Cell culture
Liver cancer cell lines (HepG2 and Huh-7) and 293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS (Thermo Fischer Scientific), 1% penicillin and streptomycin (Thermo Fisher Scientific) at 37°C, 5% CO2.
Bioinformatics analysis
One dataset (GSE113850) which contain the gene expression data liver cancer tissue and adjacent normal tissue (controls) were obtained from GEO database (https://www.ncbi.nlm.nih.gov/geo/). The relation between lncRNA/USP4 and tumor stage of liver cancer was analyzed. The survival curve was calculated using the Cancer Genome Atlas (TCGA).
Quantitative real time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from liver cancer cell lines using TRIzol reagent (TaKaRa, Tokyo, Japan) according to the manufacturer's protocol. cDNA was synthesized using the reverse transcription kit (TaKaRa, Ver.3.0) according to the manufacturer's protocol. Real-Time qPCRs were performed in triplicate under the following protocol: 2 minutes at 94°C, followed by 35 cycles (30 s at 94°C and 45 s at 55°C). The primer for LINC01234, miR-513a-5p, β-actin and U6 were obtained from GenePharma (Shanghai, China). LINC01234: forward, 5’- CAGGACCTTCTGTGGGACTC-3’ and reverse 5’-TCCAAAACTCCCCTTTCCCA-3’. MiR-513a-5p: forward, 5’- TGCGCTCAGCAAACATTTATTG-3’ and reverse 5’- CCAGTGCAGGGTCCGAGGTATT-3’. β-actin: forward, 5’-AGCGAGCATCCCCCAAAGTT-3’ and reverse 5‘-GGGCACGAAGGCTCATCATT-3’. U6: forward, 5’-CGCTTCGGCAGCACATATAC-3’ and reverse 5’- AAATATGGAACGCTTCACGA-3’. The relative fold changes were calculated using the 2-ΔΔCt method by the formula: 2-(sample ΔCt – control ΔCt), where ΔCt is the difference between the amplification fluorescent thresholds of the gene of interest and the internal reference gene (U6 or β-actin) used for normalization.
Cell transfection
Lentiviral expressing short-hairpin RNA (shRNA1 or shRNA2) directed target LINC01234 and one nontargeting sequence (negative control) were obtained from Hanbio Biotechnology Co., Ltd (Shanghai, China). Next, LINC01234 shRNA1 or shRNA2 was packaged into lentiviruses. Then the lentiviral vector DNAs were then transfected into 293T cells including lenti-LINC01234 shRNAs and negative control (NC). After transfection, the cells were incubated at 32˚C, and then the supernatant was collected. After that, supernatants of two LINC01234 shRNAs and negative control were filtered into particles. Finally, all liver cancer cells were infected with lentiviral particles according to the manufactures’ protocol. After 48 h of incubation, stable liver cancer cells were then selected by puromycin (2.5 μg/mL, Sigma Aldrich, St. Louis, MO, USA). Green fluorescence and qRT-PCR were used to verify the efficiency of transfection.
For miR-513a-5p transfection, liver cancer cells were transfected with miR-513a-5p agomir, miR-513a-5p antagomir or NC by Lipofectamine 2000 according to the previous reference (15). MiR-513a-5p agomir, miR-513a-5p antagomir and negative control RNAs were purchased from GenePharma (Shanghai, China). The efficiency of transfection was detected by q-PCR.
CCK-8 assay
Liver cancer cells were seeded in 96-well plates (5×103 per well) overnight. Then, cells were treated with negative control (NC) or LINC01234 shRNA1 for 0, 24, 48 and 72 h, respectively. 10 μl CCK-8 reagents were added to each well and further incubated for 2 h at 37°C. Finally, the absorbance of liver cancer cells was measured at 450 nm using a microplate reader (Thermo Fisher Scientific).
Cell apoptosis analysis
Liver cancer cells were trypsinized, washed with phosphate buffered saline and resuspended in Annexin V Binding Buffer, followed by staining with 5 μl FITC and 5 μl propidium (PI) in the system for 15 min. Cells were analyzed using flow cytometer (BD, Franklin Lake, NJ, USA) to test the cell apoptosis rate.
Cell invasion assay
For cell invasion analysis, transwell assay was performed in this study. The upper chamber is pre-treated with 100 μl of Matrigel. Huh7 cells were seeded into the upper chamber in media with 1% FBS, and the density was adjusted to about 1.0×106 cells per chamber. RPMI1640 medium with 10% FBS was added in the lower chamber. After 24 h of incubation at 37°C, the transwell chamber was rinsed twice with PBS (5 min per time), fixed by 5% glutaraldehyde at 4°C and stained with 0.1% crystal violet for 30 minutes. The transwell chamber was washed twice with PBS and then observed under a microscope. The number of cells invading the Matrigel was regarded to be a reflection of the invasion ability.
Dual luciferase reporter assay
The partial squences of LINC01234 and 3’-UTR of USP4 containing the putative binding sites of miR-513a-5p were synthetized and obtained from Sangon Biotech (Shanghai, China), then were cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vectors (Promega, Madison, WI, USA) to construct wild-type reporter vectors LINC01234 (WT) and USP4 (WT), respectively. The mutant LINC01234 sequences and 3’-UTR of USP4 sequences containing the putative binding sites of miR-513a-5p were performed by Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) and then cloned into pmirGLO vectors respectively, to construct mutant-type reporter vectors LINC01234 (MUT) and USP4 (MUT). The LINC01234 (WT) or LINC01234 (MUT) was transfected into 293T cells together with control, vector-control (NC) or miR-513a-5p agomir using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Similarly, the USP4 (WT) or USP4 (MUT) was transfected into 293T cells together with control, vector-control (NC) or miR-513a-5p agomir. The relative luciferase activity was analyzed by the Dual-Glo Luciferase Assay System (Promega).
RNA pull-down
For the RNA pulldown assay, the Biotin RNA Labeling Mix (Roche, Basel, Switzerland) was used to transcribe and label probe-control or probe-LINC01234 from LINC01234 shRNA lenti vector in vitro. An RNA structure buffer (Thermo, MA, USA) was used to induce secondary structure formation from the biotin-labeled RNAs. Streptavidin beads (Thermo) were washed three times with 500 μL of RNA immunoprecipitation wash buffer (Thermo) and then added to the biotinylated RNAs at 4°C overnight. The overnight mixture was separated by a magnetic field so that streptavidin bead-RNA complexes could be obtained. Then, lysates of liver cancer cells were added to the complexes and incubated on a rotator at room temperature for one hour. The incubated mixture was again separated with a magnetic field so that streptavidin bead-RNA-protein complexes could be obtained.
Wound healing assay
Huh-7 cells were plated into a 24-well Cell Culture Cluster, and were allowed to grow to 80-90% confluence. Then, cells were underlined perpendicular to the cell culture plate with a small pipette head. After washing with PBS 3 times, serum-free medium was used for further culture, and the scratch widths at 0 and 48 h were recorded under an optical microscope. The experiment was repeated 3 times.
Immunofluorescence
Liver cancer cells or tumor tissues of mice were prefixed in 4% paraform for 10 min, and fixed in pre-cold methanol for another 10 min. Next, cells were incubated with primary antibodies overnight at 4°C: anti-Ki67 (Abcam; 1:1000), anti-Smad4 (Abcam; 1:1000) and DAPI (Abcam; 1:1000). Goat anti-rabbit IgG antibody (Abcam; 1:5000) was used as the secondary antibody. The samples were visualized by fluorescence microscope (Olympus CX23, Tokyo, Japan) immediately.
Western-blot detection
Total protein was isolated from cell lysates or tumor tissues by using RIPA buffer, and quantified by BCA protein assay kit (Beyotime, Shanghai, China). Proteins were resolved on 10% SDS-PAGE, and then transferred to PVDF (Bio-Rad) membranes. After blocking, the membranes were incubated with primary antibodies at 4°C overnight, then incubated with secondary anti-rabbit antibody (Abcam; 1:5000) at room temperature for 1 h. Membranes were scanned by using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, USA). Then, the primary antibodies used in this study as follows: anti-E-cadherin (Abcam, Cambridge, MA, USA; 1:1000), anti-vimentin (Abcam; 1:1000), anti-α-SMA (Abcam; 1:1000), anti-smad2 (Abcam; 1:1000), anti-smad3 (Abcam; 1:1000), anti-USP4 (Abcam; 1:1000), anti-cleaved caspase 3 (Abcam; 1:1000), anti-Akt (Abcam; 1:1000), anti-ERK (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000). β-actin was used as an internal control.
In vivo study
18 BALB/c nude mice (6-8 weeks old) were purchased from Vital River (Beijing, China). The mice were housed within a dedicated SPF facility. Huh7 stable expressed LINC01234 shRNA1 cells were transplanted subcutaneously in each mouse according to the previous reference (16). The tumor volume was measured weekly according to the reference (17). At the end of the experiments, mice were sacrificed and the tumors were collected and weighted. The expression of p-smad2 and p-smad3 were detected by immunohistochemistry (IHC) staining as previously reported (18). All in vivo experiments were performed in accordance with National Institutes of Health guide for the care and use of laboratory animals, following a protocol approved by the Ethics Committees of East China University of Science and Technology.
TUNEL staining
Apoptosis was also determined by the TUNEL assay according to the manufacturer's instructions. Briefly, paraffin sections were washed, permeabilized, and then incubated with 50 μl TUNEL reaction mixtures in a wet box for 60 min at 37°C in the dark. For signal conversion, slides were incubated with 50 μl of peroxidase (POD) for 30 min at 37°C, rinsed with PBS, and then incubated with 50 μl diaminobenzidine (DAB) substrate solution for 10 min at 25°C. Finally, the expression of apoptotic cells was observed under an optical microscope.
Statistical analysis
Each group were performed at least three independent experiments and all data were expressed as the mean ± standard deviation (SD). Differences were analyzed using Student’s t-test (only 2 groups) or one-way analysis of variance (ANOVA) followed by Tukey’s test (more than 2 groups, Graphpad Prism7). P<0.05 was considered to indicate a statistically significant difference.