Patients and ethical statement
In total, 121 HCC patients’ tumor samples and corresponding adjacent normal liver tissues were obtained from the surgical specimen archives of the Zhongshan Hospital , Shanghai, China. The patients were informed, and signed consent forms acknowledging the use of their resected tissues for research purposes, which has been previously approved .
The HCC cell lines HepG2 and Hep1-6 were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA) and cultured following the recommended guidelines. These cells were characterized by Genewiz Inc. cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific, California, USA) with 10% fetal bovine serum (FBS) and antibiotics. Huh-7 cells were was purchased from JCRB cell bank (Tokoyo, Japan) and cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Thermo Fisher Scientific) with 10% new born calf serum.
In vivo mouse models
All studies were supervised and approved by the Shanghai University of Traditional Chinese Medicine Institutional Animal Care and Use Committee (IACUC). Female mice were used as models to study liver cancer. Power analysis indicated an n value of 5 mice per group to identify the expected effects with 90% confidence.
Total RNA was extracted from liver samples or HepG2 or Huh-7 cells with TRIzol Reagent (Thermo Fisher Scientific, California, USA). Quantitative real-time PCR (qPCR) was performed with an iQ5 machine and SYBR Premix Ex Taq™ II (TaKaRa Bio, Tokyo, Japan). Data were normalized to β-actin or IgG control (RNA pull-down assay and RIP assay). Relative genomic level of tumor tissues was compared with normal liver tissue. Primers used for RT-qPCR and RT-PCR are described in additional file 1: Table S1.
Cytoplasmic and nuclear RNA isolation
Cytoplasmic and nuclear RNA was extracted using Thermo Fisher BioReagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. QRT-PCR analysis was performed using SYBR® Green Master Mix (Invitrogen, New York, USA) to amplify the localization of LINC01132 assay and β-actin U6 were used as cytoplasmic and nucleus controls. Primers are listed in additional file 1: Table S1.
Lentivirus construction and infection
The LINC01132 sequence (listed in additional file 1: Table S1) was amplified from normal genomic RNA and cloned into the pWPXL lentiviral vector to generate pWPXL-LINC01132. Virus particles were harvested 48hrs after HEK 293T cells were transfected with pWPXL-LINC01132, with the packaging plasmid psPAX2 and the VSV-G envelope plasmid pMD2.G using Lipofectamine 2000 reagent (Invitrogen). Huh-7, HepG2 and Hep1-6, cells were infected with recombinant lentivirus-transducing units in the presence of 1 μg/ml polybrene (Sigma-Aldrich, Missouri, USA)
In vitro cell proliferation and colony formation assays
The cell proliferation assay was measured using the Cell Counting Kit-8 (CCK-8) (Dojindo, Kyushu, Japan) at 1, 3, and 5 days after LINC01132 or mock infection. Cells were seeded into the 96-well plate at a density of 103 cells/well, and 10μl of CCK-8 was added to 90μl of the cell culture medium per well. Cells were subsequently incubated at 37°C for 2hours and the optical density measured at 450 nm. For the colony formation assay, 1-1.5 *103 cells/well were plated in a 6-well plate and incubated at 37°C for 2 weeks. The colonies were fixed and stained with 0.1% crystal violet dye in 20% methanol, and the number of colonies macroscopically counted. All assays were performed in triplicate.
In vitro migration and invasion assays
Migration assays were performed in a Transwell chemotaxis 24-well chamber (BD Biosciences, Franklin Lakes, NJ). Briefly, 2×104 cells were plated in the upper chamber with a non-coated membrane. For the invasion assays, 5×104 cells were placed into the upper chamber with a Matrigel-coated membrane. After 16 hours of incubation at 37°C, migrating or invading cells were fixed and stained with 0.1% crystal violet dye in 20% methanol. Migrated or invaded cells were counted and imaged with an inverted microscope (Olympus, Tokyo, Japan).
In vivo assays for metastasis
For the in vivo metastasis assays, 2×106 Hep1-6 cells infected with the pWPXL-LINC01132 or pWPXL-GFP were resuspended in 0.2mL of serum-free DMEM and subcutaneously injected into C57BL/6 mice liver. After 40 days mice humanly euthanized and liver, lungs and intestines were collected, fixed with phosphate buffered neutral formalin and prepared for standard histological examination. The numbers of metastatic foci in liver tissue sections were counted by H&E staining under a binocular microscope (Leica, USA).
Construction of PDX mouse model of HCC
PDX model of liver cancer was established from patient's liver cancer samples. These were first sectioned into small tissue for subcutaneous tumor formation in nude mice. Two weeks later, the subcutaneous tumor bearing tissue was excised, sectioned and and transplanted again into the subcutaneous skin of nude mice. After subcutaneous tumor formation, shNC and shLINC01132 were injected subcutaneously and tumor diameter and weight were measured every 2 days. Animals were humanly euthanized after 30 days and tumor tissue removed for terminal endpoint analysis.
RNA pull-down assays
LINC01132 was transcribed in vitro with biotin RNA labelling mix and T7 RNA polymerase according to the manufacturer’s instructions (Invitrogen). In total, 40μl streptavidin-linked magnetic beads (ThermoFisher Scientific) were used to pull down the biotinylated RNA at room temperature for 2hrs. The beads-RNA-proteins were then washed with 1× binding washing buffer (5mM Tris-HCl, 1M NaCl, 0.5mM EDTA, and 0.005% Tween 20) four times. The proteins were precipitated and diluted in 60μl protein lysis buffer. Finally, the retrieved proteins were measured on SDS-PAGE gels for mass spectrometry or Western blot. Western blot in RNA pull-down assay was performed with mouse anti-NRF1 and anti-KDM5B antibodies (Cell Signaling Technology, CST, Danvers, Massachusetts, USA, 1:500) and mouse anti-β-actin antibody (CST, 1:1,000). Antibodies information is available on additional file 1: Table S2.
RNA-seq and computational analysis
RNA-seq was performed at the Sequencing and Non-Coding RNA Program at the RiboBio (Guangzhou, China) using Hiseq3000（Illumina, USA）. The hisat2 , StringTie  and Ballgown were used to align the reads to the genome, generate raw counts corresponding to each known gene, and calculate the RPKM (reads per kilobase per million) values .
LINC01132 levels were measured by northern blot using an Ambion Northern Max-Gly Kit (Austin, TX, USA). Total RNA was electrophoresed and siphoned to a positively charged nylon membrane (NC). RNA was then fixed to the NC membrane using UV cross-linking. In brief, the cross-linked membrane was then prehybridized with ULTRAhyb, and RNA was detected with an LINC01132-specific oligonucleotide probe (primers provided in additional file 1: Table S1) labeled with digoxigenin-ddUTP using a DIG Oligonucleotide 3’-End Labeling Kit (Roche Diagnostics, Indianapolis, IN, USA) in roller bottles.
Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). Non-specific binding was blocked with 5% nonfat milk and subsequently incubated with indicated primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity was visualized with chemiluminescence ECL reagents (Pierce, Rockford, IL) and imaged with ChemiDoc imaging system (12003153-s). Densitometry analysis was performed with Image-Pro Plus 6.0 (Media Cybernetics).
Mass spectrometry analysis
Specific bands were excised for proteomics screening by mass spectrometry analysis (Shanghai Applied Protein Technology, Shanghai, China). Protein identification was retrieved from the human RefSeq protein database (National Center for Biotechnology Information), using Mascot version 2.4.01 (Matrix Science, London, UK). The retrieved protein was detected by western blot.
RNA immunoprecipitation (RIP)
RIP experiments were performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Massachusetts, USA) according to the manufacturer’s instructions. The co-precipitated RNAs were detected by reverse transcription PCR. Total RNA (input controls) and normal mouse IgG controls were assayed simultaneously to validate RNA specificity to NRF1 and KDM5B (n=3 for each experiment). Gene-specific primers for LINC01132 are provided in additional file 1: Table S1).
Huh-7 and HepG2 cells infected with the lentivirus expressing LINC01132 or Huh-7 cells transfected with LINC01132 siRNA were lysed with RIPA buffer (Beyotime Biotechnology) with protease (Thermo Fisher Scientific Inc.) and RNase (Thermo Fisher Scientific Inc.) inhibitors, and then centrifuged at 16,400g for 15min. Supernatants were collected and the amount of NRF1 and DPP4 or KDM5B and DPP4 protein examined by immunoblotting to normalized for DPP4 and NRF1 loading. Supernatants were then incubated with the indicated antibody-coated Protein G Dynabeads (Life Technologies) overnight at 4°C with gentle rotation. The beads were washed five times with NT2 buffer (50mM Tris-HCL [pH 7.4], 150mM NaCl, 1mM MgCl2, 0.05% Nonidet P-40) containing protease and RNase Inhibitor, and then three times with PBS again containing protease and RNase Inhibitor. After washing, proteins were eluted and immuno-complexes were analyzed by immunoblotting (antibodies provided in additional file 1: Table S2).
Genome-wide expression of lncRNAs in HCC
Genome-wide expression of lncRNAs in HCC was collected from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). Gene expression profile of HCC was collected from GEO under the accession number GSE104310, which provided the expression of tumors and paired non-tumor tissues collected from Sun Yat-sen University Cancer Center. In total, eight normal and 12 tumor samples were included in our analysis. Moreover, genome-wide expression of liver hepatocellular carcinoma (LIHC) were obtained from TCGA project , which included 374 tumor and 50 normal samples. RNA sequencing of 21 Hepatitis B virus-HCC patients with non-neoplastic liver and tumor tissues were obtained from GSE94660 .
Identification of differentially expressed lncRNAs in HCC
Wilcoxon’s rank sum test was used to identify differentially expressed lncRNAs in HCC tumor compared to normal tissues. Only long intergenic non-coding RNAs (lincRNAs) were considered in our analyses. We identified the lincRNAs with fold changes > 2 or < 0.5 and adjusted p-values < 0.05 as differentially expressed lincRNAs.
Predict the functions of lncRNA
Spearman correlation coefficients (SCC) were used to predict the function of lncRNA and protein-coding gene expression. All genes were ranked based on SCC and subjected into Gene Set Enrichment Analysis (GSEA) . The Biocarta pathway dataset from MsigDB was used for this analysis . Pathways with normalized enrichment score > 1.96 or < -1.96 and false discovery rate (FDR) < 0.01 were identified.
Transcription regulation analysis
To identify the transcription factors (TFs) that bind to the promoter region of gene (e.g., DPP4), we queried the ChIPBase database . The TFs than can bind to the 5kb upstream to 1kb downstream of transcript start sites were identified.