Tissue specimens
The tumor and adjacent normal tissues used for immunohistochemistry were collected from 90 HCC patients who underwent surgery at Shanghai Seventh People’s Hospital between 2007 and 2009. The informed consent was obtained from every study participant. The study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the Institutional Research Ethics Committee in 2007 (No. 2007-019). The mRNA profiles used in the study were obtained from the Cancer Genome Atlas (TCGA) database (http://cancergenome.nih.gov).
Immunohistochemistry (IHC)
Tissues were fixed in 10% buffered formalin, embedded in paraffin and cut into 4 μm sections. Deparaffinized sections were treated with methanol containing 3% hydrogen peroxide for 10 min before conducting antigen retrieval using a microwave oven at 95 °C for 5 min and cooling at 25 °C for 2 h. After washing with PBS, blocking serum was applied for 20 min. The sections were incubated with primary antibodies overnight at 4 °C and biotin-marked secondary antibodies for 20 min followed by a peroxidase-marked streptavidin for an additional 20 min. The reaction was visualized by using 3, 3′-diaminobenzidine tetrahydrochloride. The nuclei were counterstained with hematoxylin. Reproducibility of staining was confirmed by reimmunostaining via the same method in multiple, randomly selected specimens.
Cell culture
The HCC cell lines used in this study included BEL-7404 cells and SMMC-7721 cells. They were obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. BEL-7404 and SMMC-7721 cells were both maintained in 1640 medium (Gibco, Gaithersburg, MD, USA), supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Gibco, Gaithersburg, MD, USA) at 37 °C in 100% air.
RNA interference
The sequences for RNA interference were 5′-TTCTCCGAACGTGTCACGT-3′ for control, and 5′- CAGTTTCTGCTCTCAGGAA-3′ for PYCR1 (GenePharma, Shanghai, China). The sequences were inserted into Age I and EcoR I points of GV115 vector. The shRNAs of GV-NC-GFP-shRNA and GV-PYCR1-GFP-shRNA were constructed as control lentivirus and shPYCR1 lentivirus, and positive clones were screened and identified. Subsequently, HCC cells were seeded in a 6-well plate at a density of 2 * 105/well for RNA interference. According to the results of preliminary experiment, HCC cells were infected with a certain amount of control lentivirus and shPYCR1 lentivirus. At 72 h post-infection, the expression of reporter gene GFP was observed by the fluorescent microscope. The fluorescence rate was the positive infective rate. If the infective rate was larger than 70% and the state of cells was normal, the further study could be continued.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA from HCC cells was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was determined by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA). A total of 20–100 ng RNA was reverse-transcribed using First-Strand cDNA Synthesis kits (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s protocol. qRT-PCR was performed on ABI 7500 System (Applied Biosystems, Foster City, CA, USA) and used to analyze the expression levels of mRNA. Relative expression level of genes was calculated using GAPDH as the internal control. Each sample was run three times. The primer pairs used in the study are listed as follows. PYCR1 forward: 5′-GGCTGCCCACAAGATAATGGC-3′; reverse: 5′-CAATGGAGCTGATGGT GACGC-3′; GAPDH forward: 5′-TGACTTCAACAGCGACACCCA-3′; reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′.
Western blot
The cells were first washed with cold PBS three times, then lysed in RIPA lysis buffer (Beyotime, Shanghai, China) containing a complete protease inhibitor cocktail (Boehringer Mannheim, USA), and cell lysates were collected. Concentrations of proteins were then determined by bicinchoninic acid (BCA) protein assay kit (Boster, Wuhan, China). About 20–50 μg of proteins were separated by 10% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. Subsequently, the membrane was incubated with primary antibodies against PYCR1 (1:200) (Abcam, UK), JUN (1:500) (Abcam, UK), IRS1 (1:500) (Abcam, UK) and GAPDH (1:2000) (Santa Cruz, Dallas, TX, USA) overnight at 4 °C, and appropriate secondary antibodies (1:2000) (Santa Cruz, Dallas, TX, USA) for 1 h at room temperature. Signals were detected by gel imaging system (ProteinSimple, San Jose, CA, USA). The ratio of interested proteins to GAPDH was analyzed by FluorChem FC3 software. Western blot experiments were performed in triplicate.
Cell proliferation assay
HCC cells infected with control lentivirus (2.5 μl, 8 × 108 TU/ml) and shPYCR1 lentivirus (6.7 μl, 3 × 108 TU/ml) were seeded in 96-well plates at a density of approximately 2–5 * 103/well in quintuplicate. Cell proliferation was performed at specific times, such as 1st day, 2nd day, 3rd day, 4th day and 5th day. The fluorescence was measured to access cell counts using Celigo Imaging Cytometer (Nexcelom Bioscience, Lawrence, MA, USA). In addition, for MTT assay, 20 μl of MTT solution was added to each well. After 4 h incubation at 37 °C, the solution was removed and DMSO was added to dissolve the formazan crystals formed. The absorbance at 490 nm was measured Glomax-Multi + Detection System (Promega, Madison, WI, USA). At least three independent experiments were performed.
Colony formation assay
Cells were seeded in 6-well plates at a concentration of 400–1000 cells per well and changes culture with media every 72 h. Colonies were fix with 4% paraformaldehyde and stained with GIEMSA solution (Dingguo, Shanghai, China) after 14 days, then colonies were visualized by fluorescence microscope and quantitated. At least three independent experiments were performed.
Cell apoptosis assay
Apoptosis assay was performed using FITC-Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacture’s instruction. Briefly, the cells were collected by trypsin, washed twice with ice-cold PBS and resuspended in 1 * binding buffer. Then, 10 μl of annexin V-FITC and propodium were added into 100 μl of cell suspensions. After incubation for 15 min, the samples were analyzed using the Beckman Counter flow cytometer.
In vivo tumor growth experiment
A total of twenty female BALB/C nude mice (4–6 weeks, 18–22 g) were purchased from Shanghai Lingchang Biotechnology (SCXK2013-0018). All of them were randomly separated into two groups, NC group (n = 10) and KD group (n = 10). They were subcutaneously inoculated with SMMC-7221 cells (5 * 106) stably expressing control lentivirus and shPYCR1 lentivirus, respectively. After the diameter of tumor was larger than 5 mm, tumor volume was determined twice per week. The tumor volume was calculated as V = (width2 * length)/2. The bioluminescence imaging was detected before sacrifice. Tumor was obtained and weighed after sacrifice. All animal studies were approved by the Ethics Committee of Shanghai Seventh People’s Hospital.
Microarray and bioinformatics analysis
Total RNA was extracted from HCC cells using Trizol reagent in shCtrl group (n = 3) and shPYCR1 group (n = 3). The qualified samples were analyzed on Affymetrix GeneChip primeview human gene expression array. Differentially expressed genes were selected with a cut-off P value of less than 0.05 based on statistical analysis and a twofold change cut-off value. Those differentially expressed genes obtained from the microarray analyses were uploaded to Ingenuity Pathway Analysis (IPA, Ingenuity Systems) and a core biologic pathway analysis was performed to identify molecular pathways.
Statistical analysis
Statistical analysis was performed by SPSS 16.0 (Chicago, IL, USA). The data were expressed as mean ± standard deviation and analyzed using Mann–Whitney test because of abnormal distribution or heterogeneity of variance. P < 0.05 was considered statistically significant. All results were confirmed by at least three independent experiments.
