Clinical specimens
Between January 2012 and October 2014, 60 pairs of CRC tumor tissues and corresponding adjacent normal tissues were collected from patients with CRC who underwent surgery at Xuzhou Medical University Affiliated Hospital (Xuzhou, China). The patients did not receive any radiotherapy or chemotherapy before the operation. Diagnoses were pathologically confirmed, and the samples were immediately placed in liquid nitrogen after collection until use. The detailed clinicopathological features are summarized and analyzed in Additional file 1: Table S1. The study was conducted according to the guidelines of the Declaration of Helsinki. The research design, study protocols and information security were approved by the Ethics Committee of the affiliated hospital of Xuzhou Medical University (Xuzhou, Jiangsu, PR China), and written informed consent was obtained from all patients before enrolling in the research program.
Cell culture and cell treatment
Human CRC cell lines (HCT116, SW620, SW480, DLD-1, LoVo) were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The human normal colorectal epithelial cell line FHC was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in an appropriate medium supplemented with 10% fetal bovine serum (FBS; ExCell Bio, NY, USA) and 1% antibiotic/antimycotic solution and maintained in an incubator at 37°C with 5% CO2 in a humidified atmosphere. DLD-1 and HT-29 cells were maintained in RPMI-1640 medium (Gibco, Thermo Fisher, Waltham, MA, USA).
Cell transfection
Negative control siRNA (si-NC) and three individual LINC01594 siRNAs (si-LINC01594#1, #2, #3) were purchased from IBSBIO (Shanghai, China). CELF6 siRNA was purchased from Gene Pharma (Shanghai, China). The siRNA sequences are listed in Additional file 2: Table S2. Cells were transfected with siRNA at 30–50% confluence using jetPRIME® Polyplus Transfection (Polyplus-transfection S.A., Illkirch, France). LINC01594 and CELF6 were amplified from human cDNA as a template and cloned into the pcDNA3.1(+) vector (Invitrogen, USA). SW480, LoVo and HCT116 cells were grown to 90% confluence before being transiently transfected with plasmids using Hieff Trans™ Liposomal Transfection Reagent (Yeasen Biotechnology, Shanghai, China) according to the manufacturer’s protocol. At 24–48 h posttransfection, cells were harvested for qPCR or Western blot analysis.
RNA extraction, reverse transcription-PCR and qRT‒PCR
Total RNA from CRC tissues and cells was isolated and quantified using TRIzol reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions. LncRNAs and mRNAs were reverse transcribed using a SweScript RT I First Strand cDNA Synthesis Kit (Servicebio, Wuhan, China) in accordance with the manufacturer’s protocol. Relative quantification of LINC01594 and CELF6 was carried out by the 2-ΔΔCT method; 18S rRNA was used as an internal control when testing relative expression in tissues, and GAPDH was used as an internal control for cell lines. The reactions were performed independently in triplicate. Quantitative PCR assays were carried out with an ABI StepOne (Carlsbad, CA, USA) using 2 × SYBR Green qPCR Master Mix (High ROX) (Servicebio, China). The primer sequences are listed in Additional file 2: Table S2.
Transwell assays
A modified two-chamber culture system with an 8-m pore size was used to test cell migration and invasion abilities using Transwell inserts (Corning Incorporated, USA) with or without Matrigel (BD Biosciences, USA) coating. Cells that had been transfected were plated into Transwell inserts, fixed with 4% paraformaldehyde solution and stained with 1% crystal violet after culture. We used an Olympus microscope to obtain images at a magnification of × 100 and ImageJ software to calculate the number of cells penetrating the pores. All experiments were carried out three times.
Wound healing assay
Cells in incubation wells (1 ×106) were wounded using a 100-µl plastic pipette tip. After 24 h of incubation, the wound sizes were assessed and imaged. The cell migration area between inscribed dashed lines in the wells was measured using ImageJ software (NIH, Bethesda) and normalized to control cells.
Immunoblotting, immunofluorescence staining (IF) and antibodies
Cells were harvested with RIPA lysis buffer (Vicmed, China) supplemented with phenylmethylsulfonyl fluoride (PMSF). Lysates were cleared by centrifugation at 14000 rpm for 15 min at 4°C. The protein content of the cell lysates was quantified, and proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose filter membrane, blocked with 5% skim milk (BD Biosciences) in Tris-buffered saline with 0.05% Tween-20, and probed with specific antibodies. Chemistar™ High-sig ECL Western blotting Substrate (Tanon, Shanghai, China) was used to detect signals.
Cells grown on glass coverslips were fixed with 4% paraformaldehyde (PFA, Sigma Aldrich), permeabilized with 0.2% Triton X-100, and stained according to standard procedures. CoraLite488-conjugated goat anti-rabbit IgG (H + L) and CoraLite594-conjugated goat anti-rabbit IgG (H + L) were purchased from Proteintech (Wuhan, China). DAPI reagent was used to stain cell nuclei. The cells were imaged using an AXION SCOPE A1 HAL100 (ZEISS, Germany).
Antibodies against the following proteins were used: CELF6 (1:500, Proteintech), DNMT1 (1;2000, ABclonal), p53 (1:5000, Proteintech, China), E-cadherin (1:5000, Proteintech), and N-cadherin (1:5000, Proteintech).
Tissue microarrays (TMAs) and RNA fluorescence in situ hybridization (RNA-FISH)
TMAs containing samples from 180 CRC patient tissues were purchased from Shanghai TUFEI Biotech Company. The Ethics Committee of Shanghai TUFEI Biotech Company approved this research. The RNA-fluorescence in situ hybridization (RNA-FISH) assay was accomplished by Servicebio (Wuhan, China). In brief, TMAs were fixed with 4% paraformaldehyde, trimmed, dehydrated, embedded, sectioned, stained, sealed and microscopically qualified in strict accordance with the SOPs for pathological laboratory examination of the Servicebio laboratory (Wuhan, China). A PANNORAMIC scanner (3DHISTECH, Hungary) was used to scan the TMAs. CaseViewer2.4 software (3DHISTECH, Hungary) was utilized for visualization, and Halo v3.0.311.314 software (Indica labs, U.S. A) was used to calculate the positive rate (%) in the target area (positive cells/the total number of cells). The relationship between LINC01594 expression and clinicopathological features in TMA specimens is listed in Additional file 3: Table S3.
FAM-labeled LINC01594 probes were designed and synthesized by Servicebio (Wuhan, China) and were combined with target genes using a FISH kit (Servicebio, Wuhan, China) according to the manufacturer’s protocol.
Methylation-specific PCR (MSP)
Cellular genomic DNA was isolated using a Genomic DNA extraction kit (TIANGEN, China), and the genomic DNA isolated was treated with bisulfite to convert unmethylated cytosine to uracil using a DNA Bisulfite Conversion Kit (TIANGEN, Beijing, China). The bisulfite-treated DNA was amplified using specific primers for methylated DNA (M-MSP) and unmethylated DNA (U-MSP) by PCR to determine CELF6 promoter methylation. The amplified DNA fragments were then subjected to 1.5% agarose gel electrophoresis, and images were taken. The MSP primers are listed in Supplemental Additional file 2: Table S2.
RNA immunoprecipitation (RIP)
Cells were collected and resuspended in 2 ml of nucleus isolation buffer (1.28 M sucrose, 40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 4% Triton X-100) and 6 ml of water for 20 min on ice. Next, 150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 100 U/ml RNase inhibitor (ABclonal, Wuhan, China) and cocktail (Vicmed, Xuzhou, China) were added, and the nuclei were sonicated using an ultrasonic cell disruptor. Antibodies were added to the supernatant, which was rotated overnight at 4°C. Magnetic beads were added for 2 h, and TRIZOL was used to purify the bead-bound RNA. RNA was detected by qPCR.
Chromatin immunoprecipitation (ChIP)
Cells were collected after formaldehyde crosslinking, resuspended in ChIP lysis buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.1% deoxycholate, 1% Triton X-100, 150 mM NaCl, 100:1 RNase inhibitor and cocktail added before use) and left on ice for 10 min. After ultrasonically shearing the cells, antibody coupling agent was added for 4 h. Subsequently, 25 µl of immunomagnetic beads was added, and incubation was continued for 1.5 hours. Chelex-100 was added, and the samples were boiled to release DNA. Then, 1 µl of proteinase K was added, and the samples were denatured at 55°C at 1300 r/min. The samples were boiled for 10 min, 10 µl of linear polyacrylamide and 250 µl of anhydrous ethanol were added to the input group, and 10 µl of 3 M sodium acetate was added. The samples were placed at -80°C for 30 min and centrifuged at 4°C at 13000 x g for 15 min. The precipitate was air-dried, and the supernatant was subjected to qPCR detection.
RNA pull-down assay
The RNA pull-down assay was carried out as follows. In brief, in vitro biotin-labeled LINC01594 was transcribed with Biotin RNA Labeling Mix (Roche Corporation, US) and T7 RNA polymerase (APExBIO, US), treated with RNase inhibitor, and purified with a clean-up kit (Promega Corporation, USA). The biotinylated LINC01594 probes were dissolved in binding and washing buffer and incubated with streptavidin agarose resin (Beyotime Biotechnology, China). Lysates of HCT116, SW480 and LoVo cells were incubated with probe-coated streptavidin beads, and products were separated by SDS‒PAGE.
Chromatin isolation by RNA purification (ChIRP)
ChIRP-qPCR experiments were performed using published protocols[18] with slight modifications. The LINC01594 probes were designed by IBSBIO, and the sequences are listed in Additional file 2: Table S2. Briefly, 5*107 SW480 cells were cross-linked with 1% formaldehyde for 10 min at RT and resuspended in lysis buffer (50 mM Tris HCl [pH 7], 10 mM EDTA, 1% SDS). and left on ice for 10 minutes. The lysate was sonicated using an ultrasonic cell disruptor (20 cycles, 30 s on/off) to obtain an average DNA size in the range from 200 to 600 bp and incubated with LINC01594 probes in hybridization buffer (500 mM NaCl, 1% SDS, 50 mM Tris–HCl, pH 7.0, 1 mM EDTA, 15% formamide, protease inhibitor cocktail, PMSF and RNase inhibitor) for 4 h at 4℃. Subsequently, chromatin complexes were purified using magnetic streptavidin beads and then subjected to stringent washes, and DNA was eluted with a cocktail of 100 µg/ml RNase A (Beyotime, Shanghai, China) and 0.1 U/µl RNase H (Beyotime, Shanghai, China). The crosslinking was reversed by incubation in the presence of 0.1 µg/µl protease K, 150 mM NaCl, and 10 mM EDTA at 50°C. DNA was then extracted with equal volumes of phenol, chloroform, and isoamyl and precipitated with ethanol at -80°C overnight.
Hematoxylin-eosin (HE) and immunochemical staining (IHC)
Sections of the CRC and adjacent tissue samples were deparaffinized in xylene and rehydrated with ethanol before paraffin embedding. All tissue samples were sectioned to produce 4-mm thick slices. To perform HE staining, the slices were stained with hematoxylin and eosin for 3 min and 5 seconds after dewaxing. For IHC, the slices were incubated in boiling 0.01 mol/l citric acid buffer for 15 min for antigen retrieval before incubation with primary antibodies. After incubation with the secondary antibody, DAB solution (Dako Denmark A/S) and hematoxylin were applied for staining before sealing.
In vivo experiments
The animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Xuzhou Medical University in Xuzhou, China. Mice were housed in a 12-h light/dark environment and had free access to a standard rodent diet and clean water. Male athymic 4-week-old BALB/c nude mice were obtained from the Animal Center of Gempharmatech Co., Ltd. (Nanjing, China) and maintained in a specific pathogen-free facility. For lung metastasis experiments, nude mice were randomly divided into two groups (n = 10 per group). HCT116 cells stably transfected with LINC01594 LV-RNA or scrambled control were harvested from 6-well plates. CRC cells (2 × 106) were administered via lateral tail vein injection (n = 10 per group). After 8 weeks, the mice were sacrificed, and the lungs were removed to observe lung metastases. The relative lung tissue anatomy was noted, and slices were used for further statistical analysis.
Analysis of online datasets
The normalized lncRNA expression values in online-available datasets were downloaded from the TCGA database and GEO database (GSE84983, GSE110715 and GSE115856), and we first searched differentially expressed lncRNAs based on the TCGA dataset. We selected upregulated lncRNAs in tumor tissues according to the TCGA dataset. We then analyzed whether these selected lncRNAs were still upregulated in the GSE84983, GSE110715 and GSE115856 datasets. Finally, lncRNAs that were upregulated in these two datasets were identified. Q-PCR was used to determine the expression of lncRNAs in cell lines and tissues. Thus, the most differentially expressed lncRNA in CRC cell lines and tumor tissues was screened out. Therefore, LINC01594 was eventually selected. The heatmaps were determined according to the normalized expression levels of lncRNAs.
Statistical analysis
GraphPad Prism 8.0.2 software (San Diego, CA, USA) was used to statistically analyze all data. Student’s t test was used when comparing differences between two groups. Correlations were assessed with Spearman’s correlation coefficient. A P value of less than 0.05 was considered to indicate statistical significance. ChIP-qPCR and qRT-PCR experiments were performed in three technical replicates. Each experiment was repeated three times at least.