Patients and tissue specimens
HCC tissues and corresponding adjacent normal tissues were obtained from Hepatic Surgery Center, Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology (HUST, Wuhan, China) between 2012 and 2015 in accordance with the Helsinki Declaration. The study was approved by the Ethics Committee of Tongji Hospital and informed consents were obtained from all patients.
Cell lines and cell culture
The human liver cancer cell lines LM3 and 97H were obtained from the Liver Cancer Institute of Fudan University, Shanghai, China. HepG2, HLF, Hep3B, PLC/PRF/5 as well as normal-type hepatocyte HL7702 cells were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). All cell lines were cultured in high-glucose Dulbecco’s modified Eagle’s medium (HyClone, USA) supplemented with 10% fetal bovine serum (Gibico, USA) at 37 °C in 5% CO2.
Cells were seeded on 6-well or 24-well plates at 50–70% confluence before transfection. SiCCDC183-AS1, miR-589-5p mimics, miR-589-5p inhibitor and the corresponding negative control were purchased from RiboBio (Guangzhou, China). Lipofectamine™ 3000 (Invitrogen, USA) was used as the transfection reagent according to the manufacturer’s protocol.
Lentivirus production and transduction
Stable CCDC183-AS1, miR-589-5p and the corresponding negative control overexpressing or knockdown cell lines were obtained by lentivirus-mediated transduction (Genechem Co., Ltd, Shanghai, China) according to the manufacturer’s protocol.
Fluorescence in situ hybridization (FISH)
FISH assay was performed to detect the location of CCDC183-AS1 in HCC cells. Cy3-labeled CCDC183-AS1 probes were ordered from RiboBio (Guangzhou, China). Hybridization steps were performed using Fluorescent In Situ Hybridization Kit (RiboBio, China) according to the manufacturer’s instructions. Confocal images were acquired on a laser scanning confocal microscope (LSM710, Carl Zeiss, Germany).
Cell proliferation assay
Cell proliferation ability was examined using Cell Counting Kit-8 (CCK-8, Dojindo Crop, Japan). A total of 1000 cells were plated in each well of a 96-well plate. At indicated time 0, 24, 48, 72 and 96 hours, each well was added with 10 μL of CCK-8 reagent and cells were incubated for 1.5 h at 37 °C, the optical density (OD) was measured at 450 nm using a microplate reader (Bio-Tek Instruments, USA). These experiments were repeated for three times.
Transwell migration and invasion assays
For the migration and invasion assays, according to the manufacturer’s protocol, HCC cells were seeded in upper chambers with 100μL of serum-free medium. The transwell chamber (8.0 μm; Corning, USA) was paved with 40 μL 1:4 mixture of matrigel (BD Biosciences, USA) and DMEM for invasion assays and paved without matrigel mix for migration assays. Meanwhile, DMEM Medium (700 μL) containing 10% fetal bovine serum was added to the lower chamber as a chemoattractant. After a 24 h incubation at 37 °C, the non-invading or non-migration cells on the upper membrane were removed mechanically. Cells on the lower surface of the membranes were fixed with 4% paraformaldehyde (Servicebio, China) for 15 min and stained with 0.1% crystal violet (Wuhan Promoter Biological Co., LTD) for 2h. For visualization, the cells were photographed and counted from five random fields. Each experiment was repeated at least three times.
RNA extraction and real-time quantitative polymerase chain reaction
The total RNAs of tissues and cell lines were extracted using TRIzol reagent (Takara, Japan) according to the manufacturer’s instructions. For lncRNA and mRNA, reverse transcriptions were performed using the HiScript® II Q RT SuperMix for qPCR (Vazyme Biotech Co., Ltd). For miRNA, reverse transcriptions were performed using the Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan). and then cDNA amplification was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd) following the manufacturer's instructions with an CFX Connect™ real time system (Bio-Rad, USA). GAPDH and U6 were used as the endogenous control. Relative quantification of lncRNA, miRNA and mRNA expression were compared to endogenous control and analyzed using the comparative CT (2-ΔΔCT) method. All reactions were performed as triplicates. The primer sequences (Sangon Biotech, Shanghai, China) were available in Additional file: Supplementary Table 1.
Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was applied for RNA immunoprecipitation assay. Briefly, cells were lysed in complete RIP lysis buffer. Next, the cell extracts were incubated with magnetic beads conjugated with anti-Argonaute 2 (AGO2) (Abcam) or anti-IgG antibody (Abcam). Then, the beads were incubated with Proteinase K buffer to digest the protein. Finally, the immunoprecipitated RNA was isolated, purified and was further subjected to qRT‐PCR analysis.
Luciferase reporter assays
The sequences of CCDC183-AS1 and 3’-untranslated region (UTR) of the SKP1 and their corresponding mutation were designed, synthesized and inserted into luciferase reporter vector psiCHECK-2 (Promega, Madison, WI, USA). HLF and 97H cells (1 × 105) were plated in 24-well plates for 24 h before transfection. Subsequently, cells were co-transfected with 50 ng of the psiCHECK-2 vector and 50 nM of the miR-589-5p or NC-mimics using Lipofectamine 3000 (Invitrogen, USA). After 48 h of co-transfection, cell lysates were prepared using Passive Lysis Buffer (Promega), and luciferase activity was examined by Dual Luciferase Assay Kit (Promega, USA) in line with the manufacturer’s protocol. The relative luciferase activity of each sample was normalized to firefly luciferase activity. Experiments were repeated three times.
Western blot (WB)
For western blot assay, cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (MedChemExpress, USA) on ice for 30 min. The protein concentrations were quantified by BCA assay. Subsequently, equal amounts of protein were extracted by 10% SDS-PAGE and transferred onto Immobilon®-P transfer membranes (Millipore, USA). Then, the membranes were blocked in 5% skim milk at 37 °C for 1 h and incubated with primary antibodies anti-SKP1 (1:1000, Cell Signaling Technology, USA), anti-cyclin D1 (1:1000, Cell Signaling Technology, USA), anti-p21 (1:1000, Cell Signaling Technology, USA), anti-N-cadherin (1:1000, BD Biosciences, USA), anti-occludin (1:1000, Boster, BioEngineering Company, Wuhan, China) and anti-GAPDH (1:5000, Proteintech, China) at 4 °C overnight. Next, the prepared membranes were incubated with secondary antibody (1:5000, Aksomics, China) at 37 °C for 1 h. Finally, the blots were visualized by ECL chemiluminescent reagent (Bio-Rad, USA) and related data was analyzed by Image Lab™ 4.0 software.
Immunohistochemistry (IHC) staining
Tumor tissues were fixed in 4% paraformaldehyde solution, embedded in paraffin and sectioned. Sections were first baked, deparaffinized in xylene and rehydrated in graded ethanol. Following washes in PBS, the samples were heated for antigen retrieval in boiling 0.01 mol/L citrate buffer (pH 6.0) for 15 min. The sections were then treated with 3% hydrogen peroxide in methanol for 10 min at room temperature. To prevent nonspecific antigen binding, the slides were blocked for 1 hour at room temperature by 5% BSA and then incubated with the primary antibodies anti-SKP1 (1:200, Proteintech, China), anti-Ki67 (1:200, Abcam, USA) at 4°C overnight. After rinses in PBS, sections were incubated for 1 h at room temperature with biotinylated anti-IgG secondary antibody. Subsequently, horseradish peroxidase-labeled streptavidin was added to the slides and incubated for 15 min. Lastly, DAB was used for chromogenic staining and counterstaining was performed with hematoxylin.
All animal care and experiments were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Tongji Hospital, affiliated to Tongji Medical College, HUST. Male BALB/c nude mice (4 weeks old) were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). To generate the subcutaneous tumor model in nude mice, 1 x 106 cells in 100μI serum free DMEM were injected into the axillary region of mice. After 4 weeks, the mice were sacrificed and the tumor tissues were detected for tumor weight, volume, qRT-PCR, WB and IHC staining. For the orthotopic model, 1 x 106 cells in 30μI serum free DMEM were injected into the left hepatic lobe of nude mice. Thirty days later, the lungs and liver were removed and underwent HE staining.
Statistical analyses were performed using GraphPad Prism 6.0 or SPSS 21.0 software. Data were compared using two-tailed Student’s t-test or one-way ANOVA test. Pearson correlation coefficient was used to analyze the linear correlations. Data are presented as mean ± SD. A p value of < 0.05 was considered statistically significant: ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001; ns = not significant.