LINC01343 targets miR-526b-5p to facilitate the development of hepatocellular carcinoma by upregulating ROBO1

Long noncoding RNAs (lncRNAs) contribute to hepatocellular carcinoma (HCC) progression and development. However, the function and molecular mechanisms of action of LINC01343 in HCC remain unclear. qRT-PCR and western blotting were performed to assess miR-526b-5p, LINC01343, and ROBO1 levels in HCC cell lines and tissue samples. Flow cytometry, transwell, and cell counting kit-8 assays were conducted in vitro to assess how LINC01343 influences the apoptosis, migration, and proliferation of HCC cells. In addition, the role of LINC01343 in the growth of tumors was verified using an in vivo xenograft tumor assay. Specific binding of miR-526b-5p to LINC01343/ROBO1 was validated using RNA immunoprecipitation and dual-luciferase reporter experiments. LINC01343 was upregulated in HCC cells and tissues. In vitro, LINC01343-knockdown Hep3B and Huh-7 cells exhibited enhanced apoptosis and suppressed proliferation and migration. An in vivo study further validated that LINC01343-knockdown repressed tumor growth. In terms of mechanisms, LINC01343 directly sponged miR-526b-5p, negatively modulating its expression. Moreover, further experiments revealed that inhibiting miR-526b-5p could counteract the tumor-suppressive effects of LINC01343-knockdown in Hep3B and Huh-7 cells. ROBO1 was identified as a direct target of miR-526b-5p. ROBO1 knockdown weakens the migratory and proliferative abilities of Hep3B and Huh-7 cells. Nonetheless, the inhibition of miR-526b-5p mitigated this effect. These findings revealed that LINC01343 serves as a vital oncogene in HCC. Moreover, the LINC01343/miR-526b-5p/ROBO1 axis may be a prospective target for HCC treatment.

qRT-PCR assay.TRIzol reagent (Thermo Fisher Scientific) was used to extract total cellular RNA according to the manufacturer's protocol.Subsequently, the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) was used for the reverse transcription of the RNAs into cDNAs.The Eco Real-time PCR System (Illumina, USA) and iTaq™ Universal SYBR® Green (BioRad, USA) were used to amplify specific cDNAs.The 2 −ΔΔCT relative quantitative approach was applied to analyze the results, with GAPDH as the internal control.
Following the kit protocol, the miRNA Isolation Kit (OMEGA) was used to extract miRNAs.The miRcute Plus miRNA First-Strand cDNA Synthesis Kit (TIANGEN, China) was used to transcribe the miRNAs into cDNA.Using the miRcute miRNA qPCR Detection Kit (SYBR Green; TIANGEN, China), cDNAs were amplified using the Eco Real-time PCR System (Illumina).The internal control used was U6.The primer sequences are listed in Table 1.
Table 1.Primer sequences used in RT-qPCR.Transwell assay.Transwell assays were performed to evaluate the migratory ability of the HCC cells.The Hep3B and Huh-7 cells were starved for 12 h.Subsequently, the top compartments of the transwell inserts were filled with 5 × 10 4 cells and incubated in the absence of FBS.The bottom compartments were loaded with 500 μL DMEM containing 10% FBS.Following a 24-h incubation, migrated cells were fixed with formalin for 10 min before treatment with 0.1% crystal violet for 20 min at room temperature.Finally, cells were viewed and counted under a microscope (Olympus, Japan).

Flow cytometry.
Apoptosis was evaluated using flow cytometry using the FITC Annexin V Apoptosis Kit (BD Biosciences).Transfected Huh-7 and Hep3B cells (5 × 10 5 ) were incubated at room temperature with 100 μL 1 × binding buffer containing 5 μL FITC Annexin V and 5 μL PI.Finally, the cells were analyzed using a flow cytometer (BD Biosciences).
Xenograft tumor formation assay.Six-week-old male nude mice weighing 18 g were acquired from National Rodent Laboratory Animal Resources (China).Each mouse was subcutaneously injected with 5 × 10 7 Huh-7 cells stably infected with either lentivirus sh-LINC01343 (n = 5; GeneCopoeia, USA) or sh-NC (n = 5).The volume of the tumors was determined weekly by applying the formula: volume = (width 2 × length) ÷ 2. Five weeks after the administration of Huh-7 cells, mice were euthanized using CO 2 before their tumors were extracted and weighed.The Ethics Committee of the First Affiliated Hospital of Chengdu Medical College (Chengdu, China) approved all the animal experiments, which were performed in accordance with the Guide for the Care and Use of Laboratory Animals.
RNA immunoprecipitation (RIP) assay.RIP assay was performed using the Magna RIP Kit (EMD Millipore, USA).Huh-7 and Hep3B cells (1 × 10 7 ) were treated with the RIP lysis buffer.The cellular extracts were exposed to magnetic beads coated overnight at 4 °C with anti-IgG antibody (EMD Millipore) or anti-Argonaute 2 antibody (AGO2, #ab32381; Abcam, USA).TRIzol (Thermo Fisher Scientific) was used for RNA extraction.The extracted RNA was analyzed using qRT-PCR.
Western blot analysis.The Total Protein Extraction Kit (KeyGEN, China) was used to extract total proteins.The BCA kit (KeyGEN, China) was used to quantify the protein concentration.Thereafter, the proteins (30 μg) were deposited on a 10% polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane.The membranes were sealed for one hour with 5% skim milk.Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies against GAPDH (1:1000, ab70744, Abcam, USA) and ROBO1 (1:1000, ab245516, Abcam, USA).This was achieved by incubation at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, 511,203, Zen-Bio, China).Finally, the protein signals were detected using the ECL Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) and further visualized using the ImageQuant LAS4000 mini (GE Healthcare, USA).

Statistical analysis.
All statistical tests were performed using GraphPad Prism 8. Results were presented as mean ± SD.Student's t-test and one-way ANOVA plus Sidak's or Tukey's test were used to test the significance of variations between and among groups, respectively.Linear regression analysis was performed to analyze the relationship between miR-526b-5p, ROBO1, and LINC01343.Statistical significance was set at P < 0.05.

Results
LINC01343 is highly expressed in HCC cells and tissues.LINC01343 levels in HCC were determined using qRT-PCR.LINC01343 expression was 3.6-fold higher in HCC tissues than in the adjacent non-tumor tissues (Fig. 1A).The expression of LINC01343 in HCC cell lines (Hep3B, Huh-7, and SNU-182) and normal liver epithelial cells (THLE2) was analyzed.We observed the upregulation of LINC01343 in HCC cell lines.Huh-7 and Hep3B cells manifested the highest expression of LINC01343, hence they were chosen for the succeeding experiments (Fig. 1B).Considering that cytoplasmic and nuclear lncRNAs have different functions 13 , we performed subcellular fractionation and FISH assays to determine the localization of LINC01343 in Hep3B and Huh-7 cells.The subcellular fractionation assay revealed that approximately 70% of LINC01343 was enriched in the cytoplasm (Fig. 1C).The results of the FISH assay further demonstrated that LINC01343 predominantly existed within the cytoplasm (Fig. 1D).Our results suggested that LINC01343 is localized in the cytoplasm and is likely to be involved in the epigenetic regulation of protein translation or transport.

Silencing LINC01343 diminished the capacities of the HCC cells to migrate and proliferate, stimulated their apoptosis, and suppressed tumor growth in vivo.
To clarify the function of LINC01343 in HCC, it was silenced in Huh-7 and Hep3B cells (Fig. 2A).We observed a decrease in LINC01343 expression in Huh-7 and Hep3B cells transfected with si-LINC01343.In vitro assays were conducted to determine whether LINC01343 directly influenced the malignant phenotypes of HCC cells.The CCK-8 assay uncovered that silencing LINC01343 in Hep3B and Huh-7 cells remarkably diminished their proliferative capacity (Fig. 2B).Moreover, transwell assays demonstrated that LINC01343 inhibition decreased the number of migratory Hep3B and Huh-7 cells by approximately 50%, indicating that LINC01343 inhibition impeded cell migration (Fig. 2C).Using flow cytometry, we observed that LINC01343-knockdown elevated the apoptosis rate 2.5fold in Hep3B and Huh-7 cells (Fig. 2D).To assess the potential effects of LINC01343 on HCC tumor growth in vivo, Huh-7 cells stably transfected with sh-LINC01343 were injected into nude mice.The sizes of the xenografts were calculated weekly.The results show that the knockdown of LINC01343 suppressed tumor volume.
The weights of the xenografts were recorded.The weights of tumors from the sh-LINC01343 group were reduced by more than 60% compared with those in the sh-NC group (Fig. 2E).Our findings indicated that LINC01343 serves as a pro-oncogenic regulator of HCC progression.
LINC01343 sponged miR-526b-5p.As LINC01343 is predominantly located in the cytoplasm of HCC cells, it may operate as a ceRNA to bind certain miRNAs and improve target gene expression.The bioinformatics tool starBase was used to predict target miRNAs putatively bound to LINC01343.Based on these results, miR-526b-5p may be a potential LINC01343 target.LINC01343/miR-526b-5p binding sites are shown in Fig. 3A.
Luciferase activity experiments were performed to validate the binding sites.We observed that the Huh-7 and Hep3B cells exhibited 50% luciferase reporter activities following their transfection with a combination of a miR-526b-5p mimic and wild-type (WT) LINC01343.By contrast, co-transfection of the LINC01343 mutant (MUT) and miR-526b-5p mimic into Huh-7 and Hep3B cells barely affected luciferase activity (Fig. 3B).These data suggested that LINC01343 and miR-526b-5p bind together.To further validate their direct binding, we performed RIP assays on Huh-7 and Hep3B cells.The transfection of the miR-526b-5p mimic increased LINC01343 levels by approximately 120-fold in RNAs enriched with AGO2.This suggested that miR-526b-5p and LINC01343 interact with each other (Fig. 3C).We examined miR-526b-5p expression in HCC cell lines and tissues.The results suggested that, unlike the control groups, the HCC tissues (Fig. 3D) and cell lines (Fig. 3E) showed downregulation of miR-526b-5p by more than 50%.miR-526b-5p expression was negatively correlated with LINC01343 expression (Fig. 3F).Taken together, these findings indicated that LINC01343 may function as a ceRNA to modulate miR-526b-5p expression.

ROBO1 was a miR-526b-5p target gene.
To further probe the molecular mechanisms underlying the biological function of miR-526b-5p in HCC, TargetScan was used to predict target genes.ROBO1 and miR-526b-5p had two predicted binding sites (Fig. 5A).miR-526b-5p's binding sites on ROBO1 were further confirmed using luciferase reporter assays in Hep3B and Huh-7 cells (Fig. 5B).The results revealed that miR-526b-5p mimics considerably repressed luciferase activity by 50% in cells that had been transfected with the WT ROBO1 vector.Nevertheless, these effects were abolished by the mutant variants.These results indicate that miR-526b-5p could directly target the ROBO1 3′UTR.We assessed ROBO1 expression in HCC cell lines and tissues.ROBO1 expression was upregulated 3.4-fold in HCC tissues (Fig. 5C) and approximately fourfold in HCC cell lines (Fig. 5D).ROBO1 expression negatively correlated with miR-526b-5p expression (Fig. 5E).These results suggested that ROBO1 serves as a miR-526b-5p target.miR-526b-5p targeted ROBO1 to suppress HCC cell migration and proliferation and stimulate apoptosis.The interaction between ROBO1 and miR-526b-5p during HCC progression was further explored.First, either miR-526b-5p or ROBO1 was silenced in the HCC cell lines.Western blot analysis demonstrated that miR-526b-5p knockdown remarkably upregulated ROBO1 expression 1.97-fold in Hep3B cells and 1.68-fold in Huh-7 cells.Furthermore, ROBO1 silencing alleviated the upregulation caused by the miR-526b-5p knockdown (Fig. 6A).Although the knockdown of ROBO1 in HCC cells suppressed proliferation, this effect was offset when the miR-526b-5p expression was inhibited (Fig. 6B).Similarly, miR-526b-5p inhibition restored migration induced by ROBO1 knockdown in Hep3B and Huh-7 cells (Fig. 6C).Additionally, when miR-526b-5p was silenced, apoptosis declined in the Hep3B and Huh-7 cells that had ROBO1 knockdown (Fig. 6D).These findings indicated that by targeting ROBO1, miR-526b-5p represses the migration and proliferation of HCC cells while promoting their apoptosis.

Discussion
HCC is one of the most common cancer types worldwide and is currently the third leading cause of cancerassociated deaths 26,27 .Despite immense efforts to reduce mortality, the exact mechanisms underlying HCC initiation and progression remain unclear 28 .This study documented the upregulation of LINC01343 in HCC cells and tissues.LINC01343 knockdown repressed the migration and proliferation of HCC cells while boosting their apoptosis.With regard to its mechanism, LINC01343 sponged miR-526b-5p to upregulate ROBO1.Overall, LINC01343 has the potential to serve as a novel biomarker for HCC prognosis and treatment.Although most lncRNAs have poorer expression levels than protein-coding genes, many are tissue-specific and dysregulated under various pathological conditions 17 .Various lncRNAs have been shown to have aberrant expression levels and contribute to HCC tumorigenesis and progression 29 .Yang et al. have reported that 107 metastasis-associated lncRNAs are deregulated in HCC 30 .Previous studies have reported that LINC01343 expression is altered in Ewing's sarcoma and oral squamous cell carcinoma.Nonetheless, the biological functions of LINC01343 remain unclear 24,25 .There have been no systematic analyses of the role of LINC01343 in tumors such as HCC.Further validation studies are required to confirm this hypothesis.In this study, we report that LINC01343 is a novel lncRNA that regulates cell proliferation, reduces apoptosis, and contributes to HCC progression.Many studies have shown that changes in lncRNA expression promote cancer progression by -TCG CTC TTG AGG GAA GCA CT-3ʹ R:5ʹ-CTC AAC TGG TGT CGT GGA -3ʹ ROBO1 F:5ʹ-GCT GGC GAC ATG GGA TCA TA-3ʹ R: 5ʹ-AATGT GGC GGC TCT TGA ACT-3ʹ GAPDH F: 5ʹ-AAG GTC ATC CCA GAG CTG AA-3ʹ R: 5ʹ-CTG CTT CAC CAC CTT CTT GA-3ʹ U6 F: 5ʹ-TCC CTT CGG GGA CAT CCG -3ʹ R: 5ʹ-AAT TTT GGA CCA TTT CTC GAT TTG T-3ʹ Committee of the First Affiliated Hospital of Chengdu Medical College (Chengdu, China) granted approval to this work [grant number: 2021CYFYIRB-HA-82].The clinical tissue specimen processing complied with the ethical standards of the Declaration of Helsinki.A written informed consent was signed by each patient.The Animal Care and Use Committee of the First Affiliated Hospital of Chengdu Medical College authorized the animal experiments.The animal studies accomplished in this work were executed in strict adherence to the ARRIVE guidelines.Consent to participate.Each patient signed a written informed consent.Copies of their written consent are available for review by the Editor-in-Chief of this journal upon request. https://doi.org/10.1038/s41598-023-42317-5

Figure 1 .
Figure 1.LINC01343 was upregulated in HCC.(A) qRT-PCR analysis of LINC01343 expression in 40 pairs of HCC tumor tissues and the adjacent normal tissues.(B) qRT-PCR analysis of LINC01343 expressions in HCC cells (Huh-7, Hep3B and SNU-182) and normal liver epithelial THLE2 cells *P < 0.05 and **P < 0.001 versus THLE2 cells group.(C) LINC01343 expression in subcellular fractions of Huh-7 and Hep3B cells detected by real-time PCR, with U6 and GAPDH as nuclear and cytoplasmic markers, respectively.(D) LINC01343 expression in subcellular fractions of Huh-7 and Hep3B cells detected by FISH assay.

Figure 2 .
Figure 2. LINC01343 downregulation inhibited HCC progression both in vitro and in vivo.(A) qRT-PCR analysis of LINC01343 expression in Huh-7 and Hep3B cells after si-LINC01343 or si-NC transfection (n = 3).(B) CCK-8 experiment was performed to evaluate the proliferative ability of Huh-7 and Hep3B cells that had been transfected with si-LINC01343 or si-NC (n = 3).(C) Transwell experiments were conducted to assess the invasive ability of Huh-7 and Hep3B cells that had been transfected with si-LINC01343 or si-NC (n = 3).(D) Cell apoptosis of Huh-7 and Hep3B cells transfected with si-NC or si-LINC01343 were examined by Annexin V-FITC/PI-labeled flow cytometry (n = 3).(E) Animal study was performed to determine the role of LINC01343 knockdown in vivo (n = 5/group).*P < 0.05 and **P < 0.001 versus si-NC or sh-NC group.

Figure 6 .
Figure 6.MiR-526b-5p inhibited proliferation and migration while promoted apoptosis of HCC via targeting ROBO1.(A) Western blot analysis of ROBO1 expressions in Huh-7 and Hep3B cells after si-DLGAP5/siRNA (si-NC) and/or miR-526b-5p inhibitor/inhibitor control (inhibitor-NC) transfection.(B) CCK-8 experiment was performed to evaluate the proliferative ability of Huh-7 and Hep3B cells that had been transfected with or without si-ROBO1 and/or miR-526b-5p inhibitor.(C) Transwell experiments were conducted to assess the migratory abilities of Hep3B and Huh-7 cells that did or did not have si-ROBO1 and/or miR-526b-5p inhibitor transfection.(D) Apoptosis of Hep3B and Huh-7 cells that had or had not been transfected with si-ROBO1 and/ or miR-526b-5p inhibitor were assessed through Annexin V-FITC/PI-labeled flow cytometry.*P < 0.05 and **P < 0.001 versus the si-NC group; # P < 0.05 and ## P < 0.001 versus the inhibitor-NC group; && P < 0.001 versus the si-ROBO1 + inhibitor group.