Liver cancer cell lines (HepG2 and Huh-7), normal liver cell lines (L-02) and 293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, United States), supplemented with 10% FBS, 1% penicillin and streptomycin (Thermo Fisher Scientific) in an incubator (37°C, 5% CO₂).

The gene expression data of liver cancer and adjacent normal tissue was obtained from dataset (GSE113850) and the Cancer Genome Atlas (TCGA). The survival curve and the correlation between tumor stage of liver cancer were calculated based on the data from TCGA.

Total RNA was extracted from liver cancer cell lines using TRIzol reagent (TaKaRa, Tokyo, Japan) according to the manufacturer’s protocol. First-strand cDNA was synthesized using the PrimeScript RT reagent Kit (Takara) according to the manufacturer’s protocol. qRT-PCR was performed in an ABI7500 system using SYBR Green methods. qRT-PCR was performed in triplicate with the following protocol: 2 min at 94°C, followed by 35 cycles (30 s at 94°C and 45 s at 55°C). The primers for all the genes were obtained from GenePharma (Shanghai, China). 2^–ΔΔCT method was used to quantify the data. The internal reference gene (U6 or β-actin) was used for normalization. The primers were obtained from GenePharma (Shanghai, China). LINC01234: forward, 5′-TCTCACCTTTCAAACGCTTGTC-3′ and reverse 5′-ACTCTCCTTCCTTTCCTCTGATTC-3′. MiR- 513a-5p: forward, 5′-AGGGAGGTGTCATCTCAACTGA-3′ and reverse 5′-CTCAACTGGTGTCGTGGAGTC-3′. β-actin: forward, 5′-GTCCACCGCAAATGCTTCTA-3′ and reverse 5′-TGCTGTCACCTTCACCGTTC-3′. USP4: forward, 5′-GGA CTATGTATTGGTCCCTACCG-3′ and reverse 5′-TCGATGG TTGCAATGGTGTC-3′. U6: forward, 5′-CTCGCTTCGGCAG CACAT-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′.

Lentiviral expressing short-hairpin RNA (shRNA1 or shRNA2) directed target LINC01234 and one non-targeting 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 37°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, United States). 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 (20). 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.

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. Subsequently, cells were treated with 10 μl CCK-8 reagents (Beyotime, Shanghai, China) 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).

Liver cancer cells were trypsinized, washed with phosphate buffered saline and resuspended in Annexin V Binding Buffer. Then, cells were stained with 5 μl FITC and 5 μl propidium (PI) in the dark for 15 min. Cells were analyzed using flow cytometer (BD, Franklin Lake, NJ, United States) to test the cell apoptosis rate.

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 invaded cells was regarded to be a reflection of the invasion ability.

The partial sequences 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, United States) to construct wild-type reporter vectors LINC01234 (WT/MT) and USP4 (WT/MT), respectively. The LINC01234 (WT/MT) or USP4 (WT/MT) were 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. The relative luciferase activity was analyzed by the Dual-Glo Luciferase Assay System (Promega). The relative luciferase activity was analyzed by the Dual-Glo Luciferase Assay System (Promega).

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, United States) was used to induce secondary structure formation from the biotin-labeled RNAs. The biotinylated LINC01234 and negative control (bio-NC) were generated via GenePharma and coated to streptavidin-conjugated magnetic beads. Liver cancer cells were lysed and then incubated with the magnetic beads for 6 h. The RNA on the beads was isolated and the enrichment level of miR-513a-5p was detected by PCR.

Huh-7 cells were plated into a 24-well Cell Culture Cluster and 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.

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). Goat anti-rabbit IgG antibody (Abcam; 1:5000) was used as the secondary antibody. The nuclei were stained with DAPI for 15 min at room temperature. The samples were visualized by fluorescence microscope (Olympus CX23, Tokyo, Japan) immediately.

Total protein was isolated from cell lysates or tissues by using RIPA buffer. The concentration of protein was detected with a BCA protein kit (Thermo Fisher Scientific). Then, proteins (40 μg per lane) were separated with 10% SDS-PAGE gel and then transferred into polyvinylidene fluoride (PVDF, Thermo Fisher Scientific) membranes. After blocking with 3% skim milk for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Subsequently, membranes were 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, United States). The primary antibodies used in this study as follows: anti-E-cadherin (Abcam, Cambridge, MA, United States; 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.

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 (21). The tumor volume was measured weekly according to the reference (22). 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 (19). 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.

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.

Each group were performed 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.

To detect differentially expressed lncRNAs between liver cancer and normal tissues, a bioinformatics analysis as performed. As indicated in Figure 1A, differentially expressed lncRNAs in GSE113850 data set (DS) were presented using Volcano Plot. In addition, differentially expressed lncRNAs among normal and tumor tissues were also presented in TCGA (Figure 1B). Among these differentially expressed lncRNAs in GSE113850 DS and TCGA, 59 were commonly downregulated, whereas 167 were commonly upregulated (Figure 1C). Moreover, 5 most upregulated overlap linRNAs in liver cancer were presented (Figure 1D). Among these 5 lncRNAs, high expression of LINC01234 was negatively correlated with the survival rate of patient with liver cancer (Figures 1E,F). These data suggested that LINC01234 might play an important role during the tumorigenesis of liver cancer. Thus, LINC01234 was selected for investigation in the following experiments.

In order to test the gene expression, qRT-PCR was used. As indicated in Figure 2A, the expression of LINC01234 in liver cancer cells was much higher than that in normal liver cells. Moreover, LINC01234 shRNA1 and shRNA2 were stably transfected into liver cancer cells (Figures 2B,C), and the expression of LINC01234 in liver cancers was significantly downregulated when transfected with LINC01234 shRNA (Figure 2D). Since LINC01234 shRNA1 exhibited better transfection efficiency, it was therefore used in subsequent experiments. Furthermore, the viability and proliferation of Huh-7 and HepG2 cells were significantly inhibited by LINC01234 knockdown (Figures 2E–H). Meanwhile, Huh-7 cells were more sensitive to LINC01234 shRNA1 than HepG2 cells. Thus, Huh-7 cells were used in the following experiments. Taken together, LINC01234 silencing significantly decreased the proliferation of liver cancer cells.

For the purpose of exploring the mechanism by which LINC01234 mediated the progression of liver cancer, Starbase¹ was performed. As shown in Figure 3A, LINC01234 had a putative miR-513a-5p targeting site. In addition, miR-513a-5p agomir/antagomir was stably transfected into Huh-7 cells (Figure 3B). Meanwhile, co-transfection of the wild-type LINC01234 vector (WT-LINC01234) with miR-513a-5p agomir, significantly reduced luciferase activities compared with mutant LINC01234 vector (MT-LINC01234) (Figure 3C). Additionally, LINC01234 bound to miR-513a-5p (Figure 3D) was verified with pull down assay. Moreover, USP4 was found to be the direct target of miR-513a-5p (Figures 3E,F). However, the difference of USP4 level between liver cancer and adjacent normal tissues is not significant (Figure 3G). Besides, the difference of USP4 expression among different stages of liver cancer is not obvious (Figure 3H). Additionally, the expression of USP4 in liver cancer cells was notably upregulated by miR-513a-5p antagomir but inhibited by miR-513a-5p agomir (Figure 3I). Altogether, LINC01234 knockdown inhibited the tumorigenesis of liver cancer via mediation of miR-513a-5p/USP4 axis.

Next, flow cytometry was performed to detect the cell apoptosis. As revealed in Figure 4A and Supplementary Figure S1, downregulation of LINC01234 notably induced the apoptosis of liver cancer cells. In addition, knockdown of LINC01234 greatly inhibited the migration and invasion of liver cancer cells (Figures 4B,C). Furthermore, the expression of E-cadherin in Huh-7 cells was significantly upregulated by LINC01234 shRNA1 (Figure 4D). In contrast, knockdown of LINC01234 greatly decreased the expression of vimentin and α-SMA in liver cancer cells (Figure 4D). To sum up, knockdown of LINC01234 significantly suppressed the progression of liver cancer in vitro.

In order to investigate the protein expressions in liver cancer cells, western blot was used. As indicated in Figures 5A,B, the expressions of USP4, p-Smad2 and p-Smad3 in Huh-7 cells were significantly downregulated in the presence of LINC01234 shRNA1, while this phenomenon was partially reversed by miR-513a-5p antagomir. In addition, the expression of Smad4 in cytoplasm of Huh-7 cells was significantly decreased by LINC01234 knockdown, which was partially reversed in the presence of miR-513a-5p antagomir (Figure 5C). This data suggested that Smad4 transferred from the cytoplasm to the nucleus in the presence of LINC01234 shRNA1. Moreover, LINC01234 shRNA1 greatly suppressed the expressions of p-Akt and p-ERK in Huh-7 cells. In contrast, the expression of cleaved caspase 3 in Huh-7 cells was notably upregulated in the presence of LINC01234 knockdown (Figures 5D,E). However, downregulation of miR-513a-5p partially reversed the effect of LINC01234 shRNA1 on these proteins (Figures 5D,E). In summary, silencing of LINC01234 notably inhibited the tumorigenesis of liver cancer via mediation of miR-513a-5p/USP4/TGF-β1 axis.

Finally, to detect the effect of LINC01234 on liver cancer in vivo, xenograft mice model was established. As shown in Figures 6A,B, tumor sizes of mice were significantly decreased by LINC01234 knockdown. Consistently, knockdown of LINC01234 notably downregulated the tumor weights of mice (Figure 6C). Meanwhile, the expression of LINC01234 in tissues of mice was greatly inhibited by LINC01234 knockdown (Figure 6D). Furthermore, Ki67 and Tunnel assays indicated tumor cell proliferation was obviously suppressed by LINC01234 shRNA1 via inducing apoptosis (Figures 6E,F). The expressions of p-Smad2 and p-Smad3 in tumor tissues were notably decreased by LINC01234 knockdown (Figures 6G,H). Altogether, knockdown of LINC01234 notably attenuated the tumor growth of liver cancer through inactivation of TGF-β signaling in vivo.