This study was approved by the Ethics Committee of the West China Hospital of Public Health. All participants were informed about the details of this study and they provided written informed consent prior to participation. A total of 54 HCC tissues and adjacent normal tissues were collected from patients at the Affiliated Hospital of Chengde Medical University. Adjacent normal tissues were obtained at least 2 cm away from HCC tissues. The inclusion criteria were as follows: (i) patients diagnosed as HCC; (ii) was not treated with radiotherapy or chemotherapy prior to surgery; and (iii) agreed to take part in the research. The exclusion criteria were as follows: (i) patients with any other clinical disorders; (ii) patients who had been treated with radiotherapy or chemotherapy before surgery; and (iii) did not agree to take part in the research. The tissues were immediately frozen in liquid nitrogen until use.

Transformed human liver epithelial-2 (THLE-2) cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in bronchial epithelial basal medium (BEGM^™; Clonetics Corporation, Walkersville, MD, USA). RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) was used for culturing the HCC cell line SNU-398 (ATCC).

The HCC cell lines SNU-182, Hep3B, and Huh-7 were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The Hep3B cell line was maintained on minimal essential media (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS, 1% GlutaMAX, 1% nonessential amino acids, 1% sodium pyruvate 100 mM solution, 100 U/mL penicillin, and 100 ng/mL streptomycin. The Huh-7 cell line was cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) added with 10% FBS, 1% GlutaMAX, and 1% nonessential amino acids. The culturing conditions for the SNU-182 cell line were the same as those for HuH7, except that the RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) was used as basal medium. Moreover, 1% penicillin–streptomycin mixture was added to all culture media. All cells were maintained at 37°C in a humidified incubator equipped with 5% CO₂.

Small interfering RNAs (siRNAs) targeting LINC01124 (si-LINC01124), FOXO3 (si-FOXO3), and negative control (NC) siRNA (si-NC) were designed and synthesized by GenePharma (Shanghai, China). LINC01124 and FOXO3 were overexpressed in cells by transfecting the LINC01124 overexpression plasmid pcDNA3.1–LINC01124 and FOXO3 overexpression plasmid pcDNA3.1–FOXO3 (Sangon Biotech Co., Ltd.; Shanghai, China), respectively. The miR-1247-5p mimic and inhibitor (RiboBio; Guangzhou, China) were used to increase and decrease endogenous miR-1247-5p expression, respectively. Furthermore, the NC miRNA mimic (NC mimic) and NC inhibitor functioned as controls for the miR-1247-5p mimic and inhibitor, respectively. For the transfection experiments, cells were seeded into 24-well plates 1 day prior to transfection. Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for transfection according to the manufacturer's protocol.

Total RNA isolation was performed using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A NanoDrop^™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) was used to assess RNA quality. For miR-1247-5p quantitation, purified RNA was reverse-transcribed into cDNA using the miScript Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany), and quantitative PCR was performed using the miScript SYBR Green PCR kit (Qiagen GmbH). The expression of miR-1247-5p was normalized to that of U6 small nuclear RNA. To analyze the expression of LINC01124 and FOXO3, reverse transcription using the PrimeScript^™ RT reagent kit with gDNA Eraser and quantitative PCR with TB Green® Premix Ex Taq^™ II (Takara, Dalian, China) were performed, respectively. Finally, the LINC01124 and FOXO3 expressions were normalized to GAPDH expression. Relative gene expression was calculated using the 2^−ΔΔCq method.

The Cell Counting Kit-8 (CCK-8) assay (Dojindo, Tokyo, Japan) was used to measure HCC cell proliferation. Transfected HCC cells were collected and seeded (1 × 10³ cells/well) into 96-well plates. The CCK-8 reagent (10 µl) was added on days 0, 1, 2, and 3 after seeding, and the cells were incubated for an additional 2 h at 37°C. The absorbance of each well at 450 nm was measured using a microplate reader (Bio Rad Laboratories, Inc., Hercules, CA, USA). The assay was performed with five replicates and repeated thrice.

Transwell filters (8 μm; BD Biosciences, Franklin Lakes, NJ, USA) were used for migration and invasion assays. For the migration assay, 5 × 10⁴ cells were suspended in 100 μl of FBS-free culture medium and added to the upper compartment of a Transwell chamber, and 500 μl of culture medium with 20% FBS was added to the lower compartment. After incubation for 24 h at 37°C, the nonmigrated cells in the upper chamber were gently removed using a wet cotton swab, and the migrated cells were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. After extensive washing, the stained cells were imaged and counted under a light microscope (×200 magnification; Olympus Corporation, Tokyo, Japan). For the invasion assay, the Transwell filters were precoated with Matrigel® (BD Biosciences). The remaining steps were the same as those used for the migration assay.

All experimental procedures involving animals were approved by the Animal Ethics Committee of West China Hospital of Public Health. Short-hairpin RNAs (shRNAs) targeting LINC01124 (sh-LINC01124) and NC shRNA (sh-NC), designed and synthesized by GenePharma, were packaged into a lentivirus. Huh-7 cells were infected with the lentivirus to obtain cells that were stably depleted of LINC01124. Totally 6 nude mice were purchased from Shanghai SLAC Laboratory Animal Company (Shanghai, China) and subcutaneously injected with stable LINC01124-knockdown Huh-7 cells (1 × 10⁶). Each group contained 3 nude mice. The longest (L) and shortest (W) diameters of the tumor xenografts were measured and recorded from days 7 to 30 with a 4-day interval. The tumor volume was calculated using the following formula:

Volume=0.5×L×W2.

At the end of the assay, all mice were euthanized by cervical dislocation, and the tumors were completely excised for further analysis.

ENCORI (https://starbase.sysu.edu.cn/index.php) was used to identify putative miRNAs that directly interacted with LINC01124. The target binding between miR-1247-5p and FOXO3 was predicted using miRDB (http://mirdb.org/) and Targetscan (http://www.targetscan.org).

The nuclear and cytoplasmic fractions of HCC cells were separated using the Cytoplasmic and Nuclear RNA Purification Kit (Norgen; Thorold, ON, Canada). Following RNA extraction, the relative ratio of LINC01124 in the nuclear and cytoplasmic fractions was determined through quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). GAPDH and U6 were used as internal controls for the cytoplasmic and nuclear RNA, respectively.

RIP assay was performed to test the binding interaction among LINC01124, miR-1247-5p, and FOXO3 via RNA-induced silencing complexes (RISCs) using the Magna RIP RNA Binding Protein Immunoprecipitation Kit (Merck Millipore, Darmstadt, Germany). Briefly, HCC cell extracts were prepared using RIP lysis buffer, followed by incubation with magnetic beads conjugated with human anti-Ago2 antibody or normal mouse IgG (Merck Millipore). Subsequently, the magnetic beads were collected and incubated with Proteinase K to remove any proteins. The immunoprecipitated RNA was measured by qRT-PCR. IgG acted as a negative control, while input functioned as the positive control.

The regions of LINC01124 and FOXO3 harboring wild-type (wt) miR-1247-5p binding sequences were ligated into the pMIR-luciferase reporter plasmid (Promega, Madison, WI, USA) to produce LINC01124-wt and FOXO3-wt reporter plasmids. Moreover, mutant (mut) segments were created and inserted into the same reporter plasmid, resulting in the formation of LINC01124-mut and FOXO3-mut reporter plasmids. To determine luciferase activity, HCC cells were seeded into 24-well plates and transfected with miR-1247-5p mimic or NC mimic along with wt or mut reporter plasmids using Lipofectamine® 2000. After 2 days, the Dual-Luciferase Reporter Assay (Promega) was used to quantify luciferase activity.

Cultured cells were resuspended in RIPA buffer (Beyotime, Jiangsu, China) to isolate the total proteins. A bicinchoninic acid protein assay kit (Beyotime) was used to quantify total protein. Equal amounts of protein were separated on 10% SDS–PAGE gels and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk (w/v) for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies against FOXO3 (ab109629; dilution 1:800) or GAPDH (ab181602; dilution 1:1000; both from Abcam, Cambridge, MA, USA). Further, horseradish peroxidase-conjugated secondary antibodies (ab205718; dilution 1:5000; Abcam) were added and incubated for 1 h at room temperature. After washing, an enhanced chemiluminescence reagent (Beyotime) was used for signal production. Protein signals were analyzed utilizing Quantity One software version 4.62 (Bio Rad Laboratories, Inc.).

All experiments were repeated for three times, and all results are presented as the mean ± standard deviation. SPSS software 19.0 (SPSS, Chicago, IL, USA) was used to perform statistical analysis. Student’s t-test was used for comparisons between two groups, and one-way analysis of variance followed by Tukey’s post hoc test was used to evaluate the differences among multiple groups. Statistical significance was set at p < 0.05.

We first analyzed the differently expressed lncRNAs in HCC using the Cancer Genome Atlas (TCGA). LINC01124 ranks 53^rd among all overexpressed lncRNAs in HCC (Fig. 1A). To thoroughly address the role of LINC01124 in HCC, its expression in HCC was determined using TCGA. Compared with normal liver tissues, HCC tissues show a significant increase in LINC01124 expression (Fig. 1B). To confirm this finding, LINC01124 expression in HCC samples from our own cohort was measured using qRT-PCR, and it was found that LINC01124 was overexpressed in HCC tissues compared with adjacent normal tissues (Fig. 1C).

Compared with THLE-2, LINC01124 was highly expressed in the HCC cell lines, particularly in Huh-7 (Fig. 2A). Accordingly, Huh-7 cells were used in the subsequent loss-of-function experiments. Next, si-LINC01124 was used to silence LINC01124 expression in Huh-7 cells (Fig. 2B), and functional experiments were performed to determine whether LINC01124 could regulate HCC behavior. si-LINC01124#1 and si-LINC01124#2 were selected as constructs for the subsequent experiments as they demonstrated the most efficient reduction in LINC01124 expression. Huh-7 cell proliferation decreased concomitantly with LINC01124 knockdown (Fig. 2C). Furthermore, the migratory (Fig. 2D) and invasive (Fig. 2E) capacities of LINC01124-depleted Huh-7 cells were inhibited compared with si-NC-transfected control cells.

We also presented the regulatory effects of LINC01124 overexpression on HCC progression. Hep3B expressed the relative lowest LINC01124 level among all tested HCC cell lines. Therefore, Hep3B was transfected with pcDNA3.1–LINC01124 (Fig. 3A) and used in gain-of-function experiments. Exogenous LINC01124 expression promoted Hep3B cell proliferation (Fig. 3B) and augmented motility (Figs. 3C and 3D). These results suggest that LINC01124 functions as a tumor promoter during HCC progression.

To determine the molecular mechanism underlying LINC01124, the subcellular localization of LINC01124 was investigated through nuclear-cytoplasmic fractionation. Our results demonstrated that LINC01124 was mostly distributed in the HCC cell cytoplasm (Fig. 4A), suggesting that LINC01124 acts as a ceRNA or miRNA sponge. Using ENCORI, a total of 12 miRNAs containing a potential binding site for LINC01124 were identified. Using the TCGA dataset, we identified six downregulated miRNAs in HCC, including miR-7-5p, miR-654-5p, miR-483-5p, miR-455-3p, miR-370-5p, and miR-1247-5p. Therefore, these miRNAs were subjected to subsequent experimental certification.

To determine the regulatory effect of LINC01124 on miRNA expression, qRT-PCR was conducted to measure miRNA expression on LINC01124-silenced or LINC01124-overexpressed HCC cells. Interference with LINC01124 resulted in miR-1247-5p upregulation in Huh-7 cells, whereas miR-1247-5p decreased in Hep3B cells in response to LINC01124 upregulation (Figs. 4B and 4C). RIP assays were used to further verify the interaction between LINC01124 and miR-1247-5p in the HCC cells. As shown in Fig. 4D, LINC01124 and miR-1247-5p were both enriched by the Ago2 antibody in HCC cells, implying that these two molecules were recruited to the RNA-induced silencing complex for potential functional interactions. Additionally, the direct binding of LINC01124 and miR-1247-5p (Fig. 4E) was confirmed in the luciferase reporter assay. The luciferase activity of LINC01124-wt decreased with miR-1247-5p overexpression, whereas that of LINC01124-mut remained unchanged after miR-1247-5p mimic cotransfection (Fig. 4F). These data collectively indicate that LINC01124 functions as a miR-1247-5p sponge in HCC cells.

Rescue experiments were designed to understand whether LINC01124 facilitates HCC progression by regulating miR-1247-5p. A significant decrease in miR-1247-5p expression was found in the miR-1247-5p inhibitor treatment Huh-7 cells. qRT-PCR also confirmed that miR-1247-5p was significantly overexpressed in miR-1247-5p mimic-transfected Hep3B cells (Fig. 5A). Functional experiments revealed the inhibition of Huh-7 cell proliferation (Fig. 5B) upon LINC01124 silencing, which was restored by inhibiting miR-1247-5p expression. Furthermore, we observed that the Hep3B cell proliferation promoted by LINC01124 overexpression was recovered when miR-1247-5p was re-expressed (Fig. 5C). Additionally, the migratory and invasive capacities of Huh-7 cells impaired by LINC01124 downregulation were offset by miR-1247-5p inhibitor treatment (Figs. 5D and 5E). Moreover, the effect of LINC01124 upregulation on Hep3B cell motility was counteracted by miR-1247-5p mimic cotransfection (Fig. 5F). Taken together, the pro-oncogenic actions of LINC01124 on HCC cells can be attributed to miR-1247-5p.

The specific role of miR-1247-5p in HCC cells was also revealed in detail. The miR-1247-5p mimic and NC mimic were transfected into HCC cells. Functional experiments indicated that transfection with the miR-1247-5p mimic resulted in a decrease in cell proliferation (Fig. 6A) in HCC cells. Additionally, overexpression of miR-1247-5p inhibited the migration and invasion (Fig. 6B) of HCC cells.

Using bioinformatics analysis, a potential binding site of miR-1247-5p was identified in FOXO3 3’-UTR (Fig. 6C). To determine whether miR-1247-5p directly targets FOXO3 in HCC cells, a luciferase reporter assay was implemented in HCC cells after transfection with miR-1247-5p mimic or NC mimic together with FOXO3-wt or FOXO3-mut. Ectopic miR-1247-5p expression significantly reduced the luciferase activity of HCC cells transfected with FOXO3-wt, whereas the luciferase activity of FOXO3-mut exhibited no change in response to ectopic miR-1247-5p expression (Fig. 6D). Furthermore, enforced miR-1247-5p expression reduced FOXO3 expression in HCC cells (Figs. 6E and 6F). The above data identified FOXO3 as a downstream target of miR-1247-5p in HCC cells.

After revealing FOXO3 as a direct target of miR-1247-5p in HCC, we next examined whether LINC01124 affects FOXO3 expression in HCC cells and investigated the underlying mechanism. FOXO3 levels were decreased in Huh-7 cells following LINC01124 depletion, whereas miR-1247-5p inhibitor cotransfection reversed this effect (Figs. 7A and 7B). Furthermore, reintroduction of LINC01124 induced a signification upregulation of FOXO3 levels in Hep3B cells, and the regulatory action was reversed by treatment of miR-1247-5p mimic (Figs. 7A and 7B). In addition, RIP assays demonstrated that LINC01124, miR-1247-5p, and FOXO3 were all enriched following immunoprecipitation with Ago2 antibody in HCC cells (Fig. 7C). In short, FOXO3 is under the control of LINC01124 through sequestration of miR-1247-5p.

Rescue experiments were also realized to address whether FOXO3 is a key indirect target of LINC01124 in HCC. The si-FOXO3 and FOXO3-overexpressing plasmid pcDNA3.1-FOXO3 were used in rescue experiments, and their transfection efficiency was demonstrated by western blot analysis (Fig. 8A). Interference with LINC01124 resulted in decreased proliferation, migration and invasion (Figs. 8B and 8C) of Huh-7 cells; nevertheless, these regulatory effects were abolished by FOXO3 upregulation. Similarly, cotransfection of si-FOXO3 was capable to abrogate the effects of pcDNA3.1-LINC01124 on Hep3B cell proliferation (Fig. 8B) and motility (Fig. 8D). Overall, the miR-1247-5p/FOXO3 axis contributes to the tumor-promoting activities of LINC01124 in HCC cells.

A xenograft tumor model was established to assess the impact of LINC01124 on HCC tumor growth in vivo. Huh-7 cells were transduced with lentivirus carrying sh-LINC01124 or sh-NC, and the cells were subcutaneously injected into nude mice. The transfection efficiency of sh-LINC01124 was measured by qRT-PCR. Transfection of sh-LINC01124 significantly decreased LINC01124 expression in Huh-7 cells (Fig. 9A). The tumor xenografts obtained from the sh-LINC01124 group exhibited reduced volume (Figs. 9B and 9C) and weight (Fig. 9D) compared with those in the sh-NC group. In addition, tumor xenografts derived from Huh-7 cells with stable LINC01124 silencing featured increased miR-1247-5p (Fig. 9E) levels in contrast to tumors in the sh-NC group. Furthermore, FOXO3 protein level was downregulated in the tumor xenografts with stable LINC01124 knockdown (Fig. 9F). Overall, LINC01124 depletion attenuated the growth of HCC cells in vivo.