 (SMMC7721) or day 35 (Huh7) post-implantation. The expression of PPP1R26 and PKM2 was evaluated by Western blot and immunohistochemistry. Animal operations were performed according to the National Institutes of Health guidelines. Studies on animals obtained the approval of the Institutional Animal Care and Use Committee of Peking University Health Science Center (Ethics Approval License: LA2020428).
SMMC7721/Huh7-Ctrl shRNA or SMMC7721/Huh7-PPP1R26 shRNA cells were subcutaneously injected into the nude mice. When tumor diameter reached up to 1 cm, mice were analyzed on a Super Nova PET/CT scanner (PINGSENG Healthcare [KunShan] Inc., Jiangsu, China). After mice had fasted overnight before the PET scans, the xenograft-bearing mice were injected with 18.5 MBq (0.5 mCi, 200 μL) of 18F-FDG via the tail vein. Scanning began 45 min after injection. The images were reconstructed using a three-dimensional ordered subsets expectation maximum algorithm (OSEM) without attenuation correction. PET images were acquired for 10 min and reconstructed with attenuation correction based on the CT data (CT-AC reconstruction).
Single-sample gene set enrichment analysis (ssGSEA)
Gene signatures of metabolism were downloaded from Reactome Pathway Database (https://reactome.org/). The identifiers of glucose metabolism, fatty acid metabolism and amino acid metabolism are R-HSA-70326, R-HSA-8978868 and R-HSA-71291, respectively. Gene expression profiles of HCC patients in the TCGA liver hepatocellular carcinoma adenocarcinoma (LIHC) dataset (n = 374) was downloaded from the TCGA Data Portal (https://portal.gdc.cancer.gov/). Based on the expression of metabolic genes, the ssGSEA algorithm of R package “GSVA” was applied to calculate the metabolic score of each sample in TCGA LIHC dataset. Metabolic level of each sample was reflected by ssGSEA scores.
Statistical analysis was performed using SPSS software version 22.0 (IBM, Armonk, NY, USA). The association between the expression of PPP1R26 and clinicopathological parameters in the HCC cohort was assessed by the chi-square test. Overall survival and its association with PPP1R26 protein expression were evaluated using the Kaplan-Meier method followed by the log-rank test. Subsequent univariate and multivariate analysis of prognostic factors was conducted using the Cox regression model. Data are presented as mean ± SEM or mean ± SD. Two-tailed Student’s t-tests were used to assess the differences between the two groups. One-way analysis of variance (ANOVA) with post hoc analysis LSD test was used to analyze differences among more than three groups. Kruskal-Wallis test was performed for nonparametric comparison. P < 0.05 was considered to indicate statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 for all analyses.