Hepatitis B virus core/capsid protein induces hepatocellular carcinoma progression via long non-coding RNA KCNQ1OT1/miR-335-5p/CDC7 axis

Introduction

Hepatocellular carcinoma (HCC) is known as the most common primary liver cancer, accounting for over 90% of cases, with an increasing prevalence worldwide (1). Despite this, the mechanisms underlying HCC progression still remain unclear (2-4). Long non-coding RNAs (lncRNAs) are a class of RNA molecules >200 nucleotides in length that do not encode proteins (5,6). Increasing evidence suggests that mutations and dysregulation of lncRNAs affect various aspects of genome function and critical biological processes (7). In many malignancies, lncRNAs are dysregulated and interact with a multitude of RNAs and proteins, influencing cancer progression (8-10). Typically, lncRNAs function as competing endogenous RNAs (ceRNAs), competitively binding with microRNAs (miRNAs) to decrease miRNAs’ regulation of their target messenger RNAs (mRNAs), as observed in HCC (11). However, the regulatory mechanisms of lncRNA-dependent gene expression in HCC require in-depth exploration to develop promising therapeutic targets and methods.

Utilizing sample data from The Cancer Genome Atlas (TCGA) database, we analyzed differentially expressed (DE) lncRNAs involved in HCC tumorigenesis, with a particular focus on the lncRNA KCNQ1 overlapping transcript 1 (KCNQ1OT1)/miR-335-5p/cell division cycle 7 (CDC7) axis. Recent studies have indicated that overexpression of KCNQ1OT1 interacts with several tumor suppressor miRNAs (including miR-148a-3p, miR-149, miR-146a-5p, miR-506, miR-504, miR-424-3p, miR-136-3, miR-139-5p, miR-223-3p and miR-375-3p) to enhance HCC progression and is associated with poor prognosis in patients (12,13). Additionally, miR-335-5p has been identified to negatively regulate critical pathways involved in the transport and utilization of essential compounds that are crucial for the rapid proliferation of HCC cells (14). Research indicates that extracellular vesicles containing miR-335-5p have the potential to decrease HCC growth and invasion both in vitro and in vivo (15). Notably, the role of KCNQ1OT1 in targeting miR-335-5p has not been documented.

The CDC7 protein plays a pivotal role in initiating DNA replication, S-phase checkpoints, and M-phase completion (16). In cancer cells, the absence of CDC7 leads to defects in S-phase progression, resulting in p53-independent apoptotic cell death (17). CDC7 forms a complex with dumbbell former 4 (DBF4) to create DBF4-dependent kinase (DDK), which is crucial for tumor cell survival (18). Highly expressed in HCC, CDC7 is significantly correlated with the survival rate of HCC patients (19). Nevertheless, studies on the association between miR-335-5p and CDC7 remain inconclusive.

In this study, we found that KCNQ1OT1 increased the expression of CDC7 by acting as a ceRNA to attract miR-335-5p, thereby promoting the proliferation and migration of HCC cells. Primary risk factors for HCC include chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), consumption of aflatoxin-contaminated foods, excessive alcohol intake, and obesity. Chronic HBV infection is a major contributor to HCC in high-risk areas (20). Based on this, our results showed that HBV and its encoded protein HBc significantly enhanced the expression of KCNQ1OT1 and CDC7 while reducing the expression of miR-335-5p. This study provides new insights into the regulatory mechanisms of HBV infection in liver cancer progression. Therefore, the KCNQ1OT1/miR-335-5p/CDC7 axis may be a promising therapeutic target in patients with HBV-related primary liver cancer. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-233/rc).

Methods

TCGA data collection and ceRNA network construction

A total of 374 HCC tissue samples and 50 normal tissue samples were downloaded from the TCGA database (https://portal.gdc.cancer.gov/). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The DESeq2 package was used to identify DE genes. The GDCRNATools package was utilized to classify these genes into the lncRNA, miRNA, and mRNA categories. The RNA interaction network and binding sites were predicted using the ENCORI/starBase database (version 2.0, https://rnasysu.com/encori/) and the RNAhybrid software (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid). The final lncRNA-miRNA-mRNA regulatory network was visualized using the Cytoscape software (http://cytoscape.github.io/). Volcano plots and heatmaps were generated using the ggplot2 package (RRID:SCR_014601). Genome Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses for DE mRNAs were conducted using the clusterProfiler package (21). The GEPIA 2 database (http://gepia2.cancer-pku.cn/#index) was used for survival analysis, and the UALCAN database (RRID:SCR_015827, https://ualcan.path.uab.edu/index.html) was used for the expression analysis of DE RNAs.

Cell culture and transfection

Human HCC cell lines HepG2 (RRID: CVCL_0027, human hepatocellular carcinoma cells) and HepG2.215 (RRID: CVCL_L855, stably transfected HBV virus HepG2 cells) were purchased from iCell Bioscience (Shanghai, China). SMMC-7721 (RRID: CVCL_0534) cells were obtained from Dr. Dequan Yang, The Eighth Affiliated Hospital, Sun Yat-sen University. HepaRG cells (RRID: CVCL_9720) were purchased from Applied Biological Materials (abm) Inc. (Vancouver, Canada). HepG2, HepG2.215 and SMMC-7721were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 µg/mL penicillin and 100 U/mL streptomycin (Gibco, USA). HepaRG cells were cultured in Williams’ E 1640 (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 2 mM L-glutamine (Gibco, USA), 5.25 µg/mL of insulin (MCE, New Jersey, USA), 50 µM hydrocortisone (MCE, USA), 100 µg/mL penicillin and 100 U/mL streptomycin (Gibco, USA). The culture was maintained at 37 ℃ and 5% CO₂. For HepaRG cells differentiation, cells were seeded at a high density, resulting in spontaneous differentiation. For cell transfection, cells were inoculated in plates and incubated for 16–24 h. When the cell density to reach 60–70%, siRNA, miRNA mimics, miRNA inhibitors, or plasmids were transfected into cells by using Lipofectamine 2000 (Thermo, Waltham, USA).

The sequences of si-NC, si-KCNQ1OT1, si-CDC7, miR-NC, miR-335-5p mimics, miR-335-5p inhibitors, and negative control (NC) inhibitors were designed and synthesized by GenePharma (Shanghai, China). Si-NC served as an NC for si-KCNQ1OT1 and si-CDC7. miR-NC functioned as an NC for miR-335-5p mimics, while NC inhibitors served as the NC for miR-335-5p inhibitors. Plasmids were obtained from MiaoLing Bio (Wuhan, China), including pcDNA3.1-CDC7 (oe-CDC7) and NC (oe-NC), HBV 1.3-mer WT replicon and its NC pGEM-4Z, HBx-pmCherry, HBp-pmCherry, HBc-pmCherry, HBs-pmCherry and the NC. All the related sequences are listed in Table S1.

Luciferase reporter assay

The sequences containing the mutated site were inserted into the luciferase reporter gene vector pmirGLO, and the mutant vectors MUT-KCNQ1OT1 and MUT-CDC7 were constructed. SMMC-7721 cells were transfected with WT-KCNQ1OT1, MUT-KCNQ1OT1, WT-CDC7, or MUT-CDC7 in combination with miR-355-5p mimics or miR-NC. After 48 h of incubation, the relative luciferase activity in each group was measured. The Dual Luciferase Reporter Gene Assay System (Beyotime, Shanghai, China) was used to measure luciferase activity.

RNA immunoprecipitation (RIP) assay

The RIP assay was conducted employing an RIP kit (Sangon Biotech, Shanghai, China), following the manufacturer’s guidelines. Briefly, cells were lysed in RIP lysis buffer, and the resultant cell lysate was incubated with magnetic beads conjugated with either immunoglobulin G (IgG) or Ago2 antibody (Proteintech, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was subsequently employed to assess the expression of KCNQ1OT1, miR-355-5p and CDC7 mRNA in the precipitates.

qRT-PCR

Total RNA was extracted from cultured cells using TRIzol reagent (Thermo, USA) according to the manufacturer’s instructions. RNA quantity was determined using a NanoDrop 2000 spectrophotometer (Thermo, USA). The ratio of absorbances at 260 and 280 nm (1.8≤ A260/A280 ≤2.0) was used to evaluate RNA purity. High-quality total RNA was reverse-transcribed using the Prime Script^TM RT Master Mix Kit (TaKaRa, Kyoto, Japan). Then, qRT-PCR was performed using TB Green^® Premix Ex Taq^TM II (TaKaRa, China), following the manufacturer’s instructions. The amplification conditions were 95 ℃ for 5 s and 60 ℃ for 30 s for a total of 40 cycles. Relative expression was calculated using the 2^−ΔΔCt method and the transcript levels were normalized to GAPDH mRNA expression levels. Reverse transcription of miRNA was performed using the miRNA 1st strand cDNA synthesis kit (Stem-loop) (Accurate Biology, Changsha, China). Quantitative PCR (QPCR) was performed with the same kit as above, and the relative expression of miRNAs was normalized to U6. The primer sequences are listed in Table S2.

Cell Counting Kit-8 (CCK-8) assay

After transfection, 5×10³ cells per well were seeded into 96-well plates and incubated for 0, 24, 48, 72, and 96 h. Before testing, 10 µL of CCK-8 reagent (Dojindo, Kumamoto, Japan) was added to the each well and cells were incubated for 3 h at 37 ℃. Subsequently, the light absorbance at 450 nm was measured. Cell proliferation ability in different groups was analyzed using the CCK-8 assay, according to the manufacturer’s protocol.

Cell colony formation assay

Different groups of transfected HCC cells were seeded in six-well plates at 1,000 cells per well. After 2 weeks, 4% paraformaldehyde was used to fix cell colonies; then, fixed colonies were stained with crystal violet for 10–30 min. After staining, the plates were washed with distilled water and air-dried. Colonies were counted for statistical analysis.

Wound healing assay

The transfected cells were distributed in a 6-well plate and allowed to grow until reaching 80–90% confluence. Scratches were created in the cell monolayer with 10 µL of sterilized pipette tips. By capturing images, the width of the scratched area was measured at 0, 24, and 48 h under a bright-field microscope without any staining. Image J software (RRID:SCR_003070) was used to analyze the migration distance.

Western blotting (WB)

Cells were lysed in RIPA buffer (Thermo, USA) supplemented with phenylmethylsulfonyl fluoride (PMSF) Protease Inhibitor (Thermo, USA). Protein concentrations were measured using the bicinchoninic acid assay (BCA) protein quantification kit (Beyotime, China). Equal amounts of protein extract were loaded onto gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedfordshire, USA), blocked with 5% non-fat milk for 2 h at 37 ℃. The membrane was incubated with indicated primary antibodies at 4 ℃ overnight, including anti-CDC7 (#ab229187, 1:1,000, Abcam, Cambridge, UK) and anti-GAPDH (#TA-08, 1:1,000, ZSGB-BIO, Beijing, China). Subsequently, secondary antibody against mouse (#ZB-2305, 1:1,000, ZSGB-BIO) or rabbit (#ZB-2301, 1:1,000, ZSGB-BIO) was incubated for 1 h at room temperature. After washing with Tris-Buffered Saline with Tween-20 (TBST), the membranes were visualized using enhanced chemiluminescence (ECL) Chemiluminescent Substrate (Thermo, USA). Data were obtained and calculated using Image Lab and ImageJ software. All blots, including all replicates with clear membrane edges, were provided in Figures S1-S10.

Immunofluorescence

Cells were seeded on coverslips and grown until reaching 50–60% confluence. Next, cells were fixed with 4% paraformaldehyde for 15 min and incubated with 0.5% Triton X-100 for 20 min. After blocked with 5% bovine serum albumin (BSA) for 45 min and incubated with anti-CDC7 (Cell Signaling Technology, #3603S, 1:100) overnight at 4 ℃, cells were then incubated with secondary fluorescence-conjugated anti-rabbit IgG H&L (Alexa Fluor^® 594) (#ab150080, 1:1,000, Abcam) and subsequently counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (#P0131, Beyotime). Images were captured using a confocal laser-scanning microscope (ZEISS LSM880, Oberkochen, Germany).

Immunohistochemistry

HCC tissue samples and adjacent normal tissue samples were post-fixed in 4% paraformaldehyde and prepared as 4-µm-thick sections in phosphate-buffered saline (PBS). The slides were then placed in an oven, baked at a temperature of 60 ℃ for a duration of 1 h. Afterward, they were deparaffinized and rehydrated. Heat-mediated antigen retrieval was performed in Tris-ethylene diamine tetraacetic acid (EDTA) buffer (pH 9.0) in a microwave oven. After cooling to room temperature, endogenous peroxidase activity was inhibited by incubating the sections with 3% hydrogen peroxide for 15 min. Next, the sections were permeabilized in 0.5% Triton X-100 for 20 min and blocked in 5% BSA for 1 h. Then, sections were incubated overnight at 4 ℃ with anti-CDC7 (Abcam, #ab229187, 1:100). After washing with PBS, each section was incubated with a mouse anti-rabbit IgG-horseradish peroxidase (HRP) (#sc-2357, 1:1,000, Santa Cruz, Dallas, USA) secondary antibody for 45 min. Each section was washed with PBST and developed with DAB solution for 5 min. Sections were restained with hematoxylin and then fixed on slides. CDC7 expression was analyzed using ImageJ software.

Statistical analysis

All statistical analyses were performed using the GraphPad Prism software (RRID:SCR_002798, version 10.0). Each experiment was repeated at least three times. Student’s t-test was applied to compare differences between two groups, and one-way analysis of variance (ANOVA) was used for comparisons among the different groups.

Results

Significant genes in HCC include KCNQ1OT1, miR-335-5p, and CDC7

To explore the roles of crucial genes in HCC progression, we first performed DE gene analyses on RNA sequencing data of 374 tumor tissues and 50 normal tissues obtained from the TCGA database. The results revealed 1,433 upregulated and 859 downregulated DE genes in the tumor samples. Among them, 136 lncRNAs, 128 miRNAs, and 2,028 mRNAs were upregulated, whereas 104 lncRNAs, 107 miRNAs, and 1,222 mRNAs were downregulated in tumor samples. Volcano plots and heatmaps displayed the expression patterns of these dysregulated lncRNAs, miRNAs, and mRNAs (Figure 1A-1C, Figure S11A-S11C). To investigate the biological functions of these DE genes and the potential pathways involved, GO annotation (Figure S11D), and KEGG enrichment analyses were performed for mRNAs to analyze possible regulatory mechanisms. The findings indicated that the cell cycle and DNA replication signaling pathways, which are closely related to the pathophysiological processes of HCC, were among the most significantly enriched pathways (Figure 1D).

Figure 1 Construction of DE lncRNA-miRNA-mRNA regulatory network for HCC. Analyses of DE lncRNAs (A), miRNAs (B), and mRNAs (C) of HCC tissues and normal tissues from the TCGA database are plotted in the volcano plots. The red dots represent the upregulated DE genes with log₂FC >0.5 and FDR <0.05, while the green dots represent downregulated genes with log₂FC <−0.5 and FDR <0.05. (D) Top 10 terms of KEGG enrichment analysis of DE mRNAs. (E) Visualization of the ceRNA network for DE lncRNA-miRNA-mRNA using the Cytoscape software. The round rectangles represent lncRNAs, ellipses represent miRNAs, and diamonds represent mRNAs. Kaplan-Meier curves analyses for high-expression and low-expression groups of KCNQ1OT1 (F) or CDC7 (G) using the GEPIA 2 database. P<0.05. Analyses of KCNQ1OT1 (H), miR-335-5p (I), and CDC7 (J) expression in different tumor grades using the UALCAN database. Grade 1, well differentiated (low grade); Grade 2, moderately differentiated (intermediate grade); Grade 3, poorly differentiated (high grade); Grade 4, undifferentiated (high grade). CDC7, cell division cycle 7; ceRNA, competing endogenous RNA; DE, differentially expressed; FC, fold change; FDR, false discovery rate; HCC, hepatocellular carcinoma; HR, hazard ratio; LIHC, liver hepatocellular carcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long non-coding RNA; miRNA, microRNA; mRNA, messenger RNA; TCGA, The Cancer Genome Atlas.

Using the ENCORI/starBase database, we predicted the interaction network of DE genes and constructed a ceRNA regulatory network comprising 3 lncRNAs, 12 miRNAs, and 21 mRNAs (Figure 1E). Unlike other potential interactions in HCC progression, the KCNQ1OT1/miR-335-5p/CDC7 axis has a unique regulatory role that could be significant in this network. Evidence shows that CDC7 plays a critical role in the cell cycle and DNA replication pathways (18). Therefore, we generated a Kaplan-Meier survival curve using the GEPIA 2 database, which indicated that patients with high expression levels of KCNQ1OT1 and CDC7 had a poor prognosis (Figure 1F,1G). To substantiate the clinical significance of this axis in HCC, expression level analyses were performed using the UALCAN database. The results revealed that both KCNQ1OT1 and CDC7 were expressed at significantly higher levels in tumor tissues than in normal tissues (Figure S11E,S11F), whereas miR-335-5p exhibited reduced expression in the HCC specimens (Figure S11G). Expression levels were closely related to and varied with tumor malignancy grade (Figure 1H-1J). These data indicated that the KCNQ1OT1/miR-335-5p/CDC7 axis is functionally significant and clinically relevant in HCC. A roadmap for the screening and validation processes of the key functional axis is shown in Figure S12.

KCNQ1OT1 promotes malignant progression of HCC by inhibiting miR-335-5p

To investigate the interaction between KCNQ1OT1 and miR-335-5p, we designed three siRNA sequences targeting different sites on KCNQ1OT1, and selected the most potent siRNA sequence, si-KCNQ1OT1-1714, as the subsequent interfering sequence (Figure S13A). Following the knockdown of KCNQ1OT1 in SMMC-7721 cells, the level of miR-335-5p was significantly increased (Figure 2A). Using the target prediction tool RNAhybrid, it was shown that miR-335-5p has binding sites on KCNQ1OT1. Consequently, we constructed WT and MUT KCNQ1OT1 plasmids; both the predicted binding sequence and the corresponding mutant sequence are shown in Figure 2B. Luciferase reporter assay indicated that the miR-335-5p mimic significantly suppressed the luciferase activity of WT-KCNQ1OT1, but not that of MUT-KCNQ1OT1, compared to the control mimic in SMMC-7721 cells (Figure 2C, Figure S13B). The RIP assay further confirmed the interaction between KCNQ1OT1 and miR-335-5p, with both accumulating in Ago2 antibody precipitates (Figure 2D). These results demonstrated that KCNQ1OT1 directly binds to and negatively regulates miR-335-5p in HCC cells.

Figure 2 KCNQ1OT1 promotes proliferation and migration of HCC cells by negatively regulating miR-335-5p. (A) The expression of miR-335-5p in SMMC-7721 cells after KCNQ1OT1 knockdown was analyzed using qRT-PCR. (B) The binding sites of KCNQ1OT1 to miR-335-5p were predicted by RNAhybrid. (C) Luciferase reporter assay was used to verify the targeted binding effect between miR-335-5p and WT-KCNQ1OT1 or MUT-KCNQ1OT1 in SMMC-7721 cells. (D) RIP assay demonstrating binding relationships between KCNQ1OT1 and miR-335-5p. (E) The CCK-8 assay of SMMC-7721 cells transfected with si-NC, si-KCNQ1OT1 or co-transfected with si-KCNQ1OT1 and miR-335-5p inhibitors (**** represents si-KCNQ1OT1 vs. si-NC; ^## and ^#### represent si-KCNQ1OT1 + miR-335-5p inhibitors vs. si-KCNQ1OT1). The cell colony formation assay (crystal violet staining) (F) and wound healing assay (bright-field without staining; scale bar: 100 µm) (G) of SMMC-7721 cells transfected with si-NC, si-KCNQ1OT1 or co-transfected with si-KCNQ1OT1 and miR-335-5p inhibitors. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ^##, P<0.01; ^####, P<0.0001. CCK-8, cell counting kit-8; HCC, hepatocellular carcinoma; IgG, immunoglobulin G; mfe, minimum free energy; MUT, mutation; NC, negative control; ns, no significance; OD, optical density; qRT-PCR, quantitative real-time polymerase chain reaction; RIP, RNA immunoprecipitation; WT, wild type.

Subsequently, CCK-8 and colony formation assays verified that KCNQ1OT1 knockdown inhibited HCC proliferation, whereas the wound healing assay demonstrated an inhibitory effect of KCNQ1OT1 knockdown on HCC migration. Furthermore, the effect of KCNQ1OT1 on the proliferation and migration in SMMC-7721 cells was counteracted by miR-335-5p inhibitors (Figure 2E-2G). Therefore, KCNQ1OT1 promoted the proliferation and migration of HCC cells by negatively regulating miR-335-5p.

MiR-335-5p inhibits HCC proliferation and migration

Given that KCNQ1OT1 may function by inhibiting miR-335-5p, we further investigated the biological role of miR-335-5p. HCC cells were transfected with miR-NC or miR-335-5p mimics, or co-transfected with miR-335-5p mimics and inhibitors. Our results indicated that miR-335-5p overexpression inhibited HCC cell proliferation and migration, an effect that was reversed by miR-335-5p inhibitors (Figure 3A-3C). Conversely, miR-335-5p inhibitors markedly enhanced cell proliferation and migration (Figure 3D-3F). Hence, the above results confirm that miR-335-5p suppresses HCC progression.

Figure 3 MiR-335-5p inhibits the proliferation and migration of HCC cells. CCK-8 assay (A), cell colony formation assay (crystal violet staining) (B), and wound healing assay (bright-field without staining; scale bar: 100 µm) (C) of SMMC-7721 cells transfected with miR-NC, miR-335-5p mimics or co-transfected with miR-335-5p mimics and inhibitors (**** represents miR-335-5p mimics vs. miR-NC; ^#, ^###, and ^#### represents miR-335-5p mimics + inhibitors vs. miR-335-5p mimics). CCK-8 assay (D), cell colony formation assay (crystal violet staining) (E), and wound healing assay (bright-field without staining; scale bar: 100 µm) (F) of SMMC-7721 cells transfected with miR-NC and miR-335-5p inhibitors. **, P<0.01; ****, P<0.0001; ^#, P<0.05; ^###, P<0.001; ^####, P<0.0001. CCK-8, cell counting kit-8; HCC, hepatocellular carcinoma; OD, optical density; NC, negative control.

CDC7 is a critical target of miR-335-5p to promote HCC progression

Next, to verify whether CDC7 is a functional target of miR-335-5p, we initially combined the results from the RNAhybrid prediction analysis with a dual-luciferase reporter gene assay (Figure 4A). The results showed that the miR-335-5p mimic inhibited the luciferase activity of WT-CDC7, but had no influence on that of MUT-CDC7 (Figure 4B, Figure S13C). The RIP assay further confirmed the interaction between CDC7 mRNA and miR-335-5p, with both accumulating in Ago2 antibody precipitates (Figure 4C). Our findings revealed that the 3'-untranslated region (UTR) of CDC7 binds to miR-335-5p. We subsequently treated SMMC-7721 cells with miR-335-5p mimics, which resulted in a significant increase in miR-335-5p levels and a notable decrease in CDC7 mRNA and protein levels. Subsequently, by combining with miR-335-5p inhibitor treatment, the expression of miR-335-5p and CDC7 in SMMC-7721 cells was successfully rescued (Figure 4D-4G). The immunofluorescence staining results were consistent with this conclusion (Figure 4H). Moreover, CDC7 expression increased at both the mRNA and protein levels when miR-335-5p inhibitors were used (Figure 4I-4K). Overall, these data suggested that CDC7 is negatively regulated by miR-335-5p.

Figure 4 The expression of CDC7 was regulated by miR-335-5p negatively. (A) The binding sites of miR-335-5p to CDC7 were predicted by RNAhybrid. (B) Luciferase reporter assay was used to verify the targeted binding effect between miR-335-5p and WT-CDC7 or MUT-CDC7 in SMMC-7721 cells. (C) RIP assay demonstrating binding relationships between miR-335-5p and CDC7 mRNA. (D) The expression of miR-335-5p in SMMC-7721 cells transfected with miR-335-5p mimics or co-transfected with miR-335-5p mimics and inhibitors was analyzed using qRT-PCR. (E) The expression of miR-335-5p in SMMC-7721 cells transfected with miR-335-5p inhibitors was analyzed using qRT-PCR. The qRT-PCR (F), WB (G), and immunofluorescence assay (scale bar: 20 µm) (H) of CDC7 level in SMMC-7721 cells transfected with miR-335-5p mimics or co-transfected with miR-335-5p mimics and inhibitors. The qRT-PCR (I), WB (J), and immunofluorescence assay (scale bar: 20 µm) (K) of CDC7 level in SMMC-7721 cells transfected with miR-335-5p inhibitors. **, P<0.01; ***, P<0.001; ****, P<0.0001. CDC7, cell division cycle 7; MUT, mutation; NC, negative control; ns, no significance; qRT-PCR, quantitative real-time polymerase chain reaction; RIP, RNA immunoprecipitation; WB, Western blotting; WT, wild type.

KCNQ1OT1 promotes HCC progression through the miR-335-5p/CDC7 axis

As previously reported, ceRNAs can competitively bind and sequester miRNAs from their original target transcripts, thereby preventing miRNA-induced degradation or suppression of target mRNAs (22). To clarify whether KCNQ1OT1 regulates the progression of HCC via the miR-335-5p/CDC7 axis, we transfected miR-335-5p inhibitors into KCNQ1OT1 knockdown HCC cells. Using qRT-PCR, WB and immunofluorescence assays, we found that CDC7 expression at the mRNA and protein levels was inhibited by KCNQ1OT1 knockdown and markedly reversed by miR-335-5p inhibitors (Figure 5A,5B). Furthermore, immunohistochemistry results showed that CDC7 expression in HCC tissues was significantly higher than that in ANT (Figure 5C). This finding further confirms that the abnormal expression of CDC7 in HCC cells is a significant factor influencing HCC progression.

Figure 5 KCNQ1OT1/miR-335-5p/CDC7 axis mediates proliferation and migration of HCC cells. qRT-PCR, WB (A), and immunofluorescence assay (scale bar: 20 µm) (B) of CDC7 expression in SMMC-7721 cells transfected with si-NC, si-KCNQ1OT1 or co-transfected with si-KCNQ1OT1 and miR-335-5p inhibitors. (C) Immunohistochemistry of CDC7 expression in HCC tissues and ANT (scale bar: 100 µm). CCK-8 assay (D), cell colony formation assay (crystal violet staining) (E), and wound healing assay (bright-field without staining; scale bar: 100 µm) (F) of SMMC-7721 cells after CDC7 knockdown. CCK-8 assay (** and **** represent oe-CDC7 vs. oe-NC; ^# and ^#### represent co-transfection with oe-CDC7 and XL413 vs. oe-CDC7) (G), cell colony formation assay (crystal violet staining) (H), and wound healing assay (bright-field without staining; scale bar: 100 µm) (I) of SMMC-7721 cells transfected with oe-NC, oe-CDC7, or co-transfected with oe-CDC7 and XL413. **, P<0.01; ***, P<0.001; ****, P<0.0001; ^#, P<0.05; ^#####, P<0.0001. ANT, adjacent normal tissues; CCK-8, cell counting kit-8; CDC7, cell division cycle 7; HCC, hepatocellular carcinoma; NC, negative control; OD, optical density; oe, overexpression; qRT-PCR, quantitative real-time polymerase chain reaction; WB, Western blotting.

To thoroughly investigate the specific mechanisms by which CDC7 affects biological processes in HCC cells, we first selected the most potent siRNA sequence for CDC7 knockdown (Figure S13D) and verified the efficiency of the CDC7 overexpression plasmid and CDC7-selective inhibitor XL413 (23) (Figure S13E-S13G). In SMMC-7721 cells, CDC7 knockdown significantly suppressed proliferation and migration (Figure 5D-5F). Moreover, XL413 effectively reversed the enhanced proliferation and migration induced by CDC7 overexpression in SMMC-7721 cells (Figure 5G-5I). Our data suggest that KCNQ1OT1 regulates CDC7 expression and promotes the malignant progression of HCC by inhibiting miR-335-5p.

Regulatory effect of KCNQ1OT1/miR-335-5p on CDC7 is induced by HBc

To deeply explore the correlation between the KCNQ1OT1/miR-335-5p/CDC7 axis and HBV-related HCC, we compared the expression levels of this axis in HepG2 and HepG2.215 cells. In HepG2.215 cells, the expression of KCNQ1OT1 and CDC7 was significantly elevated, whereas that of miR-335-5p expression was notably reduced compared to that in HepG2 cells (Figure 6A-6D). Moreover, after transfection with a 1.3-fold HBV whole genome plasmid, SMMC-7721 and differentiated HepaRG cells showed a significant increase in the expression of KCNQ1OT1 and CDC7, accompanied by a decrease in miR-335-5p levels (Figure 6E-6H). Our research demonstrated that HBV consistently elevated KCNQ1OT1 expression, reduced miR-335-5p levels, and upregulated CDC7, regardless of whether HBV viruses or complete genome plasmids were introduced stably or transiently. This suggests that HBV may have an important regulatory effect on the KCNQ1OT1/miR-335-5p/CDC7 axis.

Figure 6 Regulatory effect of HBV and its encoded proteins on the KCNQ1OT1/miR-335-5p/CDC7 axis. RNA levels of KCNQ1OT1 (A), miR-335-5p (B), and CDC7 (C) in HepG2 and HepG2.215 cells. (D) WB of CDC7 protein expression in HepG2 and HepG2.215 cells. RNA levels of KCNQ1OT1 (E), miR-335-5p (F), and CDC7 (G) in SMMC-7721 and differentiated HepaRG cells transfected with a 1.3-fold HBV whole genome plasmid. (H) WB of CDC7 protein expression in SMMC-7721 and differentiated HepaRG cells transfected with a 1.3-fold HBV whole genome plasmid. (I) WB of HBx, HBs and HBc protein expression in SMMC-7721 and differentiated HepaRG cells transfected with HBx, HBs and HBc recombinant plasmids, respectively. Expression of KCNQ1OT1 (J), miR-335-5p (K), and CDC7 (L) in SMMC-7721 and differentiated HepaRG cells transfected with NC, HBx, HBs, and HBc recombinant plasmids. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CDC7, cell division cycle 7; HBc, HBV core/capsid protein; HBV, hepatitis B virus; HBs, HBV surface protein; HBx, HBV X protein; NC, negative control; WB, Western blotting.

Studies have shown that HBV-encoded proteins are involved in numerous intracellular signaling pathways and play important roles in HBV-related HCC (2,24,25). To identify the specific HBV-encoded proteins responsible for activating this signaling axis, we transiently transfected SMMC-7721 and differentiated HepaRG cells with HBx, HBs, and HBc recombinant plasmids for 48 h. WB results showed that the HBx, HBs and HBc proteins were expressed in the cells (Figure 6I). Meanwhile, qRT-PCR results revealed that transfection with HBc substantially increased KCNQ1OT1 and CDC7 expression and decreased miR-335-5p expression compared to other HBV proteins (Figure 6J-6L, Figure S14). Consequently, the KCNQ1OT1/miR-335-5p/CDC7 axis may serve as a potential therapeutic target for HCC and offering new treatment options for patients with HBV-related HCC.

Discussion

HCC is the most prevalent form of primary liver cancer and the fourth leading cause of cancer-related mortality globally (1). Despite significant efforts, our understanding of the specific mechanisms underlying HCC progression remains limited. HBV infection, as one of the most important risk factors, affects the expression and function of specific genes, thereby contributing to liver disorders (26,27). In addition, the complex alterations caused by HBV infection are considered the primary cause of malignant progression and poor prognosis in patients with HCC (2). The mechanisms underlying HBV-mediated HCC remain a focal point for our future research.

Viral persistence in HBV infection is caused by the ability of the virus to evade the host immune system and establish an episome in the nucleus of infected cells, known as covalently closed circular double-stranded DNA (cccDNA) (3). As a transcriptional template of HBV, cccDNA encodes four overlapping open reading frames (ORFs), leading to the production of several proteins, including HBV core/capsid protein (HBc), HBeAg, envelope proteins (S, M, and L), nonstructural X protein (HBx), and P protein (HBp). Current evidence suggests that HBx plays a pathogenetic role in HBV-induced malignant transformation (4,28). The HBc protein, encoded by the C open reading frame (C ORF), is involved in nearly every stage of the HBV life cycle, including subcellular trafficking and release of the HBV genome, capsid assembly and transport, reverse transcription, and RNA metabolism (29,30). Emerging evidence indicates that HBc promotes malignant progression of HCC via various mechanisms, including epigenetic alterations (e.g., miRNA), cellular metabolic disorders, and resistance to apoptosis (2). HBc has been reported to repress the expression of the human p53 tumor suppressor gene in HCC synergistically with HBx (31). Furthermore, HBc facilitates HCC metastasis via the miR-382-5p/DLC-1 axis and may inhibit hepatocyte apoptosis induced by TRAIL by obstructing DR5 expression, contributing to the development of chronic hepatitis and HCC (32,33). However, previous studies have primarily focused on the role of the HBx protein in the molecular mechanisms of HBV malignant transformation. In the present study, we demonstrated that HBc induced the KCNQ1OT1/miR-335-5p/CDC7 axis and promoted the malignant progression of HCC, representing a significant addition to our understanding of HBV-related HCC.

Growing evidence indicates that lncRNAs play a wide range of roles in chromatin modification, transcription, and post-transcriptional regulation, acting as signals, decoys, guides, scaffolds, and ceRNAs. Moreover, recent studies have suggested that lncRNA-miRNA interactions are crucial regulators involved in diverse biological processes and carcinogenesis in HCC (34-36). LncRNAs typically interact with miRNAs as molecular sponges, modulating the binding of miRNAs to their target mRNAs (34). For instance, the highly expressed lncRNA NEAT1 functions as a ceRNA, attracting miR-362-3p to indirectly upregulate MIOX expression, thereby promoting ferroptosis in HCC cells (37). Mechanistic studies have revealed that lncRNA MIAT exerts an oncogenic function in HCC by sponging miR-22-3p to upregulate SIRT1 expression (38-40). MFI2-AS1 facilitates HCC progression through a positive feedback loop involving the MFI2-AS1/miR-134/FOXM1 axis (41). In this study, we constructed the lncRNA-miRNA-mRNA network and found that KCNQ1OT1 might play an essential role in the progression of HCC through interactions with miR-335-5p, subsequently regulating CDC7 signaling. Furthermore, our research demonstrated that the abnormal expression of this axis in HCC is regulated by HBc, which could be a significant contributor.

KCNQ1OT1, located on chromosome 11p15.5, spans a length of 91 kb (42). It is highly expressed in various malignancies and is associated with tumor growth, lymph node metastasis, survival cycle, and recurrence rate (13). Increasing evidence suggests that KCNQ1OT1 expression is significantly higher in HCC tissues and cell lines than in adjacent non-carcinoma tissues and normal cell lines (43,44). Cheng et al. discovered that KCNQ1OT1 acts as a molecular sponge for miR-149, thereby regulating S1PR1 expression and influencing HCC invasion and migration (43). Furthermore, KCNQ1OT1 regulates the expression of cyclin-dependent kinase 16 (CDK16) to mediate HCC progression by functioning as a ceRNA of miR-504 (45). Dysregulation of the KCNQ1OT1/miR-148a-3p/IGF1R axis also contributes to HCC (46).

Previous studies have shown that miR-335-5p negatively regulates the rapid proliferation of HCC cells, and extracellular vesicles carrying this miRNA can reduce cancer growth and invasion (14,15). Moreover, miR-335-5p is important in the process by which circ_0064288 and circ_0009910 promote ROCK1 expression, thereby facilitating HCC cell growth and migration (47,48). Our research has shown for the first time that miR-335-5p is inhibited by KCNQ1OT1 to regulate the expression of CDC7.

CDC7, a highly conserved serine-threonine kinase, initiates DNA replication and is activated through its interaction with its regulatory subunit DBF4 (49). Cancer cells exhibit heightened levels of replicative stress and may therefore be especially sensitive to CDC7 inhibition (49). Elevated CDC7 expression is well recognized in a wide range of cancers (including HCC, breast cancer, and colon cancer), and is strongly associated with tumor malignancy, invasiveness, and poor prognosis (50). One study has shown that combining CDC7 inhibition with ATR-CHK1 inhibition probably shows striking synergy to achieve robust suppression of proliferation in HCC cells (16). Similarly, synergistic inhibition of CDC7 and cyclin-dependent kinase 9 (CDK9) enhances the antitumor efficacy of 5-fluorouracil (5-FU) in suppressing human HCC cells (51). These studies demonstrate that CDC7 may play an integral role in the treatment of HCC. However, the specific mechanisms by which HBc modulates CDC7 activation and promotes HCC progression require further investigation.

In this study, we aimed to elucidate the specific mechanisms by which lncRNAs contribute to HBV-related HCC progression. Key findings from our study showed that HBc enhances KCNQ1OT1 expression, which in turn suppresses miR-335-5p, leading to the upregulation of CDC7. Our study targeting the HBc-induced KCNQ1OT1/miR-335-5p/CDC7 axis will be an essential addition to the exploration of HBV-related HCC treatment.

Conclusions

In summary, we verified that HBc plays a pivotal role in promoting malignant progression in HCC by modulating the KCNQ1OT1/miR-335-5p/CDC7 signaling axis. Our findings provide novel insights into the roles of KCNQ1OT1 and CDC7, which may aid in the exploration of treatments for HBV-related HCC. Future studies should aim to develop specific inhibitors for this axis and assess their efficacy in clinical settings, potentially contributing to improved outcomes in patients with HBV-related HCC.

Acknowledgments

We would like to thank Dr. Dequan Yang (Department of Clinical Laboratory, The Eighth Affiliated Hospital, Sun Yat-sen University) for providing SMMC-7721 cells. We acknowledge the Biological Laboratory of Hetao Cooperation Zone, the Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen for provision of the experimental site.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-233/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-233/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-233/prf

Funding: This research was funded by the National Natural Science Foundation of China (No. 82072267), GuangDong Basic and Applied Basic Research Foundation (No. 2024A1515220014), Shenzhen Science and Technology Innovation Program (Nos. JCYJ20240813150644056 and ZDSYS20220606100801004), Futian Healthcare Research Project (No. FTWS013), and Sun Yat-Sen Eighth Affiliated Hospital Clinical Research Program (No. PY-2023-01).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-233/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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