LncRNA938/ TAF9/TTK axis promotes EMT and serves as a therapeutic target in hepatoblastoma

Abstract

Background

Hepatoblastoma (HB) is the most common malignant liver tumor in children. The expression of TAF9 is frequently upregulated in HB; however, its underlying molecular mechanisms are not yet fully understood, and its potential as a therapeutic target warrants further investigation.

Methods

Bioinformatic analysis was performed using sequencing datas to evaluate clinical diagnostic and prognostic values of molecules. Biological functions were assessed using in vitro and in vivo experiments. Various techniques, including quantitative PCR, western blotting, immunohistochemistry, RNA immunoprecipitation, RNA pull-down, immunofluorescence, and luciferase reporter assays, were used to investigate the underlying molecular mechanisms.

Results

TAF9 was significantly overexpressed in HB tissues and correlated with poor prognosis. Both lncRNA938 and TAF9 promoted HB proliferation and metastasis. Mechanistically, lncRNA938 directly bound TAF9 and regulated its nuclear localization, while TAF9 activated TTK transcription via promoter binding. TTK inhibitors effectively reversed the epithelial-mesenchymal transition and malignant phenotypes induced by TAF9 overexpression.

Conclusion

The lncRNA938–TAF9–TTK axis is a critical driver of HB progression. Targeting this axis, particularly through TTK inhibition, represents a novel therapeutic strategy against HB.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-06809-4.

Introduction

Hepatoblastoma (HB) is a common malignant liver tumor in children, primarily affecting those aged 0–4 years [1–3]. Its global incidence ranges from 2.09 to 28.2 per 100,000 individuals. Recent advancements in treatment strategies have significantly improved the prognosis of HB [4–7]. However, challenges continue to complicate its management [8], as HB exhibits various pathological types and clinical presentations closely associated with its molecular diversity [9–11]. Consequently, investigating the molecular mechanisms underlying its progression and corresponding intervention strategies are essential.

Transcription factor II D (TFIID) is a core promoter recognition factor that is critical for the initiation of transcription by eukaryotic RNA polymerase II [12]. The protein recognizes core promoter sequences and adjacent chromatin modifications and interacts with gene-specific activators and repressors [13–15]. The TFIID complex comprises TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs) [16]. TAF9 is a crucial component of TFIID [17, 18] that plays a significant role in various processes [19–23] and protective responses after injury [24, 25]. TAF9 is also closely associated with the occurrence and progression of malignancies [26–28]. However, studies examining the role of TAF9 in HB are currently lacking. As such, exploring the molecular mechanisms of TAF9 and identifying viable diagnostic and therapeutic targets hold significant clinical and scientific value.

Dysregulation of the cell cycle is a critical mechanism underlying the onset and progression of malignant tumors [29–31]. TTK plays an indispensable role in mitosis and has garnered significant attention because of its overexpression in various malignancies [32, 33]. Overexpression of TTK disrupts the balance between cell cycle checkpoints and chromosome stability [34]. For instance, TTK enhances the expression of the E1A binding protein p300, which increases glycolysis and induces metabolic reprogramming, subsequently affecting the invasiveness of pancreatic ductal adenocarcinoma [35]. Elevated TTK expression has also been observed in the tumor tissues of patients with colorectal adenocarcinoma [36]. Although TTK has demonstrated oncogenic effects in various cancers, its role in HB remains poorly understood. This knowledge gap underscores the need for further investigation to reveal the potential role of TTK in the pathogenesis, prognosis, and therapeutic strategies of HB.

LncRNAs can regulate protein stability, modulate gene expression, and act as molecular sponges to regulate miRNAs, thereby influencing HB cell proliferation, migration, invasion, and chemotherapy resistance. For example, HOXA-AS2 is upregulated in HB, and its expression is regulated by the chromatin remodeling factor ARID1B and the transcriptional co-activator SUB1, which protects HOXA3 from degradation, thus positively regulating HOXA3 and promoting malignant biological behaviors in HB [37]. TUG1 is upregulated in HB, and its knockdown suppresses tumor growth and angiogenesis, highlighting its crucial role in HB progression and its potential as a therapeutic target [38]. LncRNA938 is a poorly understood lncRNA in terms of its expression, localization, and function in tumors. Therefore, in this study, we aimed to explore the detailed functions and molecular mechanisms of action associated with the LncRNA938/TAF9/TTK axis in HB.

Materials and methods

Gene expression analysis

RNA sequencing data from tumor and adjacent non-tumor tissues of patients with HB were retrieved from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/). Gene expression and correlation analyses were performed using the GSE104766 dataset, which includes 30 tumor tissues and 30 non-tumor samples, and the GSE133039 dataset, which includes 32 tumor tissues and 32 non-tumor samples [39, 40]. Prognostic data from the GSE75271 dataset were used for survival analysis [41].

Patients

Paired tumor and adjacent tissue samples were obtained from pediatric patients diagnosed with PRETEXT III HB who underwent surgical resection at Qingdao University Affiliated Hospital between 2022 and 2023. Before resection, all patients received neoadjuvant chemotherapy consisting primarily of cisplatin. This study was approved by the Ethics Committee of Qingdao University Affiliated Hospital (Approval No. QYFYWZLL29101). Informed consent was obtained from the parents of all children included in this study.

Cell transfection and lentiviral infection

Both blank and overexpression plasmids of TAF9 were obtained from GeneChem (Shanghai, China). TAF9 overexpression was achieved by transfecting cells with plasmids using Lipofectamine 3000 (Invitrogen, USA) to form liposomes. The three small interfering RNA (siRNA) sequences used for siRNA knockdown were sourced from GenePharma (Shanghai, China), and their sequences are shown in Table S1. siRNA was transfected into cells using Xfect™ RNA Transfection (Takara, Kyoto, Japan), and the knockdown efficiency was assessed 24 h post-transfection, followed by relevant experiments. Lentiviruses for TAF9 overexpression and lncRNA938 knockdown were purchased from GeneChem. HuH6 and HepG2 cell lines were transduced with lentiviruses, and stable cell lines with TAF9 overexpression and lncRNA knockdown were obtained after selection with puromycin for 48 h.

Cell viability and colony formation assays

Cell viability was measured using a Cell Counting Kit(CCK)-8 (MeilunBio #MA0218-5, Liaoning, China). HuH6 and HepG2 cells were seeded into 96-well plates at 1,500 and 2,000 cells per well, respectively. After allowing HuH6 cells to adhere for 8 h, the initial absorbance was measured, followed by measurements every 24 h for a total of five assessments. The CCK-8 solution was diluted 1:9 using DMEM, added to the wells, and incubated at 37 °C for 100 min before measuring the absorbance at 450 nm. For colony formation assays, 1,000 cells from each cell line were evenly plated in 6-well plates and cultured at 37 °C with 5% carbon dioxide, with the medium changed every 3 days. After 14 days, the colonies were stained with 0.1% crystal violet in methanol for 30 min, photographed, and counted.

Wound healing assay

Cells from different groups were seeded at 80% confluence in 6-well plates. After adherence, a straight line was scratched in each well using a 200-µL pipette tip. The cells were washed twice with phosphate-buffered saline (PBS) and then switched to a serum-free medium. Time zero was marked as the point of medium change. Images were captured at 0 and 48 h post-scratch. ImageJ was used to analyze the images, determine the cell migration area, and calculate the migration rate.

Cell migration and invasion assays

Processed cells from different groups were counted and resuspended in serum-free medium before being added to the upper chambers of the Transwell inserts. Complete medium was added to the lower chamber. For migration assays, 300 µL of cell suspension was used, with 100,000 HuH6 cells and 150,000 HepG2 cells in separate experiments. For invasion assays, 100 µL of a 1:8 mixture of Matrigel (BD Biosciences, USA) and serum-free medium was added to the upper chamber and allowed to solidify at 37 °C. After solidification, 200 µL of the cell suspension was added. The cells were incubated for 24–48 h and then stained with 0.1% crystal violet in methanol for 30 min. Three random regions of the inserts were imaged and quantified to evaluate cell migration and invasion.

Xenograft tumor model

Stable TAF9-overexpressing HuH6 cells and control cells (1,000,000 cells each) were subcutaneously injected into the backs of 5-week-old BALB/c nude mice (five mice per group) to establish xenograft tumor models (Beijing Vital Laboratory Animal Technology Co., Ltd., Beijing, China). The tumor size was measured every 3 days. After 4 weeks, the mice were euthanized, and the tumor tissues were embedded in paraffin and sectioned. The tumor volume was calculated using the following formula: volume (mm³) = length × width² × 0.5. This study was approved by the Animal Ethics Committee of Qingdao University Affiliated Hospital (Approval No.: AHQU-MAL20240322JC).

Quantitative reverse transcription polymerase chain reaction (qPCR)

RNA was extracted from paired tumor and adjacent tissue samples and treated cell lines using RNA Release Easy (Vayzme #027E228KA, Nanjing, China). First-strand complementary DNA was synthesized using a reverse transcription kit (Vayzme, #7E782J3). The mRNA expression was measured using qPCR on a Roche Light Cycler 480II system using the SYBR Green I method (Vayzme #Q711-02). GAPDH served as an internal control for the normalization of CT values. The primers were synthesized by Beijing Genomics Institution (Shenzhen, China), and their sequences are shown in Table S2.

Western blotting

Total protein was extracted using radioimmunoprecipitation assay (RIPA) buffer, phenylmethylsulfonyl fluoride, and 100X PIC (Beyotime) at a 100:1:1 ratio. Nuclear and cytoplasmic proteins were isolated using a Nuclear and Cytoplasmic Protein Extraction Kit (Absin# abs9346) according to the manufacturer’s instructions. Protein quantification was performed using a BCA kit (Beyotime #P0010S). Subsequently, 30 µg of protein were used for sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), separated at 120 V for 60 min, and transferred to a polyvinylidene fluoride membrane (Cytiva Amersham #10600023). The membrane was blocked using 5% non-fat milk for 2 h at room temperature and incubated overnight at 4 °C with the primary antibody. The cells were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Proteintech#RGAR001/RGAM001, 1:10,000) for 2 h. Detection was performed using ECL Western Blotting Substrate (Affinity #KF005, USA). The band intensity was analyzed with ImageJ, using β-actin as the internal control. The primary antibodies were E-cadherin (Cell Signaling Technology #3195, 1:1,000), N-cadherin (Cell Signaling Technology #13116, 1:1,000), Proliferating Cell Nuclear Antigen (PCNA) (Cell Signaling Technology #13110, 1:1,000), TAF9 (Proteintech #10544-1-AP, 1:1,000), TTK (Proteintech #10381-1-AP, 1:1,000), and β-actin (Proteintech #20536-1-AP, 1:1,000).

RNA pulldown

RNA Pulldown was performed by Genechem using Huh7 cell line. Biotin-labeled lncRNA938 (Genechem) was incubated overnight at 4 °C with streptavidin magnetic beads (Invitrogen #15942-050). After lysing cells with RIPA buffer, the lysate was mixed with the bead-RNA complex and incubated at 18–24 °C for 1 h. The beads were washed, and SDS-PAGE loading buffer (Beyotime #P0015L, Shanghai, China) was added. Samples were denatured at 95 °C for 10 min, followed by SDS-PAGE. Protein bands were stained using a silver staining kit (Beyotime #P0017S) for mass spectrometry analysis (Genechem).

RNA-binding protein immunoprecipitation (RIP)

RIP was performed using an RNA-binding protein immunoprecipitation kit (Genecreate #JKR23003, Wuhan, China). HuH6 cells were lysed in complete RNA immunoprecipitation (RIP) Lysis Buffer. IgG (Cell Signaling Technology, #2729) and Flag (Cell Signaling Technology, #D6W5B) antibodies were incubated with the magnetic beads at room temperature for 2 h. After incubation, protein lysate was added, and the mixture was incubated overnight at 4 °C. Following washing of the beads, one-third of the bead suspension was mixed with SDS loading buffer and denatured at 95 °C for 10 min for SDS-PAGE and subsequent western blotting to detect TAF9. The remaining two-thirds of the beads were treated with TRIzol reagent to extract RNA, which was then analyzed by RT-qPCR to measure lncRNA938 levels. The results were calculated as follows: fold enrichment = 2^(-ΔΔCt) (using IgG as a reference).

RNA Fluorescence In Situ Hybridization (FISH).

FISH was performed using an RNA FISH kit (GenePharma, China). Briefly, cells were plated on coverslips at a density of 60% and fixed with 4% paraformaldehyde after attachment. The lncRNA938 RNA probe was denatured at 75 °C for 10 min. A reaction mixture was prepared with 1 µL of 1 µM biotin probe, 2 µL of 1 µM SA-FITC, and 7 µL of PBS and then incubated at 37 °C for 30 min. The resulting mixture was added to the cells and incubated at 37 °C for 12 h. The excess probe was washed away, and 4′,6-diamidino-2-phenylindole staining was performed. The images were captured using laser scanning confocal microscopy and merged using ImageJ software.

Luciferase reporter assay

The TTK promoter sequence was inserted into a pGL4.10 vector containing a luciferase tag to construct a TTK reporter plasmid. HB cell lines were co-transfected with the TTK reporter plasmid and either the TAF9 overexpression plasmid or an empty vector as a control. The cells were collected and lysed 36 h post-transfection. Firefly luciferase activity was measured using a Luciferase Reporter Gene Assay Kit (11401ES60, Yeasen) according to the manufacturer’s instructions, and the luminescence values were calculated.

Immunohistochemistry

Paraffin-embedded tumor sections from subcutaneous tumors of nude mice were deparaffinized in xylene and rehydrated using a series of ethanol washes (100%, 95%, 90%, and 85% ethanol). Antigen retrieval was performed using citrate buffer (pH 6). The sections were then treated with specific antibodies against N-cadherin, E-cadherin, PCNA, Ki67, TTK, and TAF9. Endogenous peroxidase activity was blocked, and sections were incubated overnight at 4 °C. Subsequently, a polymerized HRP-conjugated secondary antibody (ZSGB-Bio #MA2522, Beijing, China) was added and incubated at 25 °C for 2 h. The reaction products were developed using 3,3’-diaminobenzidine and counterstained with hematoxylin. Finally, the slides were observed under a microscope.

Immunofluorescence.

HB cells were seeded in 24-well plates at 30% confluency and fixed with 4% paraformaldehyde for 24–48 h. Antigen retrieval was performed using Tris-EDTA buffer at 95 °C for 10 min. After washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 10 min and blocked for 30 min. The primary antibody (1:300) was applied for 1 h, followed by the FITC-conjugated secondary antibody for 1 h. The nuclei were stained with 0.5 µg/mL DAPI for 10 min. PBS washes (3 times, 5 min each) were performed between steps. Images were acquired by fluorescence microscopy, and channels were merged using ImageJ.

Statistical analysis

All experimental data were analyzed using GraphPad Prism 10 and are presented as the mean ± standard deviation. Kaplan–Meier survival analysis was performed. The patient groups were compared using chi-squared tests and t tests, with statistical significance defined as P < 0.05.

Results

TAF9 is overexpressed in HB and correlates with poor prognosis

We analyzed TAF expression in the GEO database and found that most TAFs were upregulated in HB (Fig. 1A, B). Prognostic analysis (Figure S1A–F, Fig. 1E) revealed that TAF2, TAF3, TAF4, TAF9, TAF11, TAF12, and TAF15 had hazard ratios > 1, identifying them as high-risk factors associated with poor survival. Among all TAFs, TAF9 is a target of various oncogenic factors; however, its direct oncogenic role in HB remains unclear. We found that TAF9 was highly expressed and correlated with poor survival in HB (Fig. 1C–E) and thus selected TAF9 for further investigation in subsequent validation experiments.

Fig. 1.

TAF9 is overexpressed in hepatoblastoma and correlates with poor prognosis. (A, B) Expression levels of TAF in hepatoblastoma tissues compared to normal tissues in the Gene Series Expression dataset (GSE104766 and GSE133039). (C, D) Differential expression of TAF9 in tumor and normal tissues. (E) Kaplan–Meier survival analysis showing worse prognosis for patients with high TAF9 expression. (F, G) Comparison of TAF9 mRNA and protein levels in clinical specimens (tumor and adjacent non-tumor tissues). (H) Analysis of TAF9 levels in hepatoblastoma and normal liver cell lines. (I–L) Validation of TAF9 knockdown efficiency at the mRNA and protein levels in hepatoblastoma cell lines

To confirm these findings, we measured TAF9 mRNA and protein levels in paired HB tumors and adjacent liver samples. The results revealed a significant increase in TAF9 expression in tumor tissues (Fig. 1F, G). Receiver operating characteristic curve analysis identified TAF9 as a promising diagnostic marker for HB (Figure S1G, H). Comparative analysis of TAF9 transcription levels between the normal liver cell line (7702) and HB cell lines revealed marked overexpression in HB cells (Fig. 1H). Taken together, these results indicate that TAF9 is highly expressed in HB and is associated with poor prognosis.

TAF9 promotes growth and metastasis in HB

To investigate the role and mechanism of TAF9 in HB, its expression was regulated using overexpression plasmids and siRNA knockdown in HB cell lines. After transfection, TAF9 expression levels were validated using qPCR (Fig. 1I, J) and western blotting (Fig. 1K, L), confirming successful overexpression and knockdown.

Functional assays were performed to assess the effects of TAF9 modulation on HuH6 and HepG2 cell lines. CCK-8 and colony formation assays demonstrated that TAF9 knockdown significantly inhibited cell growth (Fig. 2A–D), whereas TAF9 overexpression markedly enhanced cell proliferation (Figure S1I–L). Wound healing and Transwell assays showed that TAF9 knockdown suppressed migration and invasion capabilities (Fig. 2E, F), whereas overexpression promoted these abilities (Figure S1M, N; Figure S2A, B).

Fig. 2.

TAF9 promotes growth and metastasis in hepatoblastoma. (A, B) Colony formation assays demonstrating reduced cell proliferation following TAF9 knockdown. (C, D) Cell Counting Kit-8 assays evaluating the effects of TAF9 knockdown on cell proliferation. (E) Wound healing assays assessing the migratory capacity of cells with TAF9 knockdown. (F) Transwell assays quantifying cell invasion and migration. (G) Western blotting analysis of E-cadherin, N-cadherin, and PCNA protein levels after TAF9 knockdown. (H) Subcutaneous tumor growth in mice with TAF9 overexpression. (I) Tumor volume and size comparisons in mice with TAF9 overexpression. (J) Immunohistochemical staining of Ki-67, PCNA, E-cadherin, and N-cadherin in tumor tissues from mice

PCNA is a key marker of tumor cell proliferation. Consistent with the observed phenotypic changes in proliferation, PCNA protein levels decreased following TAF9 knockdown (Fig. 2G) and increased with TAF9 overexpression (Figure S2C, D).

The epithelial-mesenchymal transition (EMT) was also examined. TAF9 knockdown increased E-cadherin levels and reduced N-cadherin expression (Fig. 2G), consistent with the results of functional assays. TAF9 overexpression yielded opposite trends, consistent with its role in promoting EMT (Figure S2C, D).

Subsequently, a subcutaneous xenograft model was established using BALB/c nude mice. The results showed that TAF9 overexpression significantly increased the tumor volume and weight, suggesting that TAF9 plays a critical role in tumor growth (Fig. 2H–I). Immunohistochemical staining revealed that tumors overexpressing TAF9 exhibited high expression of TAF9, Ki-67, PCNA, and N-cadherin, whereas E-cadherin levels were lower than those in the control group (Fig. 2J). These findings indicate that TAF9 plays a pivotal role in HB growth and metastasis.

TAF9 activates TTK transcription by binding directly to its promoter sequence

To further explore the downstream targets of TAF9, high-throughput RNA sequencing was performed following TAF9 overexpression, revealing 1,945 differentially expressed genes (Fig. 3A, B). Based on the correlation analysis between TAF9 expression and its clinical relevance, TTK was selected as a potential downstream target for further validation. TTK is a critical component of the spindle assembly checkpoint during mitosis and is closely associated with malignant tumor proliferation. Notably, TTK inhibitors have been approved for clinical trials for various malignancies.

Fig. 3.

TAF9 activates TTK transcription by binding directly to its promoter sequence. (A, B) Volcano plot and heatmap of differentially expressed genes following TAF9 overexpression based on RNA sequencing. (C, D) Correlation between TAF9 and TTK expression in tumor and normal tissues. (E) Comparison of TTK protein levels in clinical specimens (tumor and adjacent non-tumor tissues). (F) Effects of TAF9 modulation on TTK protein levels in hepatoblastoma cells. (G, H) qPCR results showing the effects of TAF9 overexpression and knockdown on TTK expression in hepatoblastoma cell lines. (I) Luciferase reporter assay demonstrating activation of the TTK promoter region by TAF9 overexpression. (J) Cell Counting Kit-8 assay demonstrating the proliferation effects of TAF9 overexpression and TTK inhibitor treatment in hepatoblastoma cell lines. (K, L) Effects of TAF9 overexpression and TTK inhibitor treatment on cell proliferation and migration

Sequencing data revealed TTK was highly found to be expressed in HB (Figure S2E) and a positive correlation between TTK and TAF9 (Fig. 3C, D). Furthermore, TAF9 expression was higher in HuH6 cells than in HepG2 cells, with TTK expression showing a similar trend (Figure S2F). Further analysis of eight paired fresh clinical tissue samples revealed significantly higher TTK expression in tumor tissues (Fig. 3E).

In both HB cell lines, TAF9 significantly regulated TTK expression at both mRNA and protein levels (Fig. 3F–H). TAF9 directly modulates TTK expression at the transcriptional level. Next, a luciferase reporter assay was performed to determine whether TAF9 directly regulates TTK transcription through its promoter. The results showed that TTK promoter activity was significantly activated in TAF9-overexpressing cells, whereas no activation was observed in the control reporter group (Fig. 3I). These findings indicate that TAF9 promotes TTK transcription by directly activating its promoter sequence. To investigate the role and mechanism of TTK in TAF9 pathway, its expression was regulated using siRNA knockdown in HB cell lines. After transfection, TTK expression levels were validated using qPCR (Figure S2G), confirming successful knockdown. We selected si-TTK#1 for subsequent experiments, referred to it as si-TTK, and confirmed its knockdown efficiency at the protein level (Figure S2H). Genetic rescue was achieved by TAF9 overexpression combined with TTK knockdown (Figure S2I; Figure S3A, B). CCK-8 and colony formation assays showed that si-TTK reversed TAF9-induced proliferation (Figure S3C-F), whereas wound healing and Transwell assays demonstrated that TTK inhibition suppressed migration and invasion (Figure S3G, H; Figure S4A). Subsequently, we used a TTK inhibitor (CFI-402257, TargetMol #1610759-22-2) as a therapeutic agent for TAF9 overexpression. The GI50 values for the TTK inhibitor were determined in the two HB cell lines, showing values of 414.6 nM for HuH6 and 713.2 nM for HepG2 (Figure S4B). We also found that TTK expression was significantly higher in Huh6 cells than in HepG2 (Figure S2F; Figure S4C).

A low-dose TTK inhibitor (100 nM) was added to TAF9-overexpressing cell lines to assess its ability to block the oncogenic effects of TAF9. CCK-8 and colony formation assays demonstrated that the TTK inhibitor reversed the proliferation enhancement induced by TAF9 overexpression (Fig. 3J, K). Furthermore, wound healing and Transwell assays indicated that the enhanced migration and invasion capabilities were suppressed by the TTK inhibitor (Figs. 3L and 4A). We validated the expression of proliferation-, migration-, and invasion-related proteins, which showed a trend toward recovery (Fig. 4B).

Fig. 4.

Oncogenic effects of TAF9 are inhibited by TTK inhibitors. (A) Transwell assay demonstrating the effects of TAF9 overexpression and TTK inhibitor treatment on cell migration and invasion. (B) Western blotting analysis showing changes in E-cadherin, N-cadherin, and PCNA protein levels after TAF9 overexpression and TTK inhibitor treatment. (C) Subcutaneous tumor growth in mice overexpressing TAF9 treated with TTK inhibitors. (D) Comparison of tumor volume and size across different treatment groups. (E) Immunohistochemical staining of Ki-67, N-cadherin and PCNA expression in tumor tissues from treated mice

The effects of the TTK inhibitor were validated in vivo. TAF9-overexpressing HuH6 stable cell lines and control cells were subcutaneously injected into nude mice, and tumor formation was observed after 10 days. Mice bearing TAF9-overexpressing tumors were treated with control (10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline) or TTK inhibitor (6 mg/kg/day) via gavage. After 2 weeks, the mice were euthanized, the tumors were collected and weighed, and the tumor volume was measured. The TTK inhibitor inhibited tumor progression induced by TAF9 overexpression in vivo (P < 0.05) (Fig. 4C, D).

Immunohistochemical staining of tumors indicated that TAF9, Ki-67, N-cadherin, and PCNA levels were significantly higher in the TAF9-overexpression group compared to the control group. In contrast, the protein levels in the TAF9 overexpression group treated with the TTK inhibitor were reversed, resembling those in the control group (Fig. 4E). These results confirmed the oncogenic role of TAF9 in HB and highlight the potential of targeting TTK as a therapeutic strategy.

lncRNA938 interacts with TAF9 and increases the nuclear localization of TAF9

To further investigate the direct molecular targets of TAF9, we used the catRAPID database (http://s.tartaglialab.com/page/catrapid_group) to predict lncRNAs that potentially interact with TAF9 and selected five candidates for further validation (Figure S4D). We then performed RIP assays for TAF9, followed by qPCR analysis of the five lncRNAs. Among them, only lncRNA938 was successfully pulled down (Fig. 5A, Figure S4D). Additionally, RNA pulldown assays combined with mass spectrometry analysis demonstrated an interaction between lncRNA938 and TAF9 (Fig. 5B, C).

Fig. 5.

lncRNA938 interacts with TAF9 and increases nuclear localization of TAF9. (A) RIP assay followed by qPCR analysis to evaluate the binding of TAF9 and IgG to lncRNA938 in hepatoblastoma cell lines. (B) RNA immunoprecipitation showing lncRNA938 binding with proteins. (C) Venn diagram showing the results of the pulldown proteomics for the sense and antisense strands. (D) Comparison of lncRNA938 levels in clinical specimens. (E) Fluorescence in situ hybridization experiments showing the subcellular localization of lncRNA938. (F) Quantitative polymerase chain reaction results verifying the efficiency of lncRNA938 knockdown in hepatoblastoma cell lines. (G) Expression of TAF9 following lncRNA938 knockdown. (H, I) Expression of TTK following lncRNA938 knockdown. (J, K) Nuclear and cytoplasmic TAF9 protein expression after lncRNA938 knockdown. (L–N) Effects of lnc938 knockdown on proliferation, migration, and invasion. (O, P) Western blotting analysis showing changes in E-cadherin, N-cadherin, PCNA, and TTK protein levels after lncRNA938 knockdown

qPCR analysis of clinical samples indicated that the expression levels of lncRNA938 were significantly higher in HB tumors than in adjacent non-tumor tissues (Fig. 5D). To the best of our knowledge, the subcellular localization and functional role of lncRNA938 have not been explored. FISH experiments were performed to localize lncRNA938 within HB cells, revealing that it predominantly resides in the cytoplasm (Fig. 5E). Lentiviral vectors for lncRNA knockdown were constructed and transfected into HB cell lines (Fig. 5F). The knockdown of lncRNA938 did not result in changes in TAF9 levels (Fig. 5G). Further modulation of TAF9 expression revealed no significant changes in lncRNA levels.

Previously, we identified TTK as a downstream target of TAF9, and upon lncRNA knockdown, reduced TTK expression was observed (Fig. 5H, I). Therefore, we hypothesized that lncRNA938 regulates the transcriptional function of TAF9. TAF9 is primarily localized in the nucleus, and we speculated that lncRNA938 is involved in the subcellular localization of TAF9. To test this hypothesis, we examined the expression of TAF9 in the nucleus and cytoplasm following lncRNA938 knockdown. The results illustrated that lncRNA938 knockdown led to a decrease in TAF9 levels in the nucleus and an increase in its cytoplasmic levels (Fig. 5J, K), suggesting that lncRNA938 regulates the nuclear levels of TAF9. Immunofluorescence experiments further supported this conclusion (Figure S4F).

In terms of whether lncRNA938 is associated with tumor activity in HB, CCK-8 assays and colony formation experiments demonstrated that lncRNA538 knockdown inhibited the proliferation, migration, and invasion abilities of HB cell lines (Fig. 5L–N). After lncRNA knockdown, the expression of cell function-related proteins showed trends consistent with the observed changes in cell function (Fig. 5O, P). To rule out off-target effects, we successfully knocked down lncRNA938 using a targeted two siRNA sequences in HB cell lines (Figure S5A). We chose si-lncRNA938#1 for subsequent experiments, referred to it as si-lncRNA938, and performed functional assays, yielding consistent results (Figure S5B-H).These results suggest that lncRNA938 plays a significant role in HB progression.

lncRNA938 enhances HB progression via TAF9

To further validate the regulatory role of lncRNA938 in TAF9 function, rescue experiments were conducted by knocking down lncRNA938 while simultaneously overexpressing TAF9. The efficiency of lncRNA knockdown and TAF9 overexpression was verified (Fig. 6A). TAF9 overexpression restored the malignant phenotype, which was suppressed by lncRNA938 knockdown (Fig. 6B–E). The detection of proliferation-, migration-, and invasion-related proteins further supported these findings (Fig. 6F, G).

Fig. 6.

lncRNA938 enhances hepatoblastoma progression via TAF9. (A) Western blot analysis showing the effects of lncRNA938 knockdown combined with TAF9 overexpression. (B) Cell Counting Kit-8 assay demonstrating the impact on cell proliferation. (C–E) Effects of lncRNA938 knockdown combined with TAF9 overexpression on proliferation, migration, and invasion. (F, G) Western blotting analysis of E-cadherin, N-cadherin, PCNA, and TTK protein levels following lncRNA938 knockdown combined with TAF9 overexpression. (F) Schematic diagram of the mechanism

These experiments indicate that lncRNA938 exerts oncogenic effects on HB by regulating the nuclear localization of TAF9.

Discussion

The role of transcription factors in HB has attracted significant attention, particularly in high-risk patients who experience postoperative recurrence and for whom further surgery or treatment is not feasible. Moreover, these patients often exhibit poor responses to current drugs and targeted therapies. Therefore, exploring new therapeutic targets for HB is crucial for improving treatment outcomes.

In this study, we found that TAF9 is overexpressed in HB and is associated with poor prognosis. TAF9 plays an oncogenic role in HB by directly activating the transcription of its downstream target TTK. The TTK inhibitor CFI-402,257 effectively suppressed the oncogenic effects of TAF9 in vitro and in vivo. Additionally, lncRNA938 promoted carcinogenesis by increasing the nuclear localization of TAF9. The lncRNA938–TAF9–TTK axis contributes to HB progression and represents a novel therapeutic strategy.

TAF9 is closely associated with increased transcriptional activity in tumor cells. TAF9 binds to the oncogene GLI1, enhancing cellular transcriptional activity [26, 27]. However, this binding is less efficient than the interaction between TAF9 and the tumor suppressor gene TP53. In tumors with TP53 mutations, this suppression is ineffective and leads to elevated malignant transcriptional activity [26]. Additionally, the novel synthetic small molecule FN1-8 disrupts the interaction between GLI1 and TAF9, downregulating GLI1/TAF9-dependent transcriptional activity [27]. Moreover, the conserved region of HCA587/MAGEC2 binds to TAF9 in the nucleus, contributing to its oncogenic effects [28]. In this study, we found that TAF9 was highly expressed in HB tissues and associated with poor prognosis. Modulation of TAF9 expression in HB cell lines revealed that its overexpression significantly promoted HB cell proliferation, migration, and invasion. These findings confirmed that TAF9 plays a crucial oncogenic role in HB.

TTK has garnered significant attention in cancer drug development because of its crucial role in regulating mitosis and its upregulation in various tumors [42, 43]. Several TTK inhibitors have been approved for clinical trials aimed at treating advanced malignant solid tumors [34, 36, 44]. In a study focusing on highly aggressive multiple myeloma (MM), TTK inhibitors reduced MM proliferation and viability, induced DNA damage, and caused severe defects in chromosome alignment and separation, leading to aneuploidy and apoptosis [45]. Notably, TTK inhibitors have demonstrated significant antitumor activity in melanoma, colon cancer, breast cancer cells, and in organoids derived from patients with melanoma [46]. Additionally, programmed cell death 1 (PD-1)/programmed death-ligand 1 immune checkpoint activation has been observed in tumor-bearing mice treated with TTK inhibitors. This effect was further enhanced by combining a low-toxicity dose of OSU13 with anti-PD-1 checkpoint blockade, resulting in substantial STING- and CD8 + T cell-dependent tumor suppression [46]. In this study, a significant correlation was observed between TTK expression and TAF9 in clinical HB samples. Furthermore, modulation of TAF9 expression resulted in corresponding changes in TTK levels. Subsequently, luciferase reporter assays revealed that TAF9 directly activated the TTK promoter sequence, promoting its transcription. This suggests that TTK is a downstream target of the oncogenic effects of TAF9. The application of TTK inhibitors in a TAF9 overexpression model demonstrated that the oncogenic effects of TAF9 were effectively suppressed. These findings indicate that TTK inhibitors represent promising targeted therapies for HB by disrupting the TAF9-TTK axis.

Although TTK inhibitors suppress the carcinogenic activity of TAF9 in vitro and in vivo, their in vivo effects are weaker, a phenomenon also observed in pancreatic and ovarian cancer studies [47, 48]. This may be due to factors such as drug pharmacokinetics, metabolic stability, and the tumor microenvironment [49, 50]. Further research is required to assess the clinical potential of TTK inhibitors in the treatment of HB.

LncRNAs can function as either oncogenes or tumor suppressors [51] and play complex and crucial regulatory roles in tumorigenesis and progression by modulating protein function, subcellular localization, and degradation [52–55]. LncFTO-IT1 directly binds to RBM15 within the METTL3–METTL14–WTAP-RBM15 methyltransferase complex. This binding inhibits the RBM15 interaction, m6A methylation, and stability of the p53 target mRNA [56]. Similarly, lncRNA PWRN1 acts as a tumor suppressor by binding to pyruvate kinase M2 (PKM2), preventing PKM2 from entering the nucleus in its low-activity dimer form [57]. In this study, the novel lncRNA lnc938 was identified as an oncogene in HB. Lnc938 was shown to directly interact with TAF9 and regulate its nuclear localization. The reduction in malignant phenotypes observed after lnc938 knockdown was reversed by TAF9 overexpression. This suggests that lnc938 functions as an upstream regulator of TAF9, promoting its oncogenic effects through modulation of TAF9’s nuclear localization.

In conclusion, TAF9 plays a critical role in the development of HB, with the lncRNA938–TAF9-TTK axis promoting HB proliferation, invasion, and metastasis. Inhibition of TTK can effectively block the activation of this pathway, presenting a potential new therapeutic strategy for the treatment of HB. Nevertheless, the limited number of clinical samples, due to the rarity of HB, represents a major constraint of this study. Ongoing efforts to expand our patient cohort will enable more robust validation and facilitate future translational research.

Electronic supplementary material

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Author contributions

CJ, BD, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft; YX, XC, SW, FD, Writing – review and editing, Investigation, Methodology; CZ, QD, Conceptualization, Data curation, Methodology, Writing – review and editing.

Funding

The study was supported by National Natural Science Foundation of China (Grant No. 82370890), Taishan Scholars Program of Shandong Province (grant number 2019010668 and NO. tsqn202312382), Natural Science Foundation of Shandong Province (grant number ZR2021MH171, ZR2023MH243, ZR2024LZL014), Shandong Higher Education Young Science and Technology Support Program (grant number 2020KJL005 and 2023KJ224).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The use of tumor tissues in this study was approved by the Ethics Committee of Qingdao University Affiliated Hospital (No. QYFYWZLL29101). Additionally, the animal experiments were conducted in accordance with the guidelines and approved by the Animal Ethics Committee of Qingdao University Affiliated Hospital (No. AHQU-MAL20240322JC). All experiments were conducted in accordance with the approved protocol. The maximum tumor size permitted by our ethics committee is a diameter of 20 mm and a total volume of 2000 mm³. These limits were not exceeded during the study.

Consent for publication

Not applicable.

Competing interests

All authors have read the journal’s policy on disclosure of potential conflicts of interest. No potential conflict of interest was reported by the authors. The authors declare that they have no competing interests. All authors read and agree to the journal’s authorship statement.