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Potential mechanism of circKIAA1429 accelerating the progression of hepatocellular carcinoma

Infectious Agents and Cancer volume 20, Article number: 12 (2025) Cite this article

Abstract

Background

This study investigates the underlying mechanism of circKIAA1429 (hsa_circ_0084922) in hepatocellular carcinoma (HCC) progression.

Methods

circKIAA1429, SETD1A, NAP1L3, and GLIS2 expressions in HCC cells were detected by RT-qPCR or western blot. The stability of circKIAA1429 was tested after treatment with actinomycin D and Rnase R enzyme. circKIAA1429 expression was knocked down, followed by detection of cell proliferation, apoptosis, and migration/invasion using CCK-8, flow cytometry, and transwell. RIP and RNA pull-down were performed to validate the binding between circKIAA1429 and SETD1A, while ChIP analysis determined the enrichment of SETD1A and H3K4me3 or H3K27me3 on GLIS2 or NAP1L3 promoter. A nude mouse xenograft tumor model was establish to test the effect of circKIAA1429 on tumorigenicity.

Results

circKIAA1429 and NAP1L3 were highly expressed in HCC cells, while GLIS2 was poorly expressed. Knockdown of circKIAA1429 repressed cell proliferation/invasion/migration and facilitated apoptosis. Mechanistically, circKIAA1429 directly interacted with SETD1A to reduce the enrichment of SETD1A and H3K4me3 or H3K27me3 on GLIS2 or NAP1L3 promoter, thus diminishing GLIS2 expression and elevating NAP1L3 expression. In vivo, circKIAA1429 promotes tumorigenesis via GLIS2/NAP1L3.

Conclusion

circKIAA1429 interacts with SETD1A to inhibit the enrichment of H3K4me3 and H3K27me3 on GLIS2 or NAP1L3 promoter, thus inhibiting/promoting the expression of GLIS2/NAP1L3 and accelerating the progression of HCC.

Introduction

Hepatocellular carcinoma (HCC) as the most prevalent subtype of primary liver cancer in adults is notorious for significant mortality, dismal prognosis, and frequent relapse [1]. According to Global Cancer Incidence, Mortality and Prevalence (GLOBOCAN) 2022, globally HCC occupies the sixth most common cancer and the third most common cause of cancer-related deaths [2, 3]. The generally accepted risk factors for HCC mainly encompass hepatitis B and C virus infection, alcohol consumption, and aflatoxin B uptake, along with some coexisting liver diseases such as non-alcoholic fatty liver diseases [4]. Due to the rapid disease progression and insidious symptoms, approximately 80% of HCC patients have already progressed to advance stages by the time of diagnosis [5]. Despite improvements in surgery, radiofrequency ablation, and chemotherapy, the outcomes of HCC remain grim due to high relapse and metastasis rates [6]. Therefore, a more comprehensive understanding of the pathological mechanism of HCC has pivotal clinical significance.

Classical epigenetic mechanisms including DNA methylation, histone alterations, and non-coding RNAs (ncRNAs) can profoundly affect HCC tumorigenesis and progression [7]. circular RNAs (circRNAs) are newly identified endogenous ncRNA molecules with covalently closed loop structures generated from backsplicing of pre-mRNAs [8]. circRNAs display tissue-specific expression patterns and exert oncogenic or suppressive effects on HCC by modulating a broad range of biological processes [9]. has_circ_0084922 comes from KIAA1429 and named circKIAA1429, is aberrantly elevated in HCC [10]. circKIAA1429 can accelerate HCC advancement through the mechanism of m6A-YTHDF3-Zeb1 [11]. However, the detailed mechanism of circKIAA1429 in HCC development has not been clarified.

Histone methylation is considered a dynamic and reversible histone mark that involves the addition or removal of a methyl group from a lysine or arginine residue [12]. Aberrant activity of histone methylation modifying enzymes has been related to several human malignancies, including HCC [13]. For instance, a genome-wide study has pinpointed that tri-methylation of H3 on lysine 27 (H3K27me3) is frequently present in human HCC cells [14]. A high level of tri-methylation of histone H3 on lysine 4 (H3K4me3) has been associated with a poor prognosis of HCC [15]. SET domain containing protein 1 A (SETD1A) is histone lysine methyltransferase that manipulates transcriptional gene activation through H3K4 methylation [16]. SETD1A overexpression deteriorates malignant phenotypes of various cancers, such as lung cancer [17], pancreatic cancer [18], gastric cancer [19], and ovarian cancer [20]. Particularly, SETD1A is remarkably upregulated in HCC cells and tissues in relative to normal hepatocytes and paracancerous tissues [21]. Knockdown of SETD1A retrains the growth and metastasis of HCC cells [22]. SETD1A also promotes the deposition of H3K4me3 and H3K27me3, thereby activating oncogenes and inactivating tumor suppressor genes in liver cancer stem cells [23]. In this study, we propose a novel insight that circKIAA1429 interacts with SETD1A to accelerate HCC progression. Herein, the present study explores the exact mechanism of circKIAA1429-SETD1A driving HCC progression, thus conferring novel therapeutic targets for HCC.

Materials and methods

Cell culture and treatment

Human immortalized liver cell line (MIHA) and human HCC cell lines (Huh-7, HCC36, HCCLM3, MHCC97-L) were purchased from ATCC and maintained in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% heat inactivated bovine serum (GIBCO, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37℃ with 5% CO2.

Cell transfection and construction of cell lines with stable transfection

Small interfering RNA (siRNA) targeting circKIAA1429 or SETD1A or GLIS family zinc finger protein 2 (GLIS2) and negative control were designed and synthesized by GenePharm (Shanghai, China). The pcDNA3.1 vector overexpressing nucleosome assembly protein 1-like 3 (NAP1L3) was manufactured by Santa Cruz Biotechnology (Santa Cruz, CA, USA). The constructed siRNA or plasmid was transfected into Huh-7 and HCCLM3 cells, respectively, using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). After 48 h, the gene intervention efficiency was validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) or Western blot.

Short hairpin RNA (shRNA) of circKIAA1429 was designed by GeneChem (Shanghai, China) and cloned into lentiviral vector (pAdTrack-CMV, Invitrogen). Huh-7 cells were infected with lentivirus in the presence of Polybrene (Sigma-Aldrich, MO, USA). After 48 h, 2 µ g/mL puromycin was added to the culture medium for screening stable cloned cells.

Cell counting assay

The cell proliferation was measured using CCK-8 assay (Dojindo, Tokyo, Japan). In short, cells (1 × 103) were seeded into a 96-well plate and cultured in an incubator. At designated time points, 10 uL of CCK-8 solution was added and incubated for 2 h at 37 °C. Then the absorbance at 450 nm was analyzed by a microplate reader (Thomas Scientific, NJ, USA).

Apoptotic assay

The Annexin-V/Propidium Iodide apoptosis assay kit (Multiscience, Hangzhou, China) was used to quantify apoptotic cells. The cells were collected, washed with phosphate buffered saline (PBS), and resuspended in 200 µL of binding buffer containing 5 µL of Annexin-V (10 µg/mL) for 10 min. Then the cells were incubated with 10 µL of propidium iodide (PI) (20 µg/mL) and immediately analyzed by a flow cytometer (Epics XL; Beckman Coulter, Brea, CA, USA). CellQuest software was used for data collection and analysis.

Transwell invasion assay

To evaluate cell invasion ability, cells were seeded onto an 8 μm filter membrane pre-coated with Matrigel (Corning, NY, USA) in serum-free medium. The normal culture medium containing 10% fetal bovine serum was placed into the basolateral chamber. One day later, the filter was removed. The cells located in the basolateral chamber were invaded from the filter membrane. The invaded cells were fixed in 4% PFA, stained with 0.1% crystal violet, and then imaged. The detection procedures of migration ability were the same except for the absence of Matrigel.

Actinomycin D and RNase R treatment

Actinomycin D (2 mg/mL; Sigma-Aldrich) was added to the culture medium to block cell transcription. To evaluate the stability of RNA, total RNA extract (2 µg) was treated with RNase R (3 U/µg; BioVision, CA, USA) at 37 °C for 1 h, and then the levels of circKIAA1429 and linear KIAA1429 mRNA were analyzed by RT-qPCR.

RNA immunoprecipitation (RIP) assay

The binding probability of circKIAA1429 and SETD1A was predicted using the RPISeq database (http://pridb.gdcb.iastate.edu/RPISeq/) [24]. RIP was performed using RNA binding protein immunoprecipitation assay kit (Millipore, Bedford, MA, USA). The cells were lysed with buffer solution (50 mM Tris-HCl, 2.5 mM EDTA, 130 mM NaCl, 1% NP-40) supplemented with RNase and protease inhibitor (Thermo Fisher Scientific, Jiangsu, China). The lysate was incubated overnight with primary antibodies SETD1A (ab70378, Abcam, Cambridge, MA, USA) and IgG (ab172730, Abcam) at 4℃. Then the antibody bound sample was incubated with protein A Sepharose (Sigma-Aldrich) at 4℃ for 2 h. The sample was rinsed with washing buffer and then incubated with proteinase K (Sangon, Shanghai, China) for 1.5 h. RNA was extracted from the eluent using TRIzol reagent (Invitrogen) and then subjected to RT-qPCR.

RNA pull down assay

The cells were homogenized in lysis buffer, and the cell lysate was incubated with biotinylated-circKIAA1429 (generated by MEGAshortscript™ T7 kit; Sigma-Aldrich) at 4℃ for 2 h. The sample was added with streptavid-coupled dynabeads (Invitrogen) and incubated for 3 h at 4℃. The sample was washed with washing buffer and the beads were eluted with Laemmli buffer. Western blot was performed on the eluent.

Chromatin immunoprecipitation (ChIP)

Magna ChIP™ G assay kit was used for ChIP analysis. Simply put, the cells were cross-linked with 1% formaldehyde at room temperature for 10 min, and then the chromatin was sheared by ultrasound. Immunoprecipitation of chromatin-protein complexes was performed using anti-ChIP antibodies SETD1A (ab70378, Abcam), H3K4me3 (ab8580, Abcam), H3K27me3 (ab192985, Abcam), and IgG (ab172730, Abcam). The obtained DNA fragments were used as RT-qPCR templates based on specific primers (Table 1).

Table 1 PCR primer sequences
Full size table

Nude mouse xenograft tumors

All animal experiment schemes were approved by the Animal Ethics Committee of The First People’s Hospital of Tongxiang and implemented based on the Guide for the Care and Use of Laboratory Animals [25]. Twelve 4-week-old nude mice (male, 15–20 g, purchased from Beijing Vital River Laboratory) were randomly assigned to two groups, with six mice in each group. About 107 Huh-7 cells in 200 µL PBS were injected into the right side of nude mice. The tumor volume was measured every 7 days, and the tumor volume was calculated: 0.5 × length × width2. After 4 weeks of injection, mice were euthanized by intraperitoneal injection of 200 mg/kg pentobarbital sodium. Finally, the xenograft tumor tissue was removed for examination.

Immunohistochemistry

The tumor tissue was fixed in 4% formalin, embedded in paraffin, and sliced (4 μm). The tissue sections were baked at 60℃ for 2 h and de-paraffinized with xylene and gradient ethanol. Then, 3% hydrogen peroxide was used to block endogenous peroxidase for 20 min for antigen recovery. The sections were blocked with goat serum to avoid non-specific staining, and then incubated overnight with anti-Ki-67 (ab16667, Abcam), anti-E-cadherin (ab40772, Abcam), and anti-N-cadherin (ab76011, Abcam) at 4℃, followed by incubation with the secondary antibody (Ab205718, Abcam) at room temperature for 2 h. The signal was tested by 2,4-diaminobutyric acid substrate. Immunopositive staining was performed by measuring the proportion of positive cells.

After dewaxing, the sections were incubated with proteinase K working solution, washed with PBS, and then treated with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) reaction mixture at 37℃ in the dark for 60 min. TUNEL detection was performed on all sections according to the instructions of the In Situ BrdU-Red DNA Fragment (TUNEL) Detection Kit (Abcam).

RT-qPCR

The total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed into complementary DNA (cDNA) using Prime Script RT Master Mix (Takara, Dalian, China). Premix EX Taq (Takara) was used for qPCR. The primer sequences are shown in Table 1. The primers were synthesized by Generay (Shanghai, China). The relative expression of genes was calculated by the 2−ΔΔCt method [26], with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference.

Western blot analysis

The proteins were extracted from cells using cell lysate, separated by electrophoresis (10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis), and transferred onto polyvinylidene fluoride membranes. After blocking in 5% skim milk for 2 h, the membrane was incubated with the primary antibodies GLIS2 (PA5-72849, 1:1000, ThermoFisher), NAP1L3 (H00004675-D01P, 1:1000, ThermoFisher), SETD1A (ab70378, 1:10000, Abcam), and β-actin (ab8227, 1:1000, Abcam) at 4℃ overnight, and then treated with the secondary antibody (ab205718, 1:2000, Abcam) at room temperature for 2 h. The signal was detected by the enhanced chemiluminescence system (Bio-Rad, Hercules, CA, USA). The relative expression of proteins was analyzed using Image Pro Plus 6.0 software (Medical Cybernetics, Los Angeles, CA, USA).

Statistical analysis

Data analysis and map plotting were performed using the SPSS 21.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). The data were examined for normal distribution and homogeneity of variance. The t test was adopted for comparisons between two groups, and one-way or two-way analysis of variance (ANOVA) was employed for the comparisons among multiple groups, following Tukey’s multiple comparison test. A value of P < 0.05 indicated a significant difference.

Results

circKIAA1429 expression in HCC cells

To determine the circKIAA1429 expression in HCC, we cultured human liver cell line (MIHA) and human HCC cell lines (Huh-7, HCC36, HCCLM3, MHCC97-L). Compared with MIHA cells, circKIAA1429 expression was significantly elevated in HCC cell lines, and circKIAA1429 expression was relatively higher in Huh-7 and HCCLM3 cells (P < 0.01, Fig. 1A). Then, we used actinomycin D and RNase R to evaluate the stability of circKIAA1429 in various HCC cell lines. circKIAA1429 expression was more stable than linear KIAA1429 expression (P < 0.01, Fig. 1B-C).

Fig. 1
figure 1

circKIAA1429 is highly expressed in HCC cells. A: circKIAA1429 expression in various cells was detected by RT-qPCR. B-C: After treatment with actinomycin D and RNase R, the stability of circKIAA1429 in HCC cells was detected by RT-qPCR. The cell experiments were repeated 3 times independently. The data are expressed as mean ± standard deviation. The data in panel A were analyzed by one-way ANOVA, and data in panels B-C were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. **P < 0.01

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Knockdown of circKIAA1429 represses the proliferation/migration/invasion of HCC cells, but promotes apoptosis

We knocked down circKIAA1429 expression in Huh-7 and HCCLM3 cells (P < 0.01, Fig. 2A), and selected si-circKIAA1429-1 and si-circKIAA1429-2 with better transfection efficiency for subsequent experimentation. CCK-8 assay revealed that knockdown of circKIAA1429 significantly restrained HCC cell proliferation of HCC cells (P < 0.01, Fig. 2B). Flow cytometry demonstrated that knockdown of circKIAA1429 enhanced HCC cell apoptosis (P < 0.01, Fig. 2C). In addition, knockdown of circKIAA1429 significantly depressed HCC cell migration and invasion (P < 0.01, Fig. 2D).

Fig. 2
figure 2

Knockdown of circKIAA1429 represses the proliferation, migration, and invasion of HCC cells, but promotes apoptosis. si-circKIAA1429 was transfected into cells, with si-NC as a negative control. A: circKIAA1429 expression in cells was detected by RT-qPCR. B: The proliferation activity was evaluated by CCK-8 assay. C: The cell apoptosis was measured by flow cytometry. D: The cell migration and invasion were detected by transwell. The cell experiments were repeated 3 times independently. The data are expressed as mean ± standard deviation. The data in panels A-D were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. **P < 0.01

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circKIAA1429 directly interacts with SETD1A to inhibit the enrichment of H3K4me3 on the GLIS2 promoter, leading to a decrease in GLIS2 expression

We obtained the binding probability between circKIAA1429 and SETD1A through the RPISeq database (Fig. 3A). SETD1A can promote H3K4me3 [23], and GLIS2 expression is reduced in HCC [27]. Therefore, we speculate that the binding of circKIAA1429 to SETD1A leads to a decrease in GLIS2 expression. RIP results showed that SETD1A enriched more circKIAA1429, and RNA pull-down results also demonstrated that circKIAA1429 pulled down SETD1A (P < 0.01, Fig. 3B-C). ChIP analysis revealed that SETD1A and H3K4me3 were enriched on the GLIS2 promoter, and the enrichment was significantly enhanced after knockdown of circKIAA1429 (P < 0.01, Fig. 3D). GLIS2 expression was reduced in HCC cells (P < 0.01, Fig. 3E-F) and elevated after knockdown of circKIAA1429 (P < 0.01, Fig. 3G-H). However, GLIS2 expression was declined again after silencing of SETD1A (P < 0.01, Fig. 3G-J).

Fig. 3
figure 3

circKIAA1429 directly interacts with SETD1A to inhibit the enrichment of H3K4me3 on the GLIS2 promoter, leading to a decrease in GLIS2 expression. A: The binding probability between circKIAA1429 and SETD1A was predicted through the RPISeq database. B-C: The binding between circKIAA1429 and SETD1A was validated by RIP and RNA pull down. D: The enrichment of SETD1A and H3K4me3 on GLIS2 promoter was analysis by ChIP. E-H: GLIS2 expression in cells was detected by RT-qPCR and western blot. I-J: SETD1A expression in cells after transfection of si-SETD1A was detected by RT-qPCR and western blot. The cell experiments were repeated 3 times independently. The data are expressed as mean ± standard deviation. The data in panels E-F were analyzed by one-way ANOVA, and data in panels B, D, G-J were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. **P < 0.01

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circKIAA1429 directly interacts with SETD1A to inhibit the enrichment of H3K27me3 on the NAP1L3 promoter, leading to an increase in NAP1L3 expression

SETD1A can also promote the deposition of H3K27me3 [23], and NAP1L3 expression is elevated in HCC [28]. We speculate that the binding of circKIAA1429 to SETD1A leads to an increase in NAP1L3 expression. ChIP analysis revealed that SETD1A and H3K27me3 were enriched on the NAP1L3 promoter, and the enrichment was significantly enhanced after knockdown of circKIAA1429 (P < 0.01, Fig. 4A). NAP1L3 expression was elevated in HCC cells (P < 0.01, Fig. 4B-C), declined after knockdown of circKIAA1429, but increased again after silencing of SETD1A (P < 0.01, Fig. 4D-E).

Fig. 4
figure 4

circKIAA1429 directly interacts with SETD1A to inhibit the enrichment of H3K27me3 on the NAP1L3 promoter, leading to an increase in NAP1L3 expression. A: The enrichment of SETD1A and H3K4me3 on NAP1L3 promoter was analysis by ChIP. B-E: NAP1L3 expression in cells was detected by RT-qPCR and western blot. The cell experiments were repeated 3 times independently. The data are expressed as mean ± standard deviation. The data in panels B-C were analyzed by one-way ANOVA, and data in panels A, D-E were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. **P < 0.01

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Low expression of GLIS2 alleviates the anticancer effect of circKIAA1429 knockdown on HCC cells

To verify the role of circKIAA1429/GLIS2 axis in HCC cells, we silenced GLIS2 expression in Huh-7 cells and selected si-GLIS2-3 with better transfection efficiency for a combined experiment with si-circKIAA1429-1 (P < 0.01, Fig. 5A-B). Compared with knockdown of of circKIAA1429 alone, combined treatment resulted in augmented proliferation and reduced apoptosis of Huh-7 cells (P < 0.01, Fig. 5C-D). In addition, silencing of GLIS2 expression evidently promoted the migration and invasion of Huh-7 cells with low expression of circKIAA1429 (P < 0.01, Fig. 5E).

Fig. 5
figure 5

Low of GLIS2 alleviates the anticancer effect of circKIAA1429 knockdown on HCC cells. si-GLIS2 was transfected into cells, with si-NC as a negative control. A-B: GLIS2 expression in cells was detected by RT-qPCR and western blot. C: The proliferation activity was evaluated by CCK-8 assay. D: The cell apoptosis was measured by flow cytometry. E: The cell migration and invasion were detected by transwell. The cell experiments were repeated 3 times independently. The data are expressed as mean ± standard deviation. The data in panels A-B, D-E were analyzed by one-way ANOVA, and data in panel C were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. **P < 0.01

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Overexpression of NAP1L3 alleviates the anticancer effect of circKIAA1429 knockdown on HCC cells

Subsequently, to validate the role of circKIAA1429/NAP1L3 axis in HCC cells, we overexpressed NAP1L3 expression in Huh-7 cells and conducted a combined experiment with si-circKIAA1429-1 (P < 0.01, Fig. 6A-B). Compared with knockdown of circKIAA1429 alone, combined treatment resulted in enhanced proliferation, reduced apoptosis (P < 0.01, Fig. 6C-D), and augmented cell migration/invasion (P < 0.01, Fig. 6E).

Fig. 6
figure 6

Overexpression of NAP1L3 alleviates the anticancer effect of circKIAA1429 knockdown on HCC cells. NAP1L3 pcDNA3.1 (NAP1L3) was transfected into cells, with NC pcDNA3.1 (NC) as a negative control. A-B: NAP1L3 expression in cells was detected by RT-qPCR and western blot. C: The proliferation activity was evaluated by CCK-8 assay. D: The cell apoptosis was measured by flow cytometry. E: The cell migration and invasion were detected by transwell. The cell experiments were repeated 3 times independently. The data are expressed as mean ± standard deviation. The data in panel A were analyzed by t test. The data in panels B, D-E were analyzed by one-way ANOVA, and data in panel C were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. **P < 0.01

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Low expression of circKIAA1429 reduces in vivo tumorigenicity of HCC cells via GLIS2/NAP1L3

Finally, to validate our in vitro results, we evaluated the effect of circKIAA1429 on HCC tumorigenicity by subcutaneous injection of sh-circKIAA1429-infected Huh-7 cells and control cells on the right side of nude mice. Consistent with our in vitro observations, knockdown of circKIAA1429 resulted in a decrease in tumor volume and weight (P < 0.01, Fig. 7A-B). Tissue staining results showed a decrease in Ki-67 positivity rate, an increase in TUNEL positivity rate (P < 0.01, Fig. 7C-D), an increase in E-cadherin positivity rate, and a decrease in N-cadherin positivity rate (P < 0.01, Fig. 7D). In addition, molecular analysis of tumor tissue showed that compared with the sh-NC group, the expressions of circKIAA1429 and NAP1L3 were decreased in the sh-circKIAA1429 group, while the expression of GLIS2 was increased (P < 0.01, Fig. 7E-F).

Fig. 7
figure 7

Low expression of circKIAA1429 reduces in vivo tumorigenicity of HCC cells via GLIS2/NAP1L3. A: The tumor volume was recorded every week. B: After euthanizing nude mice on the 28th day, the tumor tissue was weighed and representative images were taken. C: The cell apoptosis in the tissue was detected by TUNEL assay. D: The positive rates of Ki-67, E-cadherin, and N-cadherin were detected by immunohistochemistry. E-F: circKIAA1429, GLIS2, or NAP1L3 expression was detected by RT-qPCR and western blot. N = 6. The data are expressed as mean ± standard deviation. The data in panels B-C were analyzed by t test. The data in panels A, D-F were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. **P < 0.01

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Discussion

HCC is a fatal disease, and considerable patients diagnosed at an advanced stage are not eligible for the current therapies [6]. Accumulating studies have uncovered the significant contribution of aberrantly expressed circRNAs to HCC [29]. The present elucidates that circKIAA1429 interacts with SETD1A to accelerate HCC progression via the NAP1L3/GLIS2 axis (Fig. 8).

Fig. 8
figure 8

The effect of circKIAA1429/GLIS2/NAP1L3 on HCC progression. circKIAA1429 directly interacts with SETD1A to inhibit the enrichment of SETD1A and H3K4me3/H3K27me3 on the GLIS2 or NAP1L3 promoter, resulting in a decrease in GLIS2 expression and an increase in NAP1L3 expression, ultimately accelerating the progression of HCC

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Previous studies have revealed the high expression profile of circKIAA1429 in HCC [10, 11]. circKIAA1429 overexpression facilitates HCC migration, invasion, and epithelial-to-mesenchymal transition [11]. Silencing of circKIAA1429 restrains malignant behaviors of HCC cells via the miR-133a-3p/HMGA2 axis [10]. Consistently, we noted that circKIAA1429 was highly expressed in HCC cells, and knockdown of circKIAA1429 repressed HCC cell proliferation/migration/invasion but accelerated apoptosis. circRNA can interact with histone methyltransferases to affect the methylation levels of downstream factor promoters [30]. H3K4me is a prevalent histone modification indicative of poor prognoses of HCC patients [15]. Histone H3K4 methyltransferase SETD1A is upregulated in multiple cancers and hastens cancerous processes [17,18,19,20]. SETD1A is abundantly expressed in HCC patients, and SETD1A overexpression is associated with various malignant features of HCC cells [22]. SETD1A drives HCC stemness and development via transcription activation of histone modifying enzymes [23]. We obtained the binding probability between circKIAA1429 and SETD1A through the RPISeq database. RIP and RNA pull-down results also confirmed the binding between circKIAA1429 and SETD1A.

GLIS2 is a Krüppel-like zinc finger protein belonging to the GLIS family. GLIS2 is involved in multiple pathologies, such as cystic nephropathy, diabetes, hepatic fibrosis, acute megakaryocytic leukemia, and cancer [31]. The GLIS2 promoter region is hypermethylated in HCC cells, and GLIS2 overexpression depresses HCC growth and metastasis in vivo, implying the potential of GLIS2 as a therapeutic target for HCC [27]. In our study, ChIP analysis revealed that SETD1A and H3K4me3 could be enriched on the GLIS2 promoter, and the enrichment was significantly enhanced after knockdown of circKIAA1429. Moreover, GLIS2 expression was reduced in HCC cells, elevated after knockdown of circKIAA1429, but declined again after silencing of SETD1A. These results confirmed that circKIAA1429 directly interacted with SETD1A to inhibit the enrichment of H3K4me3 on the GLIS2 promoter, leading to a decrease in GLIS2 expression.

In addition, it is reported that SETD1A can also promote the deposition of H3K27me3 [23]. Nucleosome assembly proteins (NAPs) were initially identified as histone chaperones and chromatin-assembly factors, and subsequently, additional functions such as tissue-specific transcriptional regulation, apoptosis, and cell cycle regulation have been increasingly revealed [32]. NAP1L3 is markedly elevated in HCC tissues and cells, and NAP1L3 inhibition abrogates the oncogene effect of circGFRA1 in vivo [28]. Higher NAP1L1 expression is associated with more aggressive clinicopathological features of HCC patients and poorer overall survival [33]. Our ChIP analysis revealed that SETD1A and H3K27me3 could be enriched on the NAP1L3 promoter, and the enrichment significantly enhanced after knockdown of circKIAA1429. NAP1L3 expression was elevated in HCC cells, declined after knockdown of circKIAA1429, but elevated again after silencing of SETD1A. In short, circKIAA1429 directly interacted with SETD1A to inhibit the enrichment of H3K27me3 on the NAP1L3 promoter, leading to an increase in NAP1L3 expression. Functional rescue experiments unveiled that low of GLIS2 or overexpression of NAP1L3 abolished the anticancer effect of circKIAA1429 knockdown on HCC cells. Finally, we validated in the xenotransplanted tumor mouse model that low expression of circKIAA1429 reduced the in vivo tumorigenicity of HCC cells via GLIS2/NAP1L3.

In conclusion, circKIAA1429 directly interacts with SETD1A to inhibit the enrichment of SETD1A and H3K4me3/H3K27me3 on the GLIS2/NAP1L3 promoter, which results in decreased GLIS2 expression and increased NAP1L3 expression, thus accelerating the progression of HCC. However, this study also has several limitations. This study only explores the downstream mechanism of circKIAA1429 and has not yet detected the upstream mechanism of circKIAA1429. Furthermore, we only investigated the role of a single mechanism in two cell lines, and it is unclear whether the mechanism we discovered works in other cells. Whether circKIAA1429 can affect the expression of NAP1L3 or GLIS2 through ceRNA mechanisms or RNA binding proteins remains unclear. Our study is currently in the exploratory stage, and more experimental evidence is needed to apply our findings to clinical practice. In the future, we will determine whether circKIAA1429 can also affect the expression of NAP1L3 or GLIS2 through ceRNA mechanisms or RNA binding proteins, further interpret the impact of circKIAA1429 in HCC, and provide new theoretical knowledge for the treatment of HCC.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

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Funding

This study was not funded.

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Author notes

  1. Yiting Yuan and Junwei Huang contributed equally to this work.

Authors and Affiliations

  1. Department of General Surgery, The First People’s Hospital of Tongxiang, No. 1918, Xiaochang East Road, Wutong Street, Tongxiang City, Zhejiang Province, 314500, PR China

    Yiting Yuan, Junwei Huang, Guifen Wei, Guang Hu, Hongmei Yu & Yiming Tao

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  1. Yiting Yuan
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  2. Junwei Huang
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  3. Guifen Wei
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  4. Guang Hu
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  5. Hongmei Yu
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  6. Yiming Tao
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Contributions

YTY, JWH, and YMT conceived the study and experiments. YTY, GFW and GH performed experiments. YTY, JWH and HMY performed and analyzed the experiments. YMT supervised the manuscript.

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Correspondence to Yiming Tao.

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All animal experiment schemes were approved by the Animal Ethics Committee of The First People's Hospital of Tongxiang and implemented based on the Guide for the Care and Use of Laboratory Animals [25].

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Yuan, Y., Huang, J., Wei, G. et al. Potential mechanism of circKIAA1429 accelerating the progression of hepatocellular carcinoma. Infect Agents Cancer 20, 12 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13027-025-00645-3

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13027-025-00645-3

Keywords

Infectious Agents and Cancer

ISSN: 1750-9378

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