Patient tissue samples
A consecutive series of 216 HCC patients (≥ 18 years) who underwent curative hepatectomy at the Guangxi Medical University Cancer Hospital (Nanning, China) between January 2014 and September 2014 were included in the study. Diagnoses of HCC was confirmed by postoperative histopathology. Patients who received neoadjuvant therapies were excluded. Fresh HCC tissues and corresponding adjacent normal tissues were obtained from our tumor tissue bank [12, 21]. All procedures were performed according to the Declaration of Helsinki (2013 version). The study was approved by the Ethics Committee of Guangxi Medical University Cancer Hospital. The requirement for written informed consent was waived because of the retrospective nature of the study.
Cell cultures and M1/M2 phenotype identification
U937 cells (ATCC, CRL-1593.2, USA) were plated in 24-well plates at a density of 2 × 106 cells per well and cultured with fresh RPMI-1640 medium (A1049101, Thermo Fisher Scientific, USA) supplemented with 5% fetal bovine serum (0024012DJ, Thermo Fisher Scientific, USA). To induce the M2 phenotype, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 100 ng/mL, P1585, Sigma-Aldrich, USA) for 24 h, followed by the addition of interleukin (IL)-4 (20 ng/mL, PHC0044, Thermo Fisher Scientific, USA) and IL-13 (20 ng/mL, PHC0135, Thermo Fisher Scientific, USA) for 12 h. The switch from the M2 to the M1 phenotype was obtained by pretreatment with PMA (100 ng/mL) for 24 h, followed by incubation with lipopolysaccharide (LPS; 100 ng/mL, L2630, Sigma-Aldrich, USA) and interferon (IFN)-γ (20 ng/mL, GF305, Sigma-Aldrich, USA). Cellular phenotypes were identified by analyzing the expression of transforming growth factor (TGF)-β, arginase 1 (Arg-1), and tumor necrosis factor α (TNFα) by quantitative PCR (qPCR) analysis. Western blotting analysis was used to quantify the expression of Arg-1 and inducible nitric oxide synthase (iNOS) protein. An enzyme-linked immunosorbent assay (ELISA) was used to assay secreted IFN-γ, TGF-β, and IL-10. Duplicate samples of the identified M1 and M2 cells were stored at − 80 °C for subsequent analysis.
Microarray analysis
Total RNA was isolated from M1 and M2 macrophages using TRIzol® reagent according to the manufacturer’s instructions. The mRNA was further purified using an mRNA-ONLY™ Eukaryotic mRNA Isolation kit (Epicentre, Madison, WI, USA) according to the manufacturer’s instructions. RNA quantity was examined using a NanoDrop® ND-1000 (Thermo Scientific, Scotts Valley, CA, USA), and RNA integrity was evaluated by standard denaturing agarose gel electrophoresis. Sample labeling and Agilent array hybridizations were conducted according to the manufacturer’s instructions for one-color microarray-based gene expression analysis (Agilent Technology, Santa Clara, CA, USA). Each sample was randomly primed using an Arraystar Flash RNA Labeling Kit (Arraystar, Rockville, MD, USA) and transcribed as fluorescently labeled complementary RNA (cRNA), which was then purified using a RNeasy® Mini Kit (Qiagen, Hilden, Germany). Each labeled cRNA sample (1 μg) was fragmented by adding 5 µL of blocking agent (Qiagen, Hilden, Germany) and 1 µL of fragmentation buffer, followed by heating at 60 °C for 30 min and then diluting the samples with 25 µL of GE hybridization buffer. Next, 50 mL of hybridization solution were added to the gasket slide and then lncRNA microarray slides were assembled. All slides were then incubated at 65 °C for 17 h in an Agilent hybridization oven. Finally, the hybridized arrays were washed, fixed, and scanned using the Agilent DNA Microarray Scanner (G2565BA; Agilent). lncRNA expression profiles were analyzed using the SBC Human (4 × 180 K) lncRNA Microarray version 3.0 (Arraystar LncRNA microarray, USA). The obtained lncRNA expression data were then analyzed using GeneSpring software (Agilent Technologies, USA). Fold change was used to identify lncRNAs and mRNAs differentially expressed between M1 and M2 cells. Multiple tests were used to calculate adjusted P values (q value).
Cell lines, cell migration, invasion, and proliferation assays
SMMC-7721, QGY-7703, HepG2, HL-7702, and Huh-7 cell lines were used to detect the expression of lnc-Ma301. Cell migration and invasion assays using SMMC7721 cells were performed in 24-well chambers with 8-µm transwell inserts (Falcon). The cells were seeded in serum-free Dulbecco’s Modified Eagle medium (DMEM; GIBCO, USA) added to the top chamber. For invasion assays, the chambers were precoated with Matrigel (catalogue no.354230, BD) that had been diluted with DMEM at a 1:3 ratio, and then left to solidify for 30 min at 37 °C. SMMC7721 cells (2 × 104) were suspended in 100 µL DMEM and 0.2% BSA, then added to the upper chamber. The cells were left to migrate for 4 h or invade for 24 h at 37 °C. Quantification was performed by counting the mean number of cells in five random fields per chamber using a light microscopy (TS100-F, Nikon, Japan). Cell proliferation assays were performed using the CCK8 method. After transfection, SMMC7721 cells (100 μL, 1 × 104 cells) were seeded in 96-well plates and cultured for 48 h.
ELISA
IL-12 and IL-10 levels in supernatants of M1 and M2 cultures were quantified using an ELISA kit (Invitrogen, UK) according to the manufacturer’s instructions. Briefly, plates were coated with capture anti-IL-10 (MA5-23796, Invitrogen, USA) and anti-IL-12 (PA5-18741, Invitrogen, USA) antibodies in 50 µL/well of carbonate buffer and incubated overnight at 4 °C. A standard curve was prepared using two-fold serial dilutions of the initial concentrations of IL-10 (10 ng/mL) or IL-12 (50 ng/mg) in 1% phosphate-buffered saline (PBS)-BSA in a total volume of 50 µL/well. The samples were then incubated at room temperature with biotinylated antibodies at final concentrations of 2 µg/mL for anti-IL-10 antibody or 1 µg/mL for anti-IL-12 antibody followed by addition of AMDEX streptavidin-peroxidase (Sigma, UK). The reaction was stopped by adding 20 µL of 1 mM H2SO4 once the standard wells showed an intense blue color, and the plates were read at 450 nm using a spectrophotometer (V-5000H, Shanghai Metash Instruments Co., Ltd).
Western blot analysis
M1 and M2 macrophages were harvested, washed, re-suspended in 100 μL of lysis buffer [20 mmol/L HEPES (pH 7.4), 0.5% Nonidet P-40 (v/v), 1 mmol/L EDTA, 2 mmol/L dithiothreitol, 1 mmol/L PMSF, 100 mmol/L NaCl, 2 mmol/L Na3VO4], mixed with NuPAGE LDS buffer (Life Technologies, Carlsbad, CA, USA) and resolved by SDS-PAGE on a NuPAGE 4–12% gel (Life Technologies). The samples were then transferred onto a Trans-Blot nitrocellulose membrane (BioRad, Hercules, CA, USA) for western blotting. Membranes were washed and incubated for 1 h in 5% skimmed milk in TBS and then incubated with primary anti-Arg-1 (1:1000), anti-iNOS (1:500) or anti-β-actin (1:200; Abcam, UK) overnight at 4 °C. Next, samples were incubated with goat-anti-rabbit horseradish peroxidase-conjugated IgG (1:1000) for 1 h and visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to the manufacturer’s protocol. Other antibodies included E-cadherin (catalog no. 20874-1-AP, Proteintech, China) (1:5000), vimentin (10366-1-AP, Proteintech) (1:2000), matrix metalloproteinase 9 (MMP9) (10375-2-AP, Proteintech) (1:1000), slug (ab27568, Abcam, UK) (1:1000), Ki67 (27309-1-AP, Proteintech) (1:1000), anti-AKT1 (ab179463, Abcam) (1:1000), anti-ERK-3 (ab53277, Abcam) (1:1000), caprin-1 (ab241071, Abcam) (1:1000), and GAPDH (ab9484, Abcam) (1:5000) were used to detect relevant proteins.
Quantitative real-time PCR (qRT-PCR)
qRT-PCR was performed using a SYBR Premix Ex Taq kit (RR820A, Takara, Japan) according to the manufacturer’s instructions. The primer sequences are listed in Additional file 1: Table S1. Data were collected and analyzed using a LightCycler 480 instrument (Roche, USA) with the 2ΔΔCt method.
Cell lines over- and underexpressing lnc-Ma301 and caprin-1
The construct pLCDH-lnc-Ma301 (pLCDH is abbreviated as pL in context) was prepared by inserting the lnc-Ma301 sequence (primer sequences in Additional file 1: Table S1) into the polylinker region of pL-CMV-MCS-EF1-GFP + Puro (CD513B-1; Geneseed Biotech, Guangzhou, China) using XbaI and EcoRI restriction enzymes. The full-length sequence of human EST031.1 was obtained by rapid amplification of cDNA ends using RACE and extended. The plasmid was sequenced to confirm that the target sequence had been inserted correctly. Endotoxin-free plasmid was extracted using the E.Z.N.A. ® Endo-free Plasmid Mini Kit I (Omega) and stored at − 20 °C.
The plasmid was packaged into lentivirus using 293T cells (ATCC CRL-11268). Cells stably expressing lnc-Ma301 were harvested and analyzed by qRT-PCR, western blotting, and cell functional assays. As a control to verify the effects of lnc-Ma301, it was targeted specifically using a small interfering RNA (siRNA), siR-lnc-Ma301 (5′-CCAGUGUGAGUGAUGUUUATT-3′). As a negative control, the scrambled sequence siR-NC (5′-UUCUCCGAACGUGUCACGUTT-3′) was prepared.
We performed analogous steps to under- and overexpress caprin-1. The construct pcDNA3.1-caprin-1 (pcDNA3.1 is abbreviated as pC in context) was prepared by inserting the caprin-1 sequence (forward primer, 5′-CTGCACAGCCTATGAATCCAAC-3′; reverse primer, 5′-TTGAGATGCTGTGTACCCCTC-3′) (2148 bp) between the BamHI (GGATCC) and XhoI restriction enzymes in pcDNA3.1. The plasmid was amplified in Top 10 cells. The siRNA siR-caprin-1 (5′-GGAGCAGCUUAUGAGAGAATT-3′) was prepared, as well as the negative control siR-NC (5′-UUCUCCGAACGUGUCACGUTT-3′).
RNA pull-down assay
The MEGAscript™ T7 High Yield Transcription kit (Invitrogen, USA) was used to transcribe biotin-labelled RNAs in vitro. Bio-16-UTP (10 mM, Ambion) was used for transcription. After adding 2 µL of Dnase I, the Eppendorf tube was incubated at 37 °C for 15 min to remove the DNA, then 2 µL of 0.2 M EDTA (pH 8.0) was added. In order to allow the RNA to form secondary structure, 1 µg of biotinylated RNA in RNA structure buffer was heated at 95 °C for 2 min, put on ice for 3 min, then left at room temperature for 30 min. Magnetic beads (Invitrogen, USA) were used to bind and enrich the RNAs. Folded RNA was then mixed with cytoplasmic extract from liver cancer cells in 500 µL RIP wash buffer. The magnetic beads were re-suspended in 50 µL RIP wash buffer, then the suspension was added to Dynabeads M-280 Streptavidin (60210, Invitrogen) and incubated at 4 °C. The suspension was centrifuged for 1 min, and the supernatant was discarded. Magnetic beads were washed briefly with RIP wash buffer for six times and boiled in SDS buffer. The retrieved proteins were detected by western blot and mass spectrometry. RNA probes were as follows: lnc-Ma301 sense: taatacgactcactatagggGGAGAGTTTGGGTCACAGGAGC, lnc-Ma301 antisense: CCTACTTGTTTTTTTTATTTTGG.
RNA immunoprecipitation
RNA immunoprecipitation was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation kit (17–700, Millipore) according to the manufacturer’s instructions. Antibody against caprin-1 (ab241071, Abcam) was used. The proteins enriched by the probe were collected, digested, and identified by FT-ICR-MS (solariX XR ESI, Bruker, USA) followed by the confirmation based on the protein data searching.
RACE assay
Total RNA was isolated using TRIzol Plus RNA Purification Kit (Invitrogen), according to the manufacturer’s instructions. RACE was conducted using the Smart RACE cDNA Amplification Kit (Cat. No. 634923, Clontech) according to the manufacturer’s instructions. The pEASY-Blunt Simple Cloning Kit (Catalogno. CB111-01, TransGen Biotech, China) was used to clone the amplified lnc-Ma301. All selected clones were sequenced and identified. All primers used in the study are listed in Additional file 1: Table S1.
Fluorescent in situ hybridization (FISH)
HepG2 cells were allowed to attach onto the slides, washed with PBS and fixed in 4% paraformaldehyde. All slides were treated with protease reagent, incubated with prehybridization buffer at 42 °C for 4 h and hybridized with digoxin-labeled probe overnight at 42 °C. The slides were incubated with biotin-conjugated anti-digoxin antibody (anti-digoxin-FITC) at 37 °C for 1 h and washed three times. DAPI was used to stain the cell nucleus. The images were captured using a confocal microscope (TCS SP2 AOBS). The probe sequence is listed in Additional file 1: Table S1.
Lung metastasis in mice
Six-week-old male athymic nude mice were purchased from the Experimental Animal Center of Guangxi Medical University (Nanning, China) and housed under standard conditions at the animal care facility of the same center. Animal procedures were approved by the Guangxi Medical University Animal Care and Use Committee, and they complied with all relevant ethical regulations regarding animal research. SMMC-7721 HCC cells transfected with pL or pL-lnc-Ma301 were suspended in 200 μL PBS and injected in the tail vein of the mice (n = 5 per group, one for metastasis detection). The overexpression of lnc-Ma301 was confirmed by qRT-PCR before injection. Mice euthanized were inhalation of CO2 for 5 min. The CO2 exposure used a gradual fill method with a displacement rate about 50% of the chamber volume/min.
All mice were sacrificed 8 weeks later. Photographs were taken to assess lung metastasis, and lung sections were stained using hematoxylin and eosin to assess lung colonization. Levels of E-cadherin, MMP9, and Ki67 in lung tissues were quantified by qRT-PCR and validated by immunohistochemistry and western blotting. Each experiment was performed in triplicate and performed multiple times.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 5.01 or SPSS21.0. The Kaplan–Meier method was used to calculate the overall survival, and the significance of survival differences was determined by log-rank tests. Data from three independent experiments were presented as mean ± standard deviation (SD). Differences were evaluated for significance using two-tailed Student’s t test. P < 0.05 was considered statistically significant.