Abstract
Background
DDX18, a member of the DEAD-box RNA helicase family, plays a pivotal role in ribosome biogenesis and RNA metabolism and is thus extensively implicated in tumorigenesis. Although its oncogenic functions have been well-documented across various malignancies, the precise molecular mechanisms underlying DDX18-driven progression in hepatocellular carcinoma (HCC) remain largely undefined. This study systematically elucidates the pathological contributions of DDX18 to HCC through a combination of comprehensive in vitro experiments and in vivo analyses.
Methods
We assessed the expression and effects of DDX18 on HCC tissues and cellular functions through bioinformatics analyses, wound healing assays, colony formation assays, transwell assays, and flow cytometry. Western blot analysis revealed that mitogen-activated protein kinase (MAPK) signaling pathways were associated with protein levels involved in the epithelial-mesenchymal transition (EMT). Co-immunoprecipitation (Co-IP) and immunoFluorescence colocalization experiments confirmed the interaction between DDX18 and REXO4. Additionally, we evaluated the functional roles of DDX18 and REXO4 using a series of in vitro assays and nude mouse xenograft models.
Results
Our data demonstrated elevated expression of DDX18 in HCC tissues. DDX18 significantly increased cell proliferation, invasion, and migration, and activated EMT and MAPK signaling pathways in vitro. Mechanistically, DDX18 interacts with REXO4, thereby promoting tumor growth and metastasis by regulating the EMT process and MAPK signaling. Furthermore, overexpression of REXO4 reversed the inhibitory effects of DDX18 knockdown both in vitro and in vivo.
Conclusions
This study provides evidence that the DDX18/REXO4 axis plays a critical role in HCC development and may represent a novel therapeutic target or diagnostic biomarker for patients with HCC.
Keywords: Hepatocellular carcinoma (HCC), DDX18, REXO4, metastasis, mitogen-activated protein kinase (MAPK)
Highlight box.
Key findings
• This study aimed to elucidate the precise role of DDX18 in hepatocellular carcinoma (HCC) progression and to investigate its involvement in cell growth and metastasis.
What is known and what is new?
• Our results demonstrated that DDX18 overexpression significantly promoted tumor proliferation, invasion, and migration, while facilitating metastasis through epithelial-mesenchymal transition (EMT) and mitogen-activated protein kinase (MAPK) pathway activation.
• Mechanistically, DDX18 interacts with REXO4 to promote tumor growth and metastasis via EMT regulation and MAPK signaling. Additionally, REXO4 overexpression reversed the inhibitory effects of DDX18 knockdown in both settings.
What is the implication, and what should change now?
• These findings establish DDX18 as a promising therapeutic target for HCC intervention, especially in patients exhibiting metastatic progression, and underscore its dual role in both tumor growth and dissemination pathways.
Introduction
Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and continues to be a leading cause of cancer-related mortality worldwide due to its insidious progression and limited therapeutic options (1,2). Despite advancements in surgical and systemic treatments, the molecular mechanisms driving HCC metastasis and chemoresistance remain poorly understood (3). Furthermore, current therapeutic approaches for HCC have not yielded satisfactory outcomes (4,5), highlighting the urgent need to identify novel diagnostic and therapeutic targets to improve HCC prognosis (6).
DDX18, a critical regulator of RNA metabolism and chromatin organization, has emerged as a multifunctional oncogene implicated in cell cycle progression, stem cell pluripotency, and tumorigenesis across various cancers (7-9). For instance, DDX18 promotes lung adenocarcinoma progression by directly upregulating CDK4-mediated cell cycle signaling (10), while its role in nuclear phase separation underscores its capacity to modulate chromatin architecture and transcriptional programs (11). However, the regulatory mechanisms and functional significance of DDX18 in HCC remain unknown.
This study aims to investigate the oncogenic role of DDX18 in HCC, addressing this knowledge gap. We hypothesize that DDX18 drives HCC metastasis by activating epithelial-mesenchymal transition (EMT) and mitogen-activated protein kinase (MAPK) signaling pathways, potentially through interactions with downstream effectors such as REXO4. Our data show that DDX18 is overexpressed in HCC tissues and correlates with poorer clinical outcomes, underscoring its clinical relevance. By integrating bioinformatics, functional assays, and mechanistic studies, we uncover a DDX18/REXO4 axis that orchestrates EMT and MAPK activation, providing a novel framework for understanding HCC aggressiveness. This work not only expands the functional repertoire of RNA helicases in cancer but also identifies actionable targets for therapeutic intervention in HCC. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-339/rc).
Methods
Tissue specimens and cell culture
HCC tissues and corresponding normal liver tissues were collected from patients at the Affiliated Hospital of Nantong University between 2022 and 2023. None of the patients had received radiotherapy, chemotherapy, or other neoadjuvant therapies before surgery. This study was approved by the Ethics Committee of the Affiliated Hospital of Nantong University (No. 2025-L107), and informed consent was obtained from all participants. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.
HCC cell lines Huh7 and HCCLM3 were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37 ℃ in a humidified incubator with 5% CO2. DDX18 and REXO4 expression was silenced using short hairpin RNA (shRNA)-containing pLKO.1-EGFP-Puro lentiviral vectors, while overexpression was achieved using pCDH-DDX18 and pCDH-REXO4 lentiviral plasmids. The sequences of the shRNAs used were as follows: scramble shRNA: 5'-GAATTGCACAAGATAGGGTAA-3', shREXO4: 5'-CGCTCTGCATAATGACCTAAA-3', shDDX18: 5'-GGAGATGTATCTGAAGAAACA-3'. All lentiviral vectors were obtained from Genechem (Shanghai, China). Cells were seeded in six-well plates and incubated for 24 hours to reach approximately 70% confluence before subsequent experiments.
Western blot analysis
Total protein was extracted from tissue samples or cellular lysates for Western blot analysis. Protein concentrations were determined using a bicinchoninic acid (BCA) assay kit (Beyotime Biotechnology, Shanghai, China). Equivalent amounts of protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). β-actin served as a loading control. Detection was performed using horseradish peroxidase (HRP)-conjugated secondary antibodies, followed by visualization of protein bands with enhanced chemiluminescence (ECL) detection reagents (Millipore). The primary antibodies used were: DDX18 (ab128197, Abcam, Boston, MA, USA), REXO4 (18890-1-AP, Proteintech, Wuhan, China), E-cadherin (20874-1-AP, Proteintech), vimentin (60330-1-Ig, Proteintech), extracellular regulated protein kinase (ERK)1/2 (11257-1-AP, Proteintech), phosphorylated-ERK1/2 (28733-1-AP, Proteintech), c-Jun N-terminal kinase (JNK) (80024-1-RR, Profit, Wuhan, China), mitogen-activated extracellular signal-regulated kinase (MEK)1/2 (11049-1-AP, Proteintech), phosphorylated-MEK1/2 (ab278723, Abcam).
Immunofluorescence staining
For immunofluorescence staining, a previously documented method was adopted. In summary, primary antibodies against DDX18 and REXO4 were incubated overnight at 4 ℃. Subsequent immunofluorescence labeling involved the application of fluorescent secondary antibodies for 1 hour at 37 ℃, followed by counterstaining of the sections with 4',6-diamidino-2-phenylindole (DAPI).
Colony formation assay
Cells were seeded in six-well plates at a density of 500 cells/well, and cultivated at 37 ℃ with 5% CO2. After a 14-day incubation period, the cells were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and stained with a 0.1% crystal violet solution. Photographs were taken to record the stained colonies, which were then measured.
Cell apoptosis assay
Following a 48-hour transfection period, cells were washed with PBS, fixed with pre-cooled 70% ethanol at −20 ℃ for 1 hour, and stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC)-conjugated Annexin V at 4 ℃ for 10 minutes. The extent of apoptosis was determined by assessing the proportion of apoptotic cells using the BD FACS Calibur system.
Wound-healing assay
The migratory capacity of cells was assessed via a wound-healing assay. Transfected cells were scraped using a 200 µL sterile pipette to create a cell-free zone, washed with PBS, and a fresh culture medium was added. Cells were photographed immediately (0 hours) and again after 24 hours, with intercellular distance measured to gauge migratory ability. Each experiment was repeated three times.
Transwell assay
Cell migration and invasion were assessed using a transwell assay. The upper compartments of the transwell plates were either coated or left uncoated with Matrigel (BD Biosciences, Paramus, NJ, USA), and transfected cells (approximately 1×105) were placed in 200 µL of serum-free medium. The lower chamber contained 500 µL DMEM supplemented with 10% FBS. After a 2-day incubation period at 37 ℃, invasive or migratory cells were fixed, stained with 0.1% crystal violet, and counted under an inverted microscope (Olympus, Tokyo, Japan) in five randomly selected fields.
Tumor growth in vivo assay
Female BALB/c nude mice (8 weeks old) were obtained from the Laboratory Animal Center of Nantong University. Cells stably transfected with control vector (n=5), DDX18 knockdown construct (n=5), and REXO4 overexpression in the DDX18 knockdown group (n=5) were subcutaneously injected into the right flank of the mice. Tumor volumes were measured weekly for 4 weeks, and tumor growth curves were subsequently plotted. The final tumor volume was calculated using the formula: tumor volume (mm3) = length (mm) × width (mm)2/2.
Animal experiments were performed under a project license (No. P20250226-065) approved by Animal Ethics Committee of Nantong University, in compliance with Nantong University guidelines for the care and use of animals. A protocol was prepared before the study without registration.
Statistical analysis
Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9.0 software. Differences between the two groups were assessed using a two-tailed Student’s t-test, while comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA). A significance level of P<0.05 was established.
Results
DDX18 was highly expressed in HCC
We initially evaluated the expression levels of DDX18 in liver hepatocellular carcinoma (LIHC) tissues using data from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). Analysis of the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia2.cancer-pku.cn/) revealed that DDX18 expression was significantly higher in tumor tissues compared to normal tissues (Figure 1A). Kaplan-Meier survival analysis further demonstrated that elevated DDX18 expression was associated with an unfavorable prognosis in LIHC patients (Figure 1B). Additionally, Western blot analysis confirmed that DDX18 protein levels were markedly increased in HCC tissues relative to adjacent non-tumor tissues (Figure 1C). Collectively, these findings indicate that DDX18 is upregulated in HCC and strongly correlated with poor patient outcomes. To investigate the functional role of DDX18 in HCC, we transfected Huh7 and HCCLM3 cells with DDX18-overexpression plasmids or shDDX18 constructs. Western blot analysis verified that both overexpression and silencing of DDX18 were highly effective (Figure 1D,1E), confirming the successful manipulation of DDX18 expression levels.
Figure 1.
Expression analysis of DDX18 in HCC tissues and cells. (A) Comparison of DDX18 expression levels between LIHC tissues and normal tissues using data from the GEPIA platform. (B) Kaplan-Meier survival analysis with a log-rank test was conducted to evaluate the prognostic significance of DDX18 expression in LIHC patients stratified by low versus high expression levels. (C) Western blot validation of DDX18 expression in four randomly selected paired tumor and non-tumor specimens. (D,E) Western blot assessment of DDX18 protein expression in HCC cells following transfection with OV-DDX18 or shDDX18. GEPIA, Gene Expression Profiling Interactive Analysis; HCC, hepatocellular carcinoma; HR, hazard ratio; LIHC, liver hepatocellular carcinoma; N, normal; NC, negative control; OV, overexpression; sh, short hairpin; T, tumor; TPM, transcripts per million.
DDX18 promoted HCC proliferation, migration, and invasion in vitro
To elucidate the role of DDX18 in the proliferation of HCC cells, we conducted colony formation assays to evaluate the impact of DDX18 expression on HCC cell proliferation. Specifically, Huh7 and HCCLM3 cells were transfected with DDX18-overexpression plasmids or shDDX18 constructs, respectively. Knockdown of DDX18 significantly reduced colony formation in both Huh7 and HCCLM3 cells (Figure 2A,2B), while overexpression of DDX18 led to a notable increase in colony numbers (Figure 2A,2B).
Figure 2.
DDX18 influences HCC cell proliferation, migration, and invasion in vitro. (A,B) Colony formation assays were carried out in HCC cells transfected with OV-DDX18 and shDDX18#2 to evaluate colony-forming efficiency. The colonies were stained with 1% crystal violet. (C,D) Wound-healing assays were conducted to assess the migratory capacity of HCC cells, with the percentage of wound closure quantified. Scale bar: 100 µm. (E,F) Transwell assays were employed to analyze the migratory and invasive capabilities of HCC cells transfected with OV-DDX18 and shDDX18#2. Crystal violet staining. Scale bar: 50 µm. **, P<0.01. HCC, hepatocellular carcinoma; NC, negative control; OV, overexpression; sh, short hairpin.
To further investigate whether DDX18 contributes to the aggressive progression of HCC, we performed wound healing and transwell assays following DDX18 overexpression or knockdown in Huh7 and HCCLM3 cells. Our results demonstrated that overexpression of DDX18 significantly enhanced the migratory and invasive capabilities of these cells, whereas reducing DDX18 levels markedly inhibited their migratory and invasive potential (Figure 2C-2F).
DDX18 influenced the apoptosis and modulated the EMT process as well as the MAPK signaling pathway in HCC cells
To further elucidate the mechanisms by which DDX18 promotes cell proliferation, we conducted flow cytometry analysis to evaluate apoptosis in HCC cells transfected with DDX18-overexpression plasmids or shDDX18. Our results demonstrated that overexpression of DDX18 significantly decreased the apoptotic cell ratio in both Huh7 and HCCLM3 cells. Conversely, silencing DDX18 increased the apoptotic cell ratio in these two cell lines (Figure 3A,3B). These findings suggest that DDX18 exerts an inhibitory effect on apoptosis in HCC cells. Therefore, our data indicate that DDX18 contributes to the tumor-promoting effects observed in HCC development and progression.
Figure 3.
DDX18 influences apoptosis, the EMT process, and the MAPK signaling pathway in HCC cells. (A,B) Flow cytometry was employed to evaluate the percentage of apoptotic HCC cells following transfection with an empty vector (NC), OV-DDX18, control shRNA (NC), or shDDX18#2. The X-axis represents annexin V-FITC fluorescence, while the Y-axis indicates PI staining. **, P<0.01. (C) Western blot analysis was conducted to examine the levels of ERK, p-ERK, JNK, and p-JNK in HCC cells transfected with different constructs. (D) Western blot analysis revealed the expression levels of E-cadherin, N-cadherin, and vimentin in HCC cells after transfection with various constructs. EMT, epithelial-mesenchymal transition; ERK, extracellular regulated protein kinase; FITC, fluorescein isothiocyanate; HCC, hepatocellular carcinoma; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NC, negative control; OV, overexpression; p-, phosphorylated-; PI, propidium iodide; sh, short hairpin; shRNA, short hairpin RNA.
Building on our previous observations regarding the impact of DDX18 on cell proliferation and migration, we subsequently investigated its role in the EMT process and MAPK pathway activation during HCC progression. We evaluated the expression levels of key proteins associated with the MAPK pathway using Western blot analysis. The data revealed that DDX18 knockdown in Huh7 and HCCLM3 cells significantly reduced the levels of phosphorylated-ERK1/2 (p-ERK1/2) and phosphorylated-JNK (p-JNK), while the levels of total ERK1/2 and JNK remained unchanged (Figure 3C). Additionally, we examined the expression of various EMT markers. Western blot experiments showed that DDX18 knockdown increased the protein levels of E-cadherin and simultaneously decreased the levels of N-cadherin and vimentin in both Huh7 and HCCLM3 cells (Figure 3D).
DDX18 promotes HCC proliferation, migration, and invasion through regulating REXO4
Utilizing the GEPIA database, we conducted an in-depth analysis of REXO4 mRNA expression levels in LIHC tissues, revealing a significant increase compared to adjacent normal tissues, as illustrated in the comparative expression profiles (Figure 4A). Additionally, Kaplan-Meier survival curves were employed to establish a clear correlation between elevated REXO4 expression and reduced overall survival probabilities in patients with LIHC (Figure 4B). To validate the potential association between DDX18 and REXO4, a co-immunoprecipitation (Co-IP) experiment was performed, confirming the interaction between these proteins (Figure 4C). Subsequent immunofluorescence studies provided consistent evidence, demonstrating the cytoplasmic co-localization of DDX18 and REXO4 within the Huh7 and HCCLM3 cell lines (Figure 4D).
Figure 4.
DDX18 interacts with REXO4. (A) Box plots illustrate that REXO4 mRNA levels were significantly elevated in LIHC tissues compared to normal tissues. (B) Kaplan-Meier curve analysis assessed the overall survival rates of HCC patients with high versus low REXO4 expression. (C) Co-IP experiments confirmed the interaction between endogenous DDX18 and REXO4 in HCC cells. (D) Immunofluorescence staining detected the expression of DDX18 (green) and REXO4 (red) in HCC cells. Original magnification ×200. Co-IP, co-immunoprecipitation; DAPI, 4',6-diamidino-2-phenylindole; HCC, hepatocellular carcinoma; HR, hazard ratio; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; LIHC, liver hepatocellular carcinoma; mRNA, messenger RNA; N, normal; T, tumor; TPM, transcripts per million.
Functional validation experiments were conducted to substantiate the role of the DDX18-REXO4 axis in the progression of HCC. The colony formation assay demonstrated that forced expression of DDX18 enhanced cellular viability, which was partially mitigated by the silencing of REXO4 in Huh7 and HCCLM3 cells (Figure 5A). Furthermore, when compared to the shDDX18 group, restoration of cell proliferation to baseline levels was observed in the shDDX18 + REXO4 cohort (Figure 5B). In the wound healing assay, the wound healing level was quantified. The results demonstrated that overexpression of DDX18 enhanced cell migration capacity, whereas this effect was partially reversed upon downregulation of REXO4 in Huh7 and HCCLM3 cells (Figure 5C). Besides, cell migration ability was restored in the shDDX18 + REXO4 group compared to the shDDX18 group (Figure 5D). Furthermore, Transwell assays demonstrated that forced expression of DDX18 enhanced the migration and invasion abilities of cells, and downregulation of REXO4 in Huh7 and HCCLM3 cells partially reversed these effects (Figure 5E). Similarly, the migration and invasion abilities were restored in the shDDX18 + REXO4 group compared with the shDDX18 group alone (Figure 5F). These cumulative data implicate REXO4 as a downstream effector of DDX18 in the pathogenesis of HCC.
Figure 5.
Knockdown of REXO4 abrogates DDX18-enhanced growth, migration, and invasion of HCC cells in vitro. (A,B) Colony formation assays evaluated the impact of REXO4 on colony formation induced by DDX18 in HCC cells. The colonies were stained with 1% crystal violet. (C,D) Wound-healing assays demonstrated the effects of REXO4 on DDX18-induced wound healing in HCC cells. Scale bar: 100 µm. (E,F) Transwell assays investigated the influence of REXO4 on DDX18-induced migration and invasion of HCC cells. Crystal violet staining. Scale bar: 50 µm. **, P<0.01. HCC, hepatocellular carcinoma; OV, overexpression; sh, short hairpin.
DDX18 interacts with REXO4 through EMT and MAPK signaling pathway
The aforementioned findings corroborate that DDX18 interacts with REXO4, thereby modulating the EMT pathway. It is essential to investigate the mechanism by which DDX18 induces EMT in a manner contingent upon REXO4. Our Western blotting experiments demonstrated that the introduction of shREXO4 plasmids into DDX18-overexpressing Huh7 and HCCLM3 cells partially mitigated the stimulatory influence of DDX18 on the EMT process (Figure 6A). Subsequently, we explored the role of DDX18 in the activation of the ERK/MAPK pathway, as mediated by REXO4 in vitro. Our observations revealed that overexpression of DDX18 could partially restore the diminished phosphorylation of ERK and MEK, which resulted from shREXO4 plasmid transfection (Figure 6B,6C). In conclusion, our collective data affirm that DDX18 acts as a positive regulator of the ERK/MAPK signaling pathways in a manner dependent on REXO4.
Figure 6.
DDX18 regulates the EMT process and MAPK signaling pathway in a REXO4-dependent manner in HCC cells. (A) Western blot analysis determined the effects of REXO4 siRNA on the EMT process induced by DDX18 overexpression in HCC cells. (B,C) Western blot analysis elucidated the role of REXO4 in DDX18-mediated activation of the ERK/MAPK pathway in HCC cells. EMT, epithelial-mesenchymal transition; ERK, extracellular regulated protein kinase; HCC, hepatocellular carcinoma; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated extracellular signal-regulated kinase; siRNA, small interfering RNA; OV, overexpression; p-, phosphorylated-; sh, short hairpin.
REXO4 reverses the effects of DDX18 depletion in vivo
To further investigate the impact of DDX18 on tumor progression in vivo, we established a xenograft model using nude mice. The DDX18 knockdown group exhibited a significant reduction in both tumor weight and volume compared to the negative control group. Notably, the overexpression of REXO4 in the DDX18-depleted group led to an increase in tumor weight and volume relative to the DDX18 knockdown group alone (Figure 7A-7C). Collectively, these findings demonstrate that DDX18 influences tumor growth in vivo through its downstream effector REXO4, thereby modulating the progression of HCC cells.
Figure 7.
DDX18 modulates HCC growth in vivo. (A) Representative images of five mice from the shNC, shDDX18#2, or shDDX18#2 + OV-REXO4 groups are shown. (B,C) Subcutaneous tumor volumes and weights were measured. **, P<0.01. HCC, hepatocellular carcinoma; NC, negative control; OV, overexpression; sh, short hairpin.
Discussion
The development of HCC constitutes a multifaceted process, orchestrated by the interplay among numerous molecules governed by pivotal genes (12,13). An inquiry into the regulatory frameworks of these genes may pave the way for precision therapeutic approaches tailored for HCC patients (14,15). DDX18 has garnered prominence as a pivotal element in the sphere of cancer biology, especially concerning tumorigenesis, immune system circumvention, and therapeutic resistance (7,16). Recent scholarly works have underscored the heightened expression of DDX18 across various neoplasms (17), including pancreatic ductal adenocarcinoma (18), head and neck squamous cell carcinoma (19), and gastric cancer (20), suggesting its potential utility as a therapeutic target.
Our investigation has elucidated that DDX18 serves as a catalyst for the enhanced migration, invasiveness, and proliferative capacity of HCC cells, accomplishing this through the activation of EMT and MAPK signaling cascades. These discoveries are in concordance with antecedent literature delineating DDX18’s oncogenic contributions to diverse types of cancer, such as its modulation of CDK4 in lung adenocarcinoma (10) and its role in nuclear phase transitions within stem cells (11). Nonetheless, our research uniquely characterizes DDX18 as a nodal entity in the progression of HCC, linking its RNA helicase function to the restructuring of the cytoskeleton and the activation of signaling pathways. The noted association between the overexpression of DDX18 and suboptimal survival rates further validates its prognostic significance, corroborating its relevance in the context of other malignancies.
At the mechanistic level, the interaction between DDX18 and REXO4 elucidates a heretofore unappreciated pathway in the pathogenesis of HCC. REXO4, an ortholog of the RNA exonuclease 4 family, has garnered prominence as a pivotal molecule in the sphere of cancer biology. Recent scholarly inquiries have demonstrated that REXO4 is frequently overexpressed in a multitude of cancer types, potentially aiding in the onset and progression of neoplasms (21). The mechanistic dissection of REXO4’s role discloses its participation in the governance of RNA metabolism, thus affecting fundamental cellular processes including proliferation, apoptosis, and responses to stress. This suggests that REXO4 may enhance the viability and proliferation of cancer cells by manipulating the stability and degradation of particular mRNAs implicated in oncogenic signaling cascades (22,23).
The findings from our study intimate that DDX18 orchestrates the recruitment of REXO4 to modulate the expression of EMT indicators and the phosphorylation of MAPK pathways (ERK/JNK), uncovering a bipartite regulatory framework. This finding is in concordance with recent evidence associating RNA helicases with EMT, such as the involvement of DDX3X in TGF-β signaling (24), while uniquely positioning DDX18 due to its reliance on REXO4. Nevertheless, certain limitations persist. Initially, although our in vivo models confirm the pro-tumorigenic influence of DDX18, the therapeutic implications of targeting the DDX18/REXO4 axis in clinical scenarios necessitate additional investigation. Furthermore, the upstream controllers of DDX18 in HCC, including epigenetic or post-translational modifiers, have yet to be delineated. Notably, the nuclear functions of DDX18 in phase separation hint at potential interactions with chromatin remodeling complexes (11), an area ripe for subsequent research. Finally, the efficacy of combining DDX18 inhibitors with MAPK pathway inhibitors, such as MEK inhibitors, for therapeutic purposes deserves preclinical assessment.
Conclusions
In summary, this study identifies DDX18 as a critical oncogene in HCC, promoting tumor growth and metastasis via REXO4-mediated activation of the EMT and MAPK signaling pathways. The DDX18/REXO4 axis represents a novel molecular mechanism that underlies HCC aggressiveness, with significant implications for both prognosis and therapeutic strategies. Our findings not only enhance the understanding of RNA helicases in cancer biology but also provide a compelling rationale for targeting DDX18 or its downstream effectors in HCC treatment. Future research should investigate the translational potential of DDX18 inhibitors and their synergistic effects with existing therapies to improve clinical outcomes for HCC patients.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
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. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by ethics committee of the Affiliated Hospital of Nantong University (No. 2025-L107) and informed consent was taken from all the patients. Animal experiments were performed under a project license (No. P20250226-065) approved by Animal Ethics Committee of Nantong University, in compliance with Nantong University guidelines for the care and use of animals.
Footnotes
Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-339/rc
Funding: This research was supported by the the Research Project of the Affiliated Hospital of Nantong University (to X.Y.) (No. Tfh2313) and the Science and Technology Projects of Yancheng (to X.X.) (No. YCBE202417).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-339/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-339/dss
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