UHRF1 Drives Hepatocellular Carcinoma Progression via Epigenetic Repression of SFMBT2

UHRF1 Drives Hepatocellular Carcinoma Progression via Epigenetic Repression of SFMBT2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article UHRF1 Drives Hepatocellular Carcinoma Progression via Epigenetic Repression of SFMBT2 Zekai Hu, Qi Sun, Weiliang Xia, Zenglei He This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7981198/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Background Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality, necessitating the identification of novel therapeutic targets. UHRF1 is an epigenetic regulator implicated in various cancers, but its precise mechanistic role in HCC progression remains incompletely understood. This study investigates the epigenetic mechanisms by which UHRF1 promotes HCC pathogenesis, with a focus on its interaction with SFMBT2. Methods A multifaceted approach integrating clinical sample analysis, bioinformatics, in vitro cell culture experiments, and in vivo xenograft models was employed. UHRF1 expression was assessed in HCC tissues and cell lines. Functional roles were investigated through overexpression and knockdown models, evaluated by MTT, Transwell, and Western blot assays. ChIP and luciferase reporter assays were used to examine promoter binding and transcriptional regulation. Results UHRF1 was significantly upregulated in HCC tissues and correlated with advanced tumor grade and poor survival. UHRF1 promoted HCC cell proliferation, migration, and invasion by activating the ERK/AKT/NF-κB signaling pathways. Mechanistically, UHRF1 bound to CpG islands in the SFMBT2 promoter, epigenetically repressing its expression. SFMBT2 overexpression reversed UHRF1-driven oncogenic effects in vitro and in vivo. Furthermore, UHRF1-mediated SFMBT2 downregulation led to increased HOXB13 expression, which transcriptionally activated LTK. Conclusions This study identifies a novel UHRF1/SFMBT2 epigenetic axis critical for HCC progression. UHRF1 represses SFMBT2 to activate ERK/AKT/NF-κB and HOXB13/LTK pathways, driving tumor aggressiveness. Restoration of SFMBT2 counteracts these effects, highlighting its tumor-suppressive role. These findings position UHRF1 as a promising prognostic biomarker and therapeutic target in HCC. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Molecular biology Health sciences/Oncology UHRF1 SFMBT2 hepatocellular carcinoma epigenetics HOXB13 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Liver cancer is now the sixth most common malignancy worldwide and the third leading cause of cancer-related deaths, with hepatocellular carcinoma (HCC) accounting for approximately 75%–85% of primary liver cancers [ 1 ] . Current research indicates that ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) acts as an epigenetic regulator, influencing gene expression through mechanisms such as DNA methylation and histone modification [ 2 ] . Emerging evidence establishes UHRF1 as a critical epigenetic regulator in various cancers, including pancreatic, colorectal, lung, and breast cancers [ 3 – 7 ] . UHRF1 plays essential roles in tumour cell proliferation, migration, invasion, metastasis, and drug resistance [ 6 , 8 , 9 ] , and elevated expression has been found to correlate significantly with poor patient prognosis [ 10 ] . Furthermore, UHRF1 represents a potential target for anticancer drug development; compounds such as shikonin, hinokitiol, and dihydroartemisinin, which target UHRF1, have shown therapeutic effects in various cancers [ 11 – 13 ] . UHRF1 promotes tumour progression through multiple distinct targets and signalling pathways, including KRAS-mediated mechanisms in lung adenocarcinoma [ 5 ] and the KISS1/PI3K/NF-κB signalling axis in colorectal cancer [ 14 ] . In HCC, studies have shown that UHRF1 facilitates tumour progression through epigenetic modification of genes such as maternally expressed gene 3 ( MEG3 ) and colony-stimulating factor 1 ( CSF1 ) [ 9 , 15 ] , as well as by inducing immune cell infiltration [ 16 ] . However, the precise molecular mechanisms by which UHRF1 contributes to HCC progression remain to be fully elucidated. Scm-like with four mbt domains 2 (SFMBT2), another epigenetic regulator, is involved in embryonic development and the pathogenesis of multiple diseases through its intronic microRNA (miRNA) clusters and circular RNA variants [ 17 – 20 ] . Previous studies have demonstrated that SFMBT2 exerts pleiotropic effects in cancer progression via distinct molecular mechanisms. This study was performed to address the current knowledge gap by investigating the signalling pathways and gene interactions associated with UHRF1, with particular focus on its relationship with SFMBT2. To this end, a multifaceted research approach was employed, integrating clinical sample analysis, bioinformatics, and both in vitro and in vivo studies. These methodologies enabled a comprehensive examination of UHRF1’s epigenetic regulatory mechanisms, expression patterns, functional roles, and interactions with other key genes implicated in HCC. In addition, the UHRF1–SFMBT2 interaction network and SFMBT2’s potential as a prognostic biomarker and therapeutic target were explored, thereby highlighting its clinical significance in HCC management. Materials and methods Clinical sample collection This research was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University, School of Medicine ([2025B] IIT Ethics Approval No. 0483). All patients provided written informed consent. Matched HCC tissue specimens, comprising cancerous and adjacent non-cancerous tissues, were collected from 16 patients who underwent radical resection. Cancerous tissue was defined as tissue within 1 cm of the tumour edge without necrosis, and non-cancerous tissue was defined as tissue located more than 2 cm beyond the tumour edge. The patient inclusion criteria were no prior treatment before surgery, pathological confirmation of cancerous tissue as HCC, and confirmation that the surgical margin was free of residual carcinoma. Bioinformatics analysis Liver cancer RNA-sequencing data and corresponding clinical data were downloaded from TCGA database ( https://gdc.cancer.gov/ ). The dataset was divided into two groups: primary tumour tissue (371 samples) and healthy tissue (50 samples). Using R software and the ggplot2 package, a box plot was generated to illustrate the expression levels of UHRF1 in primary cancer versus healthy samples. Clinical data were further extracted for tumour grade, and using R software and the ggplot2 package, a box plot was created to display UHRF1 expression levels across healthy tissue, G1, G2, G3, and G4. To assess the clinical relevance of UHRF1 in HCC, TCGA gene data were also analysed using the dataset available through the UALCAN ( http://ualcan.path.uab.edu ). Cell culture LO2, Hep3B, HepG2, Hu7, SK-Hep-1, and PLC cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. All cell lines were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, CA, USA) supplemented with 10% foetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, in a humidified atmosphere containing 5% CO 2 at 37°C. Cells in the exponential growth phase were harvested at 80%–90% confluence for passage or experimental use. Plasmids and cell transfection Short hairpin RNA for LncSNHG1, negative control short hairpin RNA, miR-181b-5p inhibitor, and the corresponding negative control vector were all designed and synthesised by HanBio Company (China). Huh7 and HepG2 cells were cultured in six-well plates. When the cell density reached 70%–80% confluence, 2.5 µg of the plasmid vector was transfected into the cells using Lipofectamine 3000 (Invitrogen, CA, USA) according to the manufacturer’s protocol. Transfection efficiency was assessed by fluorescence microscopy, qRT-PCR, and WB. Antibodies, plasmids, and primers Details of the antibodies, plasmids, and primers used for RNA and ChIP analyses by qRT-PCR are provided in Tables 1 – 3 . Table 1 List of antibodies used in this study Target proteins Source Catalogue no. UHRF1 abcam ab213223 Flag CST #14793 SFMBT2 proteintech 25256-1-AP p-ERK proteintech 28733-1-AP ERK proteintech 51068-1-AP p-AKT CST 4060S AKT proteintech 13409-1-AP p-NFκB proteintech 82335-1-RR NFκB proteintech 10745-1-AP LTK Bioss bs-15500R HOXB13 Abcam ab201682 β-actin Proteintech 20536-1- AP Table 2 List of qRT-PCR primer sequences Target gene 5′ forward primer sequence 3′ reverse primer sequence UHRF1 TCTCAACTGCTTTGCTCCCA GTCCTTCCCCTCCTTCGTC TOMM6 CTGCTGGCTCGGCTAATGA GAGGTGCCATGAGGTCAATGT SFMBT2 AGCAGAGGAAGGGGAGAAGT TTCAGCTCCATGCACTCCTG SLC27A6 GTTCGTGTTGAAGGTGGTGC TGGTTCAGGAAGACATGGGC SCML1 AGTCCAGTGCATCCCTCAGA GACCAGGTTGAAGGGTGCTT CES3 CCACAGAGGAGGAGAAGCAG TATCTTGCTGGGGAGCGTCT LTK CTGGTTCTGATGGTGGCTGT TGATGGCGAAACTTGCTGAT HOXB13 AGACTCTGGGTGCTCCT GCCTCTTGTCCTTGGTG β-actin AGCGGGAAATCGTGCGTG CAGGGTACATGGTGGTGCC Table 3 List of qRT-PCR primer sequences used for ChIP analysis Target gene 5′ forward primer sequence 3′ reverse primer sequence LTK promoter CTACCTGAAGGGAGTGGCT CTGTGATGGGGTGAAAGAC SFMBT2-Island1 CAGGTCCGTATGCCAGGTTTA CAGCCTGGTGGGTCTTAACAT SFMBT2-Island2 GAGTCCTTGAAAACCGTGTA ATCCAAAACATTATCACCTCA SFMBT2-Island3 CCGACTTCTGGTGTGACGTAG ATGAAGGATGGCGGCTCGTA RNA extraction and qRT-PCR analyses Total cellular RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Subsequently, 500 ng of RNA was reverse-transcribed into complementary DNA (cDNA) using the Verso cDNA Synthesis Kit (Thermo Scientific). The synthesised cDNA was then diluted 25-fold and used as a template for qRT-PCR analysis. qRT-PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, CA, USA) and target-specific primers. Relative RNA expression was calculated by normalising target mRNA levels to β-actin expression as the internal control. Primer sequences are provided in Table 2 . RNA interference or overexpression For UHRF1 silencing, transient transfection was performed in HepG2 and Huh7 cells using small interfering RNA (siRNA) (GenePharma, Shanghai, China). The siRNA was incubated with Lipofectamine 2000 (Invitrogen) at room temperature for 20 minutes to form a complex, and transfection was carried out at 37°C for either 24 or 48 hours. Silencing efficiency was confirmed by WB. The siRNA sequences are provided in Table 4 . Table 4: siRNA information Plasmid Sequence UHRF1 siRNA from Qing Ke Bio, China hUHRF1 siRNA-1 sense GCCAGAGUGAGUCAGACAATT hUHRF1 siRNA -1 antisense UUGUCUGACUCACUCUGGCCC hUHRF1 siRNA-2 sense AUGUGGGAUGAGACGGAAUUG hUHRF1 siRNA -2 antisense CAAUUCCGUCUCAUCCCACAU hUHRF1 siRNA -3 sense CCAGUUGUUCCUGAGUAAATT hUHRF1 siRNA -3 antisense UUUACUCAGGAACAACUGGAA siRNA NC sense UUCUCCGAACGAGUCACGUTT siRNA NC antisense ACGUGACUCGUUCGGAGAATT LTK WT\MUT luciferase reporter gene plasmids from Qing Ke Bio, China. LTK wild type plasmid sequence GGCCTGTGCTACATCACCCATTG ACCAGAGGTGCCAGTACTAGTGCT CAAGATCAATCGATCGATCGGTCTA CCTACCTATCATCTATTGACCTTCAG TGCTACTAAAAACACTCGGATCTTCT AACGTCTGGTCCAGTCTTTCACCCCA TCACAGTGAGAGGCTGTGCACAGGG GTAACACAGGCAACGGAATTATATG AGGCAAACAC LTK mutant plasmid sequence GGCCTGTGCTACATCACCCATTGACCA GAGGTGCCAGTACTAGTGCTCAAGATC AATCGATCGATCGGTCTACCTACCTATC ATCTATTGACCTTCAGTGTTTTTAGTAG CACTCGGATCTTCTAACGTCTGGTCCAG TCTTTCACCCCATCACAGTGAGAGGCTG TGCACAGGGGTAACACAGGCAACGG AATTATATGAGGCAAACAC For UHRF1 , SFMBT2 , and HOXB13 overexpression, the coding sequences of human UHRF1 mRNA, SFMBT2 mRNA, and HOXB13 mRNA were synthesised, digested with HindIII and EcoRI, and subcloned into the pcDNA3.1 vector. The integrity of the plasmid constructs was verified by DNA sequencing. Overexpression plasmids or the pcDNA3.1 vector were incubated with Lipofectamine 2000 reagent (Invitrogen) at room temperature for 20 minutes to form a complex, and transfection was performed at 37°C for 24 hours. Overexpression efficiency was confirmed by WB. Plasmid information is provided in Table 5 . Table 5 Plasmids information Plasmid Source ID The UHRF1 overexpression YouBao Bio NM_013282 The SFMBT2 overexpression YouBao Bio NM_001029880 The HOXB13 overexpression YouBao Bio NM_006361 Cell migration and invasion assay For the migration assay, transfected cells were trypsinised, resuspended in serum-free medium, and transferred to the upper chambers of Transwell inserts in 24-well plates with an 8-µm pore size and polycarbonate membrane (Corning, NY, USA). DMEM supplemented with 10% foetal bovine serum was added to the lower chambers as a chemoattractant. After 24 or 48 hours, cells remaining in the upper chamber were removed with a cotton swab, while those on the underside of the membrane were fixed with ice-cold methanol, stained with 0.1% crystal violet, and counted under a microscope (Olympus). For the invasion assay, the Transwell membrane was coated with Matrigel (BD Biosciences) before cells were added, and all subsequent steps were performed as described for the migration assay. WB analysis Tissue or cells were lysed on ice with RIPA buffer containing protease and phosphatase inhibitors. Total protein was extracted, and its concentration determined using a BCA Protein Reagent Kit (Thermo Fisher Scientific). Equal amounts of protein from each sample were separated on 10% or 12% sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, Germany). Non-specific binding was blocked with 5% bovine serum albumin, and membranes were incubated overnight at 4°C with primary antibody (1:1000), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:1000) (Abcam, Cambridge, UK) for 1 hour. After three washes using Tris-buffered saline with Tween 20, protein bands were detected and imaged using the Bio Imaging System (Bio-Rad). β-actin was used as a loading control. Details of the primary antibodies are provided in Table 1 . Protein expression levels were determined as the ratio of the target band intensity to that of β-actin. Each protein sample was measured in triplicate. IHC staining Tumour tissue specimens were fixed in 10% neutral formalin for 24–48 hours and routinely processed for paraffin embedding. IHC staining was performed as previously described [ 21 ] . UHRF1, SFMBT2, p-ERK, p-NF-κB, HOXB13, and LTK antibodies (1:200) were detected using the streptavidin–peroxidase conjugate method. Immunoreactivity was independently evaluated by two professional pathologists. For the assessment of UHRF1 protein expression in HCC, hepatocytes with light brown to dark brown nuclear staining were defined as positive cells. Cell proliferation assay Cell proliferation was measured using a MTT assay. Briefly, cells were seeded in 96-well plates and incubated overnight, and viable cell numbers were assessed at 0, 24, 48, 72, and 96 hours after transfection. MTT solution was added to each well and incubated for 3 hours at 37°C in darkness. The purple formazan crystals were dissolved in dimethyl sulfoxide, and absorbance was measured at 570 nm using a microplate reader (Molecular Devices). Luciferase reporter assay Cells were transfected with the respective plasmid constructs expressing the target proteins (as indicated) and, after 24 hours, co-transfected with either the wild-type or mutant plasmids along with the overexpression plasmid. After a further 24 hours, reporter luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) on a GloMax 20/20 Luminometer (Promega), according to the manufacturer’s protocol. ChIP analysis The cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature and quenched with 125 mM glycine for 5 minutes. After being washed with phosphate-buffered saline, the cells were lysed in ChIP lysis buffer (0.5% NP-40, 1% Triton X-100, 150 mM NaCl, 20 mM Tris–Cl, pH 7.5, 2 mM EDTA) containing protease inhibitors and incubated on ice for 10 minutes. Chromatin was sonicated to 200- to 1000-bp fragments and centrifuged (14,000 rpm, 20 minutes, 4°C). The supernatant was diluted 10× in ChIP dilution buffer (0.01% sodium dodecyl sulfate, 1.1% Triton X-100, 1.1 mM EDTA, 20 mM Tris–Cl, pH 8.0, 167 mM NaCl). Aliquots were reserved as input, and the remaining samples were incubated overnight at 4°C with UHRF1 or HOXB13 antibody, followed by Protein A/G beads for 1 hour. The immunocomplexes were washed with 800 µL wash buffer, eluted, and incubated at room temperature for 30 minutes. After magnetic separation, the eluates were de-cross-linked with 5 M NaCl at 65°C for 4 hours, then treated with Core Mix at 45°C for 1 hour. DNA was purified using a commercial kit and analysed by qRT-PCR (Bio-Rad CFX96™) to assess enrichment at target loci. Construction of stable cell lines Lentivirus was packaged using 293T cells seeded in 15-cm dishes. At 50%–60% confluence, cells were washed with phosphate-buffered saline and cultured in serum-free DMEM. A plasmid mixture containing 40 µg PP4R1, 30 µg psPAX2, and 10 µg pMD2.G (4:3:1) in 250 µL Opti-DMEM was combined with 10 µL Lipofectamine 2000 in 240 µL Opti-DMEM. After 20 minutes of incubation, the complex was added to the cells. Viral supernatants were collected every 24 hours, centrifuged (3,500 rpm, 10 minutes, 4°C), filtered (0.22 µm), and ultracentrifuged (30,000 rpm, 2 hours, 4°C). The pellets were resuspended overnight in serum-free medium and stored at − 80°C. For stable transduction, HepG2 cells (1 × 10 4 cells/well) were infected with UHRF1 lentivirus (MOI = 200, 1.0 × 10 8 TU/mL). After 48 hours, the medium was replaced and the cells were cultured for a further 24 hours before puromycin selection (1 µg/mL). Following three passages, fluorescent-positive cells were harvested for WB analysis of UHRF1 expression. UHRF1 -overexpressing cells (2 × 10 5 cells/well) were transfected with the SFMBT2 overexpression plasmid at 50%–60% confluence. After 48 hours, selection was initiated with G418 (50 µg/mL), and the cells were maintained with daily medium changes and passaged every 2–3 days. Cells were collected after three passages for WB confirmation of SFMBT2 expression. Reagents and materials are detailed in Table 6 . Table 6 Materials for stable cell lines Reagent Name Source ID Packaging plasmid psPAX2 YouBao Bio VT1444 Envelope plasmid pMD2.G YouBao Bio VT1443 G418 Solarbio IG0010 Puromycin Solarbio P8230 Animal assay The mouse experiments were approved by the Animal Ethics Committee of Zhejiang University and conducted in accordance with animal health guidelines. All procedures were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020), as well as the ethical guidelines of the Declaration of Helsinki and the relevant local legislation and institutional requirements. Nude mice were obtained from Shanghai Slake Laboratory Animal Co., Ltd., SCKK (Shanghai) 2022-0004 (Licence). The mice were randomly assigned to three groups (n = 6 per group). HepG2 cells (1 × 10 6 cells) stably transfected with sh UHRF1 , sh SFMBT2 , or sh UHRF1/SFMBT2 were injected subcutaneously into 4-week-old nude mice using a 1-mL sterile syringe. The mice were examined every 4 days after injection. When tumours in the control group reached a volume of 1,500 mm 3 , all mice were euthanised by cervical dislocation, and tumour tissues were collected. The body weights of mice in each group at the time of euthanised were 16.85 ± 1.70 g, 18.85 ± 2.57 g, and 18.83 ± 1.95 g, respectively. Tumour volumes were calculated as V = (length × width 2 ) / 2. Statistical analyses and dataset analysis Results are expressed as mean ± standard deviation. Analyses were performed using SPSS 26.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). The mRNA expression of genes in HCC was analysed using Wilcoxon’s paired test. p < 0.05 was considered statistically significant. Results UHRF1 expression is upregulated in HCC tissues qRT-PCR (quantitative reverse transcription polymerase chain reaction) analysis revealed that UHRF1 mRNA expression was significantly higher in the cancerous tissue group than in the non-cancerous tissue group (Fig. 1 A). IHC (immunohistochemistry) and WB (western blotting) further confirmed that UHRF1 protein levels were markedly elevated in HCC tissues (Fig. 1 B–D). Notably, UHRF1 protein was almost undetectable in non-cancerous tissues, suggesting a potential role in HCC pathogenesis. We next assessed UHRF1 expression in a panel of HCC cell lines (Hep3B, HepG2, Huh7, SK-Hep-1, and PLC) and a normal liver cell line (LO2). WB showed no detectable UHRF1 expression in LO2 cells. By contrast, UHRF1 was highly expressed in HepG2 and SK-Hep-1 cells, while expression was relatively low in Hep3B, Huh7, and PLC cells (Fig. 1 E and F). To validate these findings, UHRF1 expression data from TCGA (The Cancer Genome Atlas) were analysed using the UALCAN (University of Alabama at Birmingham cancer data analysis portal) dataset. Consistent with our results, UHRF1 expression levels were significantly higher in HCC tissues than in normal tissues (Fig. 2 A). Furthermore, UHRF1 expression was positively correlated with tumour grade, with higher expression observed in poorly differentiated HCC (Fig. 2 B). Importantly, survival analysis of patients with HCC using the Gene Expression Profiling Interactive Analysis (GEPIA) database ( http://gepia.cancer-pku.cn/ ) showed that elevated UHRF1 expression was associated with significantly worse overall survival (Fig. 2 C). These findings suggest that UHRF1 may serve as a prognostic biomarker in HCC. UHRF1 promotes HCC cell proliferation, migration, and invasion To investigate the functional role of UHRF1 in HCC, we first established UHRF1 overexpression and knockdown models in HepG2 and Huh7 cells. WB confirmed successful UHRF1 protein overexpression (Fig. 3 A) as well as efficient knockdown in both cell lines (Fig. 3 B). Subsequent MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays revealed that UHRF1 overexpression significantly enhanced cell proliferation in both cell lines, whereas UHRF1 knockdown suppressed proliferative capacity. These changes reached statistical significance at the 96-hour time point (Fig. 3 C). Transwell assays further demonstrated that UHRF1 overexpression markedly increased migratory and invasive capabilities in HCC cells, while UHRF1 knockdown conversely reduced these malignant behaviours, indicating a pivotal role of UHRF1 in promoting HCC cell migration and invasion (Fig. 4 A and B). It has been reported that the development and progression of many cancers, including HCC [ 22 ] , are closely associated with activation of the serine/threonine kinase (AKT). Moreover, ERK (extracellular signal-regulated kinase) and NF-κB (nuclear factor kappa-B) also play critical roles in mediating oncogenic processes in HCC [ 23 , 24 ] . Therefore, we performed WB to assess the effects of UHRF1 on the ERK/AKT/NF-κB signalling pathway in HepG2 and Huh7 cells. The results indicated that UHRF1 knockdown significantly reduced the phosphorylation levels of ERK, AKT, and NF-κB, while UHRF1 overexpression markedly increased their activation, with all changes showing statistically significant differences (Fig. 5 A and B). Overexpression of UHRF1 inhibits SFMBT2 in vivo and in vitro Next, we sequenced the UHRF1 -overexpressing HepG2 cells to identify differentially expressed genes. Successful establishment of the UHRF1 overexpression cell line was confirmed by WB (protein level) and qRT-PCR (gene level) (Fig. 6 A and B). A total of 370 genes were identified, including 163 upregulated and 207 downregulated genes. Visualisation of the sequencing data included a bar chart showing the distribution of the genes (Fig. 6 C), a volcano plot highlighting expression changes (Fig. 6 D), and a heat map clustering these genes based on expression patterns (Fig. 6 E). Gene ontology analysis revealed that the differentially expressed genes in UHRF1 -overexpressing cells were predominantly enriched in biological processes and molecular functions related to serine protease inhibitor activity and positive regulation of interleukin-4 production (Fig. 6 F). Kyoto Encyclopedia of Genes and Genomes (KEGG) is a widely used bioinformatics database resource for understanding high-level functions and utilities of the biological system [ 25 , 26 ] . KEGG pathway analysis further demonstrated significant enrichment in pathways critical to HCC progression, including retinoid metabolism, synaptic vesicle cycle, NF-κB signalling pathway, and transforming growth factor-β (TGF-β) signalling pathway (Fig. 6 G). KEGG pathway identifiers and names were retrieved from the KEGG database ( https://www.kegg.jp/kegg/kegg1.html ). qRT-PCR was then used to validate the expression of five candidate differentially expressed genes. Among these, SFMBT2 showed a significant reduction in mRNA levels in UHRF1 -overexpressing cells (p < 0.05) (Fig. 6 H). This downregulation was further corroborated in HepG2 cells at the protein level (Fig. 6 I). IHC staining of clinical HCC tissues revealed an inverse correlation between UHRF1 and SFMBT2 protein expression: HCC tissues exhibited strong nuclear immunoreactivity for UHRF1, whereas adjacent non-cancerous tissues displayed much less staining. Conversely, SFMBT2 protein expression was markedly suppressed in HCC tissues compared with non-cancerous counterparts (Fig. 6 J). UHRF1 binds to the SFMBT2 promoter, and SFMBT2 overexpression inhibits cell migration, invasion, and proliferation and the ERK/AKT/NF-κB signalling pathway We identified three CpG islands within the promoter of SFMBT2 using the MethPrimer database ( https://www.methprimer.com/index.html ) (Fig. 7 A). ChIP (chromatin immunoprecipitation) assays demonstrated that UHRF1 protein binds to all three CpG islands in HepG2 cells (p < 0.01 for island 1, p < 0.05 for islands 2 and 3) (Fig. 7 B). These results indicate that UHRF1 directly interacts with the SFMBT2 promoter, likely mediating transcriptional repression through CpG island targeting. To validate these findings, SFMBT2 -overexpressing HepG2 and Huh7 cell lines were established. WB confirmed successful overexpression at the protein level, with distinct electrophoretic bands corresponding to SFMBT2 detected (p < 0.01 for both cell lines) (Fig. 7 C). SFMBT2 overexpression significantly reduced migration and invasion in both HepG2 and Huh7 cells, supporting its tumour-suppressive role (Fig. 7 D and E). In addition, SFMBT2 overexpression significantly inhibited cell proliferation in both cell lines (Fig. 7 F and G). We next performed WB to examine the effects of SFMBT2 on the ERK/AKT/NF-κB signalling pathway. SFMBT2 overexpression significantly decreased phosphorylation levels of ERK and NF-κB in both HepG2 and Huh7 cells (p < 0.01), while total ERK and NF-κB protein levels remained unchanged, indicating selective inhibition of pathway activation (Fig. 7 H and I). Overexpression of SFMBT2 restores the tumour-promoting effect of UHRF1 both in vitro and in xenograft models To further investigate the functional interplay between UHRF1 and SFMBT2 in HCC, Huh7 cells were co-transfected with UHRF1 and SFMBT2 overexpression plasmids. WB showed that UHRF1 protein overexpression significantly increased the phosphorylation levels of ERK and NF-κB. Strikingly, SFMBT2 protein overexpression reversed these effects, restoring p-ERK (phospho-extracellular signal-regulated kinase) and p-NF-κB (phospho-nuclear factor kappa-B) levels to near baseline (Fig. 8 A). MTT assays demonstrated that UHRF1 protein overexpression significantly enhanced cell proliferation from 48 to 72 hours post-transfection, whereas co-expression of SFMBT2 protein abolished these effects (Fig. 8 B). Transwell assays revealed that UHRF1 protein overexpression increased cell migration (p < 0.05), while SFMBT2 protein co-expression restored migration to control levels. Similar effects were consistently observed in the corresponding invasion assays (Fig. 8 C–E). A subcutaneous xenograft model was established by inoculating HepG2 cells into nude mice. Quantitative analysis of tumour growth showed that UHRF1 protein overexpression significantly increased tumour volume (p < 0.05) and weight (p < 0.05) compared with vector controls at endpoint. Conversely, concomitant SFMBT2 protein overexpression reversed these oncogenic effects, reducing tumour volume and weight to levels comparable to the control group (Fig. 8 F–H). Further IHC profiling of xenograft tissues confirmed that UHRF1 protein overexpression suppressed SFMBT2 protein expression while concomitantly increasing p-ERK and p-NF-κB expression. Consistent with previous experiments, SFMBT2 protein co-overexpression reversed these changes (Fig. 8 I). UHRF1 downregulates SFMBT2 to promote HOXB13-dependent LTK transcription Previous studies have shown that SFMBT2 protein can epigenetically repress HOXB13 (homeobox B13) transcription by binding methylated histones, thereby downregulating oncogenic pathways such as LTK [ 27 ] . To investigate the functional interplay among UHRF1, SFMBT2, and HOXB13 in HCC pathogenesis, HepG2 cells were transfected with UHRF1 and SFMBT2 overexpression plasmids, and WB was performed to assess HOXB13 and LTK protein levels. UHRF1 protein overexpression significantly increased HOXB13 (p < 0.05) and LTK (p < 0.01) protein levels (Fig. 9 A), whereas SFMBT2 protein overexpression suppressed both (p < 0.05 and p < 0.01, respectively) (Fig. 9 B). In HepG2 cells with concomitant UHRF1 and SFMBT2 protein overexpression, UHRF1 significantly increased HOXB13 (p < 0.05) and LTK (p < 0.01) protein levels compared with empty vector controls. However, co-overexpression of SFMBT2 reversed these effects, reducing HOXB13 (p < 0.05) and LTK (p < 0.05) expression (Fig. 9 C). IHC analysis of HCC tissues confirmed that UHRF1 protein overexpression increased HOXB13 and LTK protein levels, whereas SFMBT2 protein co-expression reversed these oncogenic trends (Fig. 9 D and E). ChIP assays were then performed to examine enrichment of the LTK promoter in HepG2 cells and to investigate the regulatory interplay between UHRF1 and HOXB13 in LTK transcription. Following UHRF1 protein overexpression, chromatin fragments immunoprecipitated with a HOXB13-specific antibody showed significantly increased binding to LTK promoter DNA compared with the control group (p < 0.01), indicating that UHRF1 enhances HOXB13-mediated transcriptional regulation of LTK (Fig. 9 F). To further dissect HOXB13’s role in LTK regulation, a HOXB13 overexpression plasmid was constructed and transfected into HepG2 cells. WB confirmed increased HOXB13 protein levels (Fig. 9 G). Based on JASPAR database predictions, the highest-scoring HOXB13 binding site within the LTK core promoter was subjected to site-directed mutagenesis. Wild-type and mutant-type LTK promoter sequences were cloned into dual-luciferase reporter plasmids. Co-transfection of the HOXB13 overexpression plasmid with the wild-type reporter into HepG2 cells increased luciferase activity compared with the pcDNA3.1 empty vector control (p < 0.05), whereas co-transfection with the mutant-type reporter abrogated this effect, demonstrating that HOXB13 regulates LTK promoter activity via the predicted binding site (Fig. 9 H). Finally, to validate the regulatory role of SFMBT2 in LTK expression, wild-type and mutant-type LTK promoter reporter plasmids were co-transfected with SFMBT2 overexpression plasmids into HepG2 cells. SFMBT2 protein overexpression significantly suppressed wild-type promoter activity (p < 0.05) but had no significant effect on the mutant-type construct, indicating that SFMBT2 modulates LTK expression primarily through HOXB13-dependent chromatin interactions (Fig. 9 I). Discussion HCC development is driven by complex epigenetic abnormalities. In the context of early diagnosis, DNA methylation biomarkers show significant clinical potential, with combined detection of promoter hypermethylation in specific genes markedly improving HCC identification [ 28 ] . During tumour invasion and metastasis, histone modifications play a central regulatory role in modulating tumour cell invasion and in vivo dissemination [ 29 ] . In addition, metabolite–epigenetic interactions act as critical drivers: for example, acetyl-CoA accumulation induces epithelial–mesenchymal transition, thereby promoting metastasis [ 30 ] , while succinyl transferases accelerate tumour progression by enhancing glycolytic flux [ 31 ] . Environmental factors, such as alcohol, also contribute to carcinogenesis through mechanisms including acetaldehyde-mediated DNA adduct formation, oxidative stress, and alterations in DNA methylation patterns [ 32 , 33 ] . Together, these findings highlight the pivotal role of the epigenetic regulatory network in HCC and provide a theoretical basis for the development of early diagnostic biomarkers and targeted therapeutic strategies. Previous studies have shown that suppression of the AMP-activated protein kinase signalling pathway [ 7 ] , hypermethylation at the thioredoxin-interacting protein ( TXNIP ) promoter [ 34 ] , and p53 pathway inactivation [ 35 ] are involved in the tumour-suppressive effects of UHRF1 loss. In our study, we delineate a previously uncharacterised epigenetic mechanism by which UHRF1 contributes to HCC progression. Consistent with its established role as an epigenetic integrator, UHRF1 was markedly upregulated in HCC tissues and cell lines. These observations align with evidence from other malignancies, in which UHRF1 overexpression has been consistently linked to aggressive tumour phenotypes [ 36 – 38 ] . Mechanistically, we demonstrate that UHRF1 exerts transcriptional repression of the tumour suppressor gene SFMBT2 by directly binding to CpG-rich islands within its promoter region. This epigenetic silencing is consistent with UHRF1’s known ability to recruit DNA methyltransferases and modify chromatin structure, as reported in other cancers [ 39 , 40 ] . Importantly, our findings show that UHRF1-mediated repression of SFMBT2 facilitates the activation of downstream oncogenic signalling in HCC. These results position UHRF1 not only as a prognostic biomarker but also as a promising therapeutic target. Bioinformatics analyses further reinforced the clinical relevance of UHRF1 in HCC, revealing a strong correlation between UHRF1 overexpression and advanced tumour grade, as well as poorer overall survival. This suggests that UHRF1 is a robust prognostic biomarker for HCC — a role similarly observed in other malignancies, where high UHRF1 expression predicts unfavourable clinical outcomes, including reduced overall survival and increased recurrence rates [ 41 , 42 ] . Accumulating evidence shows that SFMBT2 protein participates in the regulation of tumour suppressor genes through pathways such as miR-885-3p/ CHD7 [ 19 ] , miR-107/ SLC1A5 [ 43 ] , and miR-182-5p/ CREB1 [ 44 ] . This regulatory activity modulates multiple signalling pathways, including Wnt/β-catenin and glutamine metabolism, ultimately playing a critical role in tumorigenesis, invasion, and metabolic adaptation. These findings not only highlight SFMBT2 as a pan-cancer regulatory factor but also underscore its potential as a novel diagnostic biomarker and therapeutic target. Our study, for the first time, reveals the collaborative role of SFMBT2 and UHRF1 proteins in promoting tumour progression. We establish SFMBT2 as a pivotal epigenetic regulator downstream of UHRF1, with substantial tumour-suppressive activity in HCC. SFMBT2 protein overexpression attenuated malignant phenotypes, including proliferation, migration, and invasion, while concurrently restoring ERK/AKT/NF-κB signalling pathway homeostasis. Notably, SFMBT2 protein was able to reverse the oncogenic effects induced by UHRF1 protein overexpression, both in vitro and in xenograft models. These findings underscore the functional importance of SFMBT2 in modulating UHRF1-driven transcriptional programmes. In addition to confirming UHRF1 and SFMBT2 proteins as key regulators of tumour aggressiveness, our study identifies the HOXB13/ LTK signalling cascade as a novel downstream effector axis. We show that UHRF1-mediated repression of SFMBT2 leads to upregulation of HOXB13 protein, which in turn transcriptionally activates LTK . ChIP and luciferase reporter assays confirmed direct binding of HOXB13 to the LTK promoter and subsequent transcriptional activation. Importantly, SFMBT2 overexpression significantly disrupted this interaction, resulting in diminished LTK expression. These data suggest that HOXB13 serves as a critical transcriptional mediator bridging UHRF1/SFMBT2 dysregulation and LTK -driven oncogenic signalling. The delineation of this regulatory axis offers a novel mechanistic framework for understanding HCC progression and highlights the HOXB13/ LTK pathway as a potential therapeutic target in UHRF1-dependent tumours. However, this study is not without limitations. The relatively small sample size may limit the generalisability of our findings and necessitates caution when extrapolating results to a broader HCC population. Furthermore, the lack of longitudinal clinical validation restricts our ability to assess the temporal dynamics of UHRF1 expression and its prognostic significance over time. Additionally, while bioinformatics data provide valuable insights, they may introduce inherent biases and variability related to batch effects across datasets. These limitations underscore the importance of validating our findings in larger, more diverse cohorts and suggest that further investigations are warranted to confirm the role of UHRF1 as a prognostic biomarker and therapeutic target in HCC. Conclusions In conclusion, our findings suggest a novel oncogenic circuit in which UHRF1 promotes HCC progression by epigenetically repressing SFMBT2, thereby enabling activation of the ERK/AKT/NF-κB and HOXB13/ LTK pathways. The tumour-suppressive function of SFMBT2 serves as a critical counterbalance to UHRF1 activity, and its restoration significantly attenuates tumourigenic signalling. Moreover, our identification of the HOXB13/ LTK axis as a downstream effector of this epigenetic programme introduces new potential targets for therapeutic intervention. These findings provide a comprehensive framework for future studies aimed at disrupting UHRF1-mediated epigenetic repression and reactivating tumour suppressor pathways in HCC. Abbreviations HCC, hepatocellular carcinoma LTK, leukocyte tyrosine kinase UHRF1, ubiquitin-like containing PHD and RING finger domain 1 MEG3, maternally expressed gene 3 CSF1, colony-stimulating factor 1 SFMBT2, Scm-like with four mbt domains 2 miRNA, microRNA TCGA, The Cancer Genome Atlas UALCAN, Birmingham cancer data analysis portal DMEM, Dulbecco’s Modified Eagle Medium qRT-PCR, quantitative reverse transcription polymerase chain reaction WB, western blotting ChIP, chromatin immunoprecipitation cDNA, complementary DNA siRNA, small interfering RNA HOXB13, homeobox B13 IHC, immunohistochemistry p-ERK, phospho-extracellular signal-regulated kinase p-NF-κB, phospho-nuclear factor kappa-B MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide GEPIA, Gene Expression Profiling Interactive Analysis AKT, serine/threonine kinase TXNIP, thioredoxin-interacting protein Declarations Ethics approval and consent to participate This research was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University, School of Medicine ([2025B] IIT Ethics Approval No. 0483). All patients provided written informed consent. All procedures were conducted in accordance with the ARRIVE guidelines, the AVMA Guidelines for the Euthanasia of Animals (2020), as well as the ethical guidelines of the Declaration of Helsinki and the relevant local legislation and institutional requirements. Consent for publication Not applicable. Availability of data and materials The data analysed during this study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding This study was supported by the National Natural Science Foundation of China (No. 81970552). Author contributions Zekai Hu and Qi Sun contributed equally to this work as co-first authors. Zekai Hu and Qi Sun performed the experiments and analysed. Zekai Hu drafted the manuscript. Weiliang Xia obtained funding, Weiliang Xia and Zenglei He reviewed and edited the manuscript. Acknowledgements Not applicable. References Bray, F. et al. 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Supplementary Files Figure1C.pdf Figure1E.pdf Figure3AHepG2.pdf Figure3AHuh7.pdf Figure3BHepG2.pdf Figure3BHuh7.pdf Figure5AHepG2.pdf Figure5AHuh7.pdf Figure5BHepG2.pdf Figure5BHuh7.pdf Figure6A.pdf Figure6I.pdf Figure7CHepG2.pdf Figure7CHuh7.pdf Figure7H.pdf Figure7I.pdf Figure8A.pdf Figure9A.pdf Figure9B.pdf Figure9C.pdf Figure9G.pdf tumordata20251128.xlsx Cite Share Download PDF Status: Published Journal Publication published 14 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 19 Dec, 2025 Reviews received at journal 14 Dec, 2025 Reviews received at journal 07 Dec, 2025 Reviewers agreed at journal 05 Dec, 2025 Reviewers agreed at journal 03 Dec, 2025 Reviewers invited by journal 02 Dec, 2025 Editor assigned by journal 02 Dec, 2025 Editor invited by journal 02 Dec, 2025 Submission checks completed at journal 28 Nov, 2025 First submitted to journal 28 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":259093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eUHRF1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression is upregulated in HCC tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: qRT-PCR analysis of \u003cem\u003eUHRF1\u003c/em\u003emRNA expression in surgical specimens of HCC and adjacent non-cancerous tissues from patients.\u003c/p\u003e\n\u003cp\u003eB: Quantification of WB results showing significantly increased UHRF1 protein expression in HCC tissues (n = 22).\u003c/p\u003e\n\u003cp\u003eC: WB detection of UHRF1 protein expression in surgical specimens of HCC and adjacent non-cancerous tissues.\u003c/p\u003e\n\u003cp\u003eD: Representative IHC staining images of UHRF1 in human HCC tissues and adjacent non-cancerous tissues (400× magnification).\u003c/p\u003e\n\u003cp\u003eE: UHRF1 expression in six hepatoma cell lines.\u003c/p\u003e\n\u003cp\u003eF: Quantification of 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Normal group (n = 50) vs. primary tumour group (n = 371).\u003c/p\u003e\n\u003cp\u003eB: \u003cem\u003eUHRF1\u003c/em\u003e expression in LIHC increases with tumour grade. Normal group (n = 50), G1 (n = 60), G2 (n = 205), G3 (n = 138), G4 (n = 12).\u003c/p\u003e\n\u003cp\u003eC: Overall survival curves for patients with HCC (n = 365, p = 0.003) generated using the GEPIA database (http://gepia.cancer-pku.cn/).\u003c/p\u003e\n\u003cp\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7981198/v1/cbf21375e1c6698570291f85.png"},{"id":97668279,"identity":"34b6bcbe-9ae5-4cce-bcd9-5b1b7438643e","added_by":"auto","created_at":"2025-12-08 09:25:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":203570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUHRF1 regulates cell proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: WB analysis showing efficient UHRF1 overexpression following plasmid transfection in HepG2 and Huh7 cells.\u003c/p\u003e\n\u003cp\u003eB: WB analysis showing efficient UHRF1 knockdown following siRNA treatment in HepG2 and Huh7 cells.\u003c/p\u003e\n\u003cp\u003eC: MTT assay showing changes in cell proliferation at the indicated time points after UHRF1 overexpression or knockdownin HepG2 and Huh7 cells.\u003c/p\u003e\n\u003cp\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7981198/v1/6483d5ca3da8044828132568.png"},{"id":97667792,"identity":"f70a3710-2aaf-4800-a5e7-677e3919f65e","added_by":"auto","created_at":"2025-12-08 09:24:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":809022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUHRF1 regulates cell migration and invasion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Transwell assays showing increased cell migration and invasion following UHRF1 overexpression.\u003c/p\u003e\n\u003cp\u003eB: Transwell assays showing decreased cell migration and invasion following UHRF1knockdown.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7981198/v1/7f26c9790c98e2459d7ef1f1.png"},{"id":97667094,"identity":"31aaec83-c68a-49a9-b251-efa3329ae8d0","added_by":"auto","created_at":"2025-12-08 09:22:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":294570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUHRF1 regulates ERK/AKT/NF-κB signalling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: WB results showing upregulation of p-ERK, p-AKT, and p-NF-κB following UHRF1 overexpression.\u003c/p\u003e\n\u003cp\u003eB: WB results showing downregulation of p-ERK, p-AKT, and p-NF-κB following UHRF1 knockdown.\u003c/p\u003e\n\u003cp\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7981198/v1/3b0d7bdac9255eef5a519999.png"},{"id":97667160,"identity":"aa52423e-8e5c-4cd8-b272-d6d22db6939e","added_by":"auto","created_at":"2025-12-08 09:22:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":501251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of UHRF1 inhibits SFMBT2 in vivo and in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, B: \u003cem\u003eUHRF1\u003c/em\u003e overexpression cell lines were constructed and confirmed by WB and qRT-PCR.\u003c/p\u003e\n\u003cp\u003eC–E: RNA sequencing of \u003cem\u003eUHRF1\u003c/em\u003e-overexpressing cells showing (C) bar chart of differentially expressed genes, (D) volcano plot, and (E) heat map.\u003c/p\u003e\n\u003cp\u003eF: Gene Ontology analysis revealed enrichment in serine protease inhibitor activity and positive regulation of interleukin-4 production.\u003c/p\u003e\n\u003cp\u003eG: KEGG pathway analysis indicated enrichment in retinoid metabolism, synaptic vesicle cycle, NF-κB signaling pathway, and TGF-β signalling pathway.\u003c/p\u003e\n\u003cp\u003eH, I: WB and qRT-PCR validation of five candidate genes identified \u003cem\u003eSFMBT2 \u003c/em\u003eas significantly reduced in the \u003cem\u003eUHRF1\u003c/em\u003eoverexpression group, confirmed in HepG2 cells.\u003c/p\u003e\n\u003cp\u003eJ: IHC showing high UHRF1 and low SFMBT2 expression in HCC tissues.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7981198/v1/f11294aa3d3925bb21e2fe12.png"},{"id":97666311,"identity":"438cd3f4-65c6-4b49-849b-8fe2b3eac103","added_by":"auto","created_at":"2025-12-08 09:20:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":549267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUHRF1 binds to the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSFMBT2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter, and SFMBT2 overexpression inhibits cell migration, invasion, and proliferation and the ERK/NF-κB signalling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Distributionof three CpG islands on the \u003cem\u003eSFMBT2 \u003c/em\u003epromoter.\u003c/p\u003e\n\u003cp\u003eB: ChIP assay detecting UHRF1 binding to all three CpG islands on the\u003cem\u003e SFMBT2\u003c/em\u003e core promoter in HepG2 cells.\u003c/p\u003e\n\u003cp\u003eC: WB confirmingsuccessful overexpression of SFMBT2 in HepG2 and Huh7 cells.\u003c/p\u003e\n\u003cp\u003eD, E: Transwell assays showing reducedmigration and invasion following SFMBT2 overexpression in (D) HepG2 and (E) Huh7 cells.\u003c/p\u003e\n\u003cp\u003eF, G: MTT assays demonstrating that SFMBT2 overexpression suppresses proliferation in (F) HepG2 and (G) Huh7 cells.\u003c/p\u003e\n\u003cp\u003eH, I: WB showing decreased p-ERK and p-NF-κB levels after SFMBT2 overexpression in (H) HepG2 and (I) Huh7 cells.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7981198/v1/22de02835c8345414a3f18d4.png"},{"id":97426249,"identity":"cf7cedc1-10e8-4c24-93a0-7374ff89f779","added_by":"auto","created_at":"2025-12-04 09:17:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":940785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of SFMBT2 restores the tumour-promoting effect of UHRF1 both in vitro and in xenograft models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: In Huh7 cells, co-overexpression of UHRF1 and SFMBT2 was performed to assess changes in signalling pathways. WB confirmed that SFMBT2 overexpression reversed ERK/NF-κB pathway activation induced by UHRF1 overexpression.\u003c/p\u003e\n\u003cp\u003eB: MTT assay showing that SFMBT2 overexpression reversed UHRF1-induced proliferation.\u003c/p\u003e\n\u003cp\u003eC–E: Transwell assays demonstrating that SFMBT2 overexpression reversed UHRF1-induced migration and invasion in Huh7 cells.\u003c/p\u003e\n\u003cp\u003eF: Xenograftexperiments in nude mice showing that UHRF1 overexpression increased tumour volume and weight, while SFMBT2 overexpression reversed these effects.\u003c/p\u003e\n\u003cp\u003eG, H: Quantitative bar graphs of tumour (G) volume and (H) weight.\u003c/p\u003e\n\u003cp\u003eI: IHC of xenograft tissues showing that UHRF1 overexpression reduced SFMBT2 expression and increased p-ERK and p-NF-κB, while SFMBT2 overexpression reversed these changes.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7981198/v1/f987167dd6f7c487670f8edc.png"},{"id":97667097,"identity":"c728ee77-9b3e-4502-aca3-8e6f4f57dbc2","added_by":"auto","created_at":"2025-12-08 09:22:43","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":623906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUHRF1 promotes HOXB13-mediated transcriptional regulation of LTK by downregulating SFMBT2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic representation of the experimental findings showing that UHRF1 binds CpG islands in the \u003cem\u003eSFMBT2\u003c/em\u003e promoter, reducing its transcription. Lower SFMBT2 levels are associated with increased HOXB13 expression and enhanced binding of HOXB13 to the \u003cem\u003eLTK\u003c/em\u003e promoter, leading to elevated LTK expression.\u003c/p\u003e\n\u003cp\u003eA: WB analysis of HOXB13 and LTK expression levels in HepG2 cells following UHRF1 overexpression.\u003c/p\u003e\n\u003cp\u003eB: WB analysis of HOXB13 and LTK expression levels in HepG2 cells following SFMBT2 overexpression.\u003c/p\u003e\n\u003cp\u003eC: WB analysis of UHRF1, SFMBT2, HOXB13, and LTK expression levels in HepG2 cells co-overexpressing UHRF1 and SFMBT2.\u003c/p\u003e\n\u003cp\u003eD: IHC analysis of HOXB13 expression in tumour tissues. HOXB13 expression was elevated in the UHRF1-overexpression group compared with the control group, while SFMBT2 co-overexpression reversed this upregulation.\u003c/p\u003e\n\u003cp\u003eE: IHC analysis of LTK expression in tumour tissues. LTK expression was increased in the UHRF1-overexpression group compared with the control group, while SFMBT2 co-overexpression reversed this phenotype.\u003c/p\u003e\n\u003cp\u003eF: ChIP assay evaluating \u003cem\u003eLTK\u003c/em\u003e promoter enrichment in HepG2 cells. After UHRF1 overexpression, chromatin precipitated with HOXB13 antibody showed significantly increased binding of HOXB13 to the \u003cem\u003eLTK \u003c/em\u003epromoter DNA.\u003c/p\u003e\n\u003cp\u003eG: WB confirming successful overexpression of HOXB13 in HepG2 cells after transfection with HOXB13-expressing plasmids.\u003c/p\u003e\n\u003cp\u003eH: Dual-luciferase reporter assay in HepG2 cells co-transfected with wild-type or mutant \u003cem\u003eLTK\u003c/em\u003epromoter constructs (mutating the highest-scoring HOXB13 binding site predicted by the JASPAR database) and HOXB13-expressing plasmids.\u003c/p\u003e\n\u003cp\u003eI: Dual-luciferase reporter assay in HepG2 cells co-transfected with wild-type or mutant\u003cem\u003eLTK\u003c/em\u003e promoter constructs and SFMBT2-expressing plasmids.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7981198/v1/f08f8451e4c1bf9d5ba957c7.png"},{"id":107351793,"identity":"71ca2fb7-235a-4d0e-9967-d9b8308e2ed3","added_by":"auto","created_at":"2026-04-20 16:12:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5071639,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7981198/v1/9027f772-e31c-4e79-8e78-e89985f2f510.pdf"},{"id":97426231,"identity":"5be84f1a-39a3-45a4-b940-be90ca5d890e","added_by":"auto","created_at":"2025-12-04 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the third leading cause of cancer-related deaths, with hepatocellular carcinoma (HCC) accounting for approximately 75%\u0026ndash;85% of primary liver cancers\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Current research indicates that ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) acts as an epigenetic regulator, influencing gene expression through mechanisms such as DNA methylation and histone modification\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Emerging evidence establishes UHRF1 as a critical epigenetic regulator in various cancers, including pancreatic, colorectal, lung, and breast cancers\u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. UHRF1 plays essential roles in tumour cell proliferation, migration, invasion, metastasis, and drug resistance\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, and elevated expression has been found to correlate significantly with poor patient prognosis\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Furthermore, UHRF1 represents a potential target for anticancer drug development; compounds such as shikonin, hinokitiol, and dihydroartemisinin, which target UHRF1, have shown therapeutic effects in various cancers\u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUHRF1 promotes tumour progression through multiple distinct targets and signalling pathways, including KRAS-mediated mechanisms in lung adenocarcinoma\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e and the KISS1/PI3K/NF-κB signalling axis in colorectal cancer\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In HCC, studies have shown that UHRF1 facilitates tumour progression through epigenetic modification of genes such as maternally expressed gene 3 (\u003cem\u003eMEG3\u003c/em\u003e) and colony-stimulating factor 1 (\u003cem\u003eCSF1\u003c/em\u003e)\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, as well as by inducing immune cell infiltration\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. However, the precise molecular mechanisms by which UHRF1 contributes to HCC progression remain to be fully elucidated.\u003c/p\u003e\u003cp\u003eScm-like with four mbt domains 2 (SFMBT2), another epigenetic regulator, is involved in embryonic development and the pathogenesis of multiple diseases through its intronic microRNA (miRNA) clusters and circular RNA variants\u003csup\u003e[\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Previous studies have demonstrated that SFMBT2 exerts pleiotropic effects in cancer progression via distinct molecular mechanisms. This study was performed to address the current knowledge gap by investigating the signalling pathways and gene interactions associated with UHRF1, with particular focus on its relationship with SFMBT2.\u003c/p\u003e\u003cp\u003eTo this end, a multifaceted research approach was employed, integrating clinical sample analysis, bioinformatics, and both in vitro and in vivo studies. These methodologies enabled a comprehensive examination of UHRF1\u0026rsquo;s epigenetic regulatory mechanisms, expression patterns, functional roles, and interactions with other key genes implicated in HCC. In addition, the UHRF1\u0026ndash;SFMBT2 interaction network and SFMBT2\u0026rsquo;s potential as a prognostic biomarker and therapeutic target were explored, thereby highlighting its clinical significance in HCC management.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eClinical sample collection\u003c/h2\u003e\u003cp\u003eThis research was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University, School of Medicine ([2025B] IIT Ethics Approval No. 0483). All patients provided written informed consent. Matched HCC tissue specimens, comprising cancerous and adjacent non-cancerous tissues, were collected from 16 patients who underwent radical resection. Cancerous tissue was defined as tissue within 1 cm of the tumour edge without necrosis, and non-cancerous tissue was defined as tissue located more than 2 cm beyond the tumour edge. The patient inclusion criteria were no prior treatment before surgery, pathological confirmation of cancerous tissue as HCC, and confirmation that the surgical margin was free of residual carcinoma.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBioinformatics analysis\u003c/h3\u003e\n\u003cp\u003eLiver cancer RNA-sequencing data and corresponding clinical data were downloaded from TCGA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gdc.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://gdc.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The dataset was divided into two groups: primary tumour tissue (371 samples) and healthy tissue (50 samples). Using R software and the \u003cem\u003eggplot2\u003c/em\u003e package, a box plot was generated to illustrate the expression levels of UHRF1 in primary cancer versus healthy samples. Clinical data were further extracted for tumour grade, and using R software and the \u003cem\u003eggplot2\u003c/em\u003e package, a box plot was created to display UHRF1 expression levels across healthy tissue, G1, G2, G3, and G4. To assess the clinical relevance of UHRF1 in HCC, TCGA gene data were also analysed using the dataset available through the UALCAN (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ualcan.path.uab.edu\u003c/span\u003e\u003cspan address=\"http://ualcan.path.uab.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eLO2, Hep3B, HepG2, Hu7, SK-Hep-1, and PLC cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. All cell lines were cultured in high-glucose Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) (Gibco, CA, USA) supplemented with 10% foetal bovine serum, 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin, in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Cells in the exponential growth phase were harvested at 80%\u0026ndash;90% confluence for passage or experimental use.\u003c/p\u003e\n\u003ch3\u003ePlasmids and cell transfection\u003c/h3\u003e\n\u003cp\u003eShort hairpin RNA for LncSNHG1, negative control short hairpin RNA, miR-181b-5p inhibitor, and the corresponding negative control vector were all designed and synthesised by HanBio Company (China). Huh7 and HepG2 cells were cultured in six-well plates. When the cell density reached 70%\u0026ndash;80% confluence, 2.5 \u0026micro;g of the plasmid vector was transfected into the cells using Lipofectamine 3000 (Invitrogen, CA, USA) according to the manufacturer\u0026rsquo;s protocol. Transfection efficiency was assessed by fluorescence microscopy, qRT-PCR, and WB.\u003c/p\u003e\n\u003ch3\u003eAntibodies, plasmids, and primers\u003c/h3\u003e\n\u003cp\u003eDetails of the antibodies, plasmids, and primers used for RNA and ChIP analyses by qRT-PCR are provided in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of antibodies used in this study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTarget proteins\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCatalogue no.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUHRF1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eabcam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eab213223\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFlag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCST\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e#14793\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSFMBT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eproteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25256-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep-ERK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eproteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28733-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eERK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eproteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51068-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep-AKT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCST\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4060S\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAKT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eproteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13409-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep-NFκB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eproteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82335-1-RR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNFκB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eproteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10745-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLTK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBioss\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ebs-15500R\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHOXB13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbcam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eab201682\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eβ-actin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20536-1- AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of qRT-PCR primer sequences\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTarget gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime; forward primer sequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u0026prime; reverse primer sequence\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUHRF1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCTCAACTGCTTTGCTCCCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGTCCTTCCCCTCCTTCGTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTOMM6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTGCTGGCTCGGCTAATGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGAGGTGCCATGAGGTCAATGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSFMBT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAGCAGAGGAAGGGGAGAAGT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTTCAGCTCCATGCACTCCTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSLC27A6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGTTCGTGTTGAAGGTGGTGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGGTTCAGGAAGACATGGGC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCML1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAGTCCAGTGCATCCCTCAGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGACCAGGTTGAAGGGTGCTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCES3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCCACAGAGGAGGAGAAGCAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTATCTTGCTGGGGAGCGTCT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLTK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTGGTTCTGATGGTGGCTGT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGATGGCGAAACTTGCTGAT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHOXB13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAGACTCTGGGTGCTCCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGCCTCTTGTCCTTGGTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eβ-actin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAGCGGGAAATCGTGCGTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCAGGGTACATGGTGGTGCC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of qRT-PCR primer sequences used for ChIP analysis\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTarget gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime; forward primer sequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u0026prime; reverse primer sequence\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLTK promoter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTACCTGAAGGGAGTGGCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCTGTGATGGGGTGAAAGAC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSFMBT2-Island1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAGGTCCGTATGCCAGGTTTA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCAGCCTGGTGGGTCTTAACAT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSFMBT2-Island2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGAGTCCTTGAAAACCGTGTA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATCCAAAACATTATCACCTCA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSFMBT2-Island3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCCGACTTCTGGTGTGACGTAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGAAGGATGGCGGCTCGTA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eRNA extraction and qRT-PCR analyses\u003c/h2\u003e\u003cp\u003eTotal cellular RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer\u0026rsquo;s protocol. Subsequently, 500 ng of RNA was reverse-transcribed into complementary DNA (cDNA) using the Verso cDNA Synthesis Kit (Thermo Scientific). The synthesised cDNA was then diluted 25-fold and used as a template for qRT-PCR analysis. qRT-PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, CA, USA) and target-specific primers. Relative RNA expression was calculated by normalising target mRNA levels to β-actin expression as the internal control. Primer sequences are provided in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eRNA interference or overexpression\u003c/h3\u003e\n\u003cp\u003eFor \u003cem\u003eUHRF1\u003c/em\u003e silencing, transient transfection was performed in HepG2 and Huh7 cells using small interfering RNA (siRNA) (GenePharma, Shanghai, China). The siRNA was incubated with Lipofectamine 2000 (Invitrogen) at room temperature for 20 minutes to form a complex, and transfection was carried out at 37\u0026deg;C for either 24 or 48 hours. Silencing efficiency was confirmed by WB. The siRNA sequences are provided in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable 4:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esiRNA\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;information\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 261px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlasmid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 261px;\"\u003e\n \u003cp\u003eUHRF1 siRNA\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003efrom Qing Ke Bio, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 261px;\"\u003e\n \u003cp\u003ehUHRF1 siRNA-1 sense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003eGCCAGAGUGAGUCAGACAATT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003ehUHRF1 siRNA -1 antisense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003eUUGUCUGACUCACUCUGGCCC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003ehUHRF1 siRNA-2 sense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003eAUGUGGGAUGAGACGGAAUUG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003ehUHRF1 siRNA -2 antisense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003eCAAUUCCGUCUCAUCCCACAU\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003ehUHRF1 siRNA -3 sense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003eCCAGUUGUUCCUGAGUAAATT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003ehUHRF1 siRNA -3 antisense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003eUUUACUCAGGAACAACUGGAA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003esiRNA NC sense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003eUUCUCCGAACGAGUCACGUTT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003esiRNA NC antisense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003eACGUGACUCGUUCGGAGAATT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003eLTK WT\\MUT luciferase reporter gene plasmids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003efrom Qing Ke Bio, China.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003eLTK wild type plasmid sequence\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003eGGCCTGTGCTACATCACCCATTG\u003cbr\u003eACCAGAGGTGCCAGTACTAGTGCT\u003cbr\u003eCAAGATCAATCGATCGATCGGTCTA\u003cbr\u003eCCTACCTATCATCTATTGACCTTCAG\u003cbr\u003eTGCTACTAAAAACACTCGGATCTTCT\u003cbr\u003eAACGTCTGGTCCAGTCTTTCACCCCA\u003cbr\u003eTCACAGTGAGAGGCTGTGCACAGGG\u003cbr\u003eGTAACACAGGCAACGGAATTATATG\u003cbr\u003eAGGCAAACAC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003eLTK mutant plasmid sequence\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 307px;\"\u003e\n \u003cp\u003eGGCCTGTGCTACATCACCCATTGACCA\u003cbr\u003eGAGGTGCCAGTACTAGTGCTCAAGATC\u003cbr\u003eAATCGATCGATCGGTCTACCTACCTATC\u003cbr\u003eATCTATTGACCTTCAGTGTTTTTAGTAG\u003cbr\u003eCACTCGGATCTTCTAACGTCTGGTCCAG\u003cbr\u003eTCTTTCACCCCATCACAGTGAGAGGCTG\u003cbr\u003eTGCACAGGGGTAACACAGGCAACGG\u003cbr\u003eAATTATATGAGGCAAACAC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\u003c/br\u003e\u003cp\u003eFor \u003cem\u003eUHRF1\u003c/em\u003e, \u003cem\u003eSFMBT2\u003c/em\u003e, and \u003cem\u003eHOXB13\u003c/em\u003e overexpression, the coding sequences of human \u003cem\u003eUHRF1\u003c/em\u003e mRNA, \u003cem\u003eSFMBT2\u003c/em\u003e mRNA, and \u003cem\u003eHOXB13\u003c/em\u003e mRNA were synthesised, digested with HindIII and EcoRI, and subcloned into the pcDNA3.1 vector. The integrity of the plasmid constructs was verified by DNA sequencing. Overexpression plasmids or the pcDNA3.1 vector were incubated with Lipofectamine 2000 reagent (Invitrogen) at room temperature for 20 minutes to form a complex, and transfection was performed at 37\u0026deg;C for 24 hours. Overexpression efficiency was confirmed by WB. Plasmid information is provided in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePlasmids information\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlasmid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eID\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThe UHRF1 overexpression\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYouBao Bio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_013282\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThe SFMBT2 overexpression\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYouBao Bio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_001029880\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThe HOXB13 overexpression\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYouBao Bio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_006361\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eCell migration and invasion assay\u003c/h3\u003e\n\u003cp\u003eFor the migration assay, transfected cells were trypsinised, resuspended in serum-free medium, and transferred to the upper chambers of Transwell inserts in 24-well plates with an 8-\u0026micro;m pore size and polycarbonate membrane (Corning, NY, USA). DMEM supplemented with 10% foetal bovine serum was added to the lower chambers as a chemoattractant. After 24 or 48 hours, cells remaining in the upper chamber were removed with a cotton swab, while those on the underside of the membrane were fixed with ice-cold methanol, stained with 0.1% crystal violet, and counted under a microscope (Olympus). For the invasion assay, the Transwell membrane was coated with Matrigel (BD Biosciences) before cells were added, and all subsequent steps were performed as described for the migration assay.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eWB analysis\u003c/h2\u003e\u003cp\u003eTissue or cells were lysed on ice with RIPA buffer containing protease and phosphatase inhibitors. Total protein was extracted, and its concentration determined using a BCA Protein Reagent Kit (Thermo Fisher Scientific). Equal amounts of protein from each sample were separated on 10% or 12% sodium dodecyl sulfate\u0026ndash;polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, Germany). Non-specific binding was blocked with 5% bovine serum albumin, and membranes were incubated overnight at 4\u0026deg;C with primary antibody (1:1000), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:1000) (Abcam, Cambridge, UK) for 1 hour. After three washes using Tris-buffered saline with Tween 20, protein bands were detected and imaged using the Bio Imaging System (Bio-Rad). β-actin was used as a loading control. Details of the primary antibodies are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Protein expression levels were determined as the ratio of the target band intensity to that of β-actin. Each protein sample was measured in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIHC staining\u003c/h2\u003e\u003cp\u003eTumour tissue specimens were fixed in 10% neutral formalin for 24\u0026ndash;48 hours and routinely processed for paraffin embedding. IHC staining was performed as previously described\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. UHRF1, SFMBT2, p-ERK, p-NF-κB, HOXB13, and LTK antibodies (1:200) were detected using the streptavidin\u0026ndash;peroxidase conjugate method. Immunoreactivity was independently evaluated by two professional pathologists. For the assessment of UHRF1 protein expression in HCC, hepatocytes with light brown to dark brown nuclear staining were defined as positive cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCell proliferation assay\u003c/h2\u003e\u003cp\u003eCell proliferation was measured using a MTT assay. Briefly, cells were seeded in 96-well plates and incubated overnight, and viable cell numbers were assessed at 0, 24, 48, 72, and 96 hours after transfection. MTT solution was added to each well and incubated for 3 hours at 37\u0026deg;C in darkness. The purple formazan crystals were dissolved in dimethyl sulfoxide, and absorbance was measured at 570 nm using a microplate reader (Molecular Devices).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e\u003cp\u003eCells were transfected with the respective plasmid constructs expressing the target proteins (as indicated) and, after 24 hours, co-transfected with either the wild-type or mutant plasmids along with the overexpression plasmid. After a further 24 hours, reporter luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) on a GloMax 20/20 Luminometer (Promega), according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eChIP analysis\u003c/h2\u003e\u003cp\u003eThe cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature and quenched with 125 mM glycine for 5 minutes. After being washed with phosphate-buffered saline, the cells were lysed in ChIP lysis buffer (0.5% NP-40, 1% Triton X-100, 150 mM NaCl, 20 mM Tris\u0026ndash;Cl, pH 7.5, 2 mM EDTA) containing protease inhibitors and incubated on ice for 10 minutes. Chromatin was sonicated to 200- to 1000-bp fragments and centrifuged (14,000 rpm, 20 minutes, 4\u0026deg;C). The supernatant was diluted 10\u0026times; in ChIP dilution buffer (0.01% sodium dodecyl sulfate, 1.1% Triton X-100, 1.1 mM EDTA, 20 mM Tris\u0026ndash;Cl, pH 8.0, 167 mM NaCl). Aliquots were reserved as input, and the remaining samples were incubated overnight at 4\u0026deg;C with UHRF1 or HOXB13 antibody, followed by Protein A/G beads for 1 hour. The immunocomplexes were washed with 800 \u0026micro;L wash buffer, eluted, and incubated at room temperature for 30 minutes. After magnetic separation, the eluates were de-cross-linked with 5 M NaCl at 65\u0026deg;C for 4 hours, then treated with Core Mix at 45\u0026deg;C for 1 hour. DNA was purified using a commercial kit and analysed by qRT-PCR (Bio-Rad CFX96\u0026trade;) to assess enrichment at target loci.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eConstruction of stable cell lines\u003c/h2\u003e\u003cp\u003eLentivirus was packaged using 293T cells seeded in 15-cm dishes. At 50%\u0026ndash;60% confluence, cells were washed with phosphate-buffered saline and cultured in serum-free DMEM. A plasmid mixture containing 40 \u0026micro;g PP4R1, 30 \u0026micro;g psPAX2, and 10 \u0026micro;g pMD2.G (4:3:1) in 250 \u0026micro;L Opti-DMEM was combined with 10 \u0026micro;L Lipofectamine 2000 in 240 \u0026micro;L Opti-DMEM. After 20 minutes of incubation, the complex was added to the cells. Viral supernatants were collected every 24 hours, centrifuged (3,500 rpm, 10 minutes, 4\u0026deg;C), filtered (0.22 \u0026micro;m), and ultracentrifuged (30,000 rpm, 2 hours, 4\u0026deg;C). The pellets were resuspended overnight in serum-free medium and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. For stable transduction, HepG2 cells (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were infected with \u003cem\u003eUHRF1\u003c/em\u003e lentivirus (MOI\u0026thinsp;=\u0026thinsp;200, 1.0 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e TU/mL). After 48 hours, the medium was replaced and the cells were cultured for a further 24 hours before puromycin selection (1 \u0026micro;g/mL). Following three passages, fluorescent-positive cells were harvested for WB analysis of UHRF1 expression. \u003cem\u003eUHRF1\u003c/em\u003e-overexpressing cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were transfected with the SFMBT2 overexpression plasmid at 50%\u0026ndash;60% confluence. After 48 hours, selection was initiated with G418 (50 \u0026micro;g/mL), and the cells were maintained with daily medium changes and passaged every 2\u0026ndash;3 days. Cells were collected after three passages for WB confirmation of SFMBT2 expression. Reagents and materials are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMaterials for stable cell lines\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReagent Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eID\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePackaging plasmid psPAX2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYouBao Bio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVT1444\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnvelope plasmid pMD2.G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYouBao Bio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVT1443\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG418\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSolarbio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIG0010\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePuromycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSolarbio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP8230\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eAnimal assay\u003c/h2\u003e\u003cp\u003eThe mouse experiments were approved by the Animal Ethics Committee of Zhejiang University and conducted in accordance with animal health guidelines. All procedures were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020), as well as the ethical guidelines of the Declaration of Helsinki and the relevant local legislation and institutional requirements. Nude mice were obtained from Shanghai Slake Laboratory Animal Co., Ltd., SCKK (Shanghai) 2022-0004 (Licence). The mice were randomly assigned to three groups (n\u0026thinsp;=\u0026thinsp;6 per group). HepG2 cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) stably transfected with sh\u003cem\u003eUHRF1\u003c/em\u003e, sh\u003cem\u003eSFMBT2\u003c/em\u003e, or sh\u003cem\u003eUHRF1/SFMBT2\u003c/em\u003e were injected subcutaneously into 4-week-old nude mice using a 1-mL sterile syringe. The mice were examined every 4 days after injection. When tumours in the control group reached a volume of 1,500 mm\u003csup\u003e3\u003c/sup\u003e, all mice were euthanised by cervical dislocation, and tumour tissues were collected. The body weights of mice in each group at the time of euthanised were 16.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 g, 18.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.57 g, and 18.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 g, respectively. Tumour volumes were calculated as V = (length \u0026times; width\u003csup\u003e2\u003c/sup\u003e) / 2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses and dataset analysis\u003c/h2\u003e\u003cp\u003eResults are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Analyses were performed using SPSS 26.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). The mRNA expression of genes in HCC was analysed using Wilcoxon\u0026rsquo;s paired test. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eUHRF1\u003c/b\u003e \u003cb\u003eexpression is upregulated in HCC tissues\u003c/b\u003e\u003c/p\u003e\u003cp\u003eqRT-PCR (quantitative reverse transcription polymerase chain reaction) analysis revealed that \u003cem\u003eUHRF1\u003c/em\u003e mRNA expression was significantly higher in the cancerous tissue group than in the non-cancerous tissue group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). IHC (immunohistochemistry) and WB (western blotting) further confirmed that UHRF1 protein levels were markedly elevated in HCC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;D). Notably, UHRF1 protein was almost undetectable in non-cancerous tissues, suggesting a potential role in HCC pathogenesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next assessed \u003cem\u003eUHRF1\u003c/em\u003e expression in a panel of HCC cell lines (Hep3B, HepG2, Huh7, SK-Hep-1, and PLC) and a normal liver cell line (LO2). WB showed no detectable \u003cem\u003eUHRF1\u003c/em\u003e expression in LO2 cells. By contrast, \u003cem\u003eUHRF1\u003c/em\u003e was highly expressed in HepG2 and SK-Hep-1 cells, while expression was relatively low in Hep3B, Huh7, and PLC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F).\u003c/p\u003e\u003cp\u003eTo validate these findings, \u003cem\u003eUHRF1\u003c/em\u003e expression data from TCGA (The Cancer Genome Atlas) were analysed using the UALCAN (University of Alabama at Birmingham cancer data analysis portal) dataset. Consistent with our results, \u003cem\u003eUHRF1\u003c/em\u003e expression levels were significantly higher in HCC tissues than in normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, \u003cem\u003eUHRF1\u003c/em\u003e expression was positively correlated with tumour grade, with higher expression observed in poorly differentiated HCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Importantly, survival analysis of patients with HCC using the Gene Expression Profiling Interactive Analysis (GEPIA) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) showed that elevated \u003cem\u003eUHRF1\u003c/em\u003e expression was associated with significantly worse overall survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings suggest that \u003cem\u003eUHRF1\u003c/em\u003e may serve as a prognostic biomarker in HCC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eUHRF1 promotes HCC cell proliferation, migration, and invasion\u003c/h2\u003e\u003cp\u003eTo investigate the functional role of UHRF1 in HCC, we first established \u003cem\u003eUHRF1\u003c/em\u003e overexpression and knockdown models in HepG2 and Huh7 cells. WB confirmed successful UHRF1 protein overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) as well as efficient knockdown in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Subsequent MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays revealed that UHRF1 overexpression significantly enhanced cell proliferation in both cell lines, whereas UHRF1 knockdown suppressed proliferative capacity. These changes reached statistical significance at the 96-hour time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTranswell assays further demonstrated that UHRF1 overexpression markedly increased migratory and invasive capabilities in HCC cells, while UHRF1 knockdown conversely reduced these malignant behaviours, indicating a pivotal role of UHRF1 in promoting HCC cell migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIt has been reported that the development and progression of many cancers, including HCC\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, are closely associated with activation of the serine/threonine kinase (AKT). Moreover, ERK (extracellular signal-regulated kinase) and NF-κB (nuclear factor kappa-B) also play critical roles in mediating oncogenic processes in HCC\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Therefore, we performed WB to assess the effects of UHRF1 on the ERK/AKT/NF-κB signalling pathway in HepG2 and Huh7 cells. The results indicated that UHRF1 knockdown significantly reduced the phosphorylation levels of ERK, AKT, and NF-κB, while UHRF1 overexpression markedly increased their activation, with all changes showing statistically significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eOverexpression of UHRF1 inhibits SFMBT2 in vivo and in vitro\u003c/h2\u003e\u003cp\u003eNext, we sequenced the \u003cem\u003eUHRF1\u003c/em\u003e-overexpressing HepG2 cells to identify differentially expressed genes. Successful establishment of the \u003cem\u003eUHRF1\u003c/em\u003e overexpression cell line was confirmed by WB (protein level) and qRT-PCR (gene level) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). A total of 370 genes were identified, including 163 upregulated and 207 downregulated genes. Visualisation of the sequencing data included a bar chart showing the distribution of the genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), a volcano plot highlighting expression changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), and a heat map clustering these genes based on expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGene ontology analysis revealed that the differentially expressed genes in \u003cem\u003eUHRF1\u003c/em\u003e-overexpressing cells were predominantly enriched in biological processes and molecular functions related to serine protease inhibitor activity and positive regulation of interleukin-4 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Kyoto Encyclopedia of Genes and Genomes (KEGG) is a widely used bioinformatics database resource for understanding high-level functions and utilities of the biological system \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. KEGG pathway analysis further demonstrated significant enrichment in pathways critical to HCC progression, including retinoid metabolism, synaptic vesicle cycle, NF-κB signalling pathway, and transforming growth factor-β (TGF-β) signalling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). KEGG pathway identifiers and names were retrieved from the KEGG database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.kegg.jp/kegg/kegg1.html\u003c/span\u003e\u003cspan address=\"https://www.kegg.jp/kegg/kegg1.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eqRT-PCR was then used to validate the expression of five candidate differentially expressed genes. Among these, \u003cem\u003eSFMBT2\u003c/em\u003e showed a significant reduction in mRNA levels in \u003cem\u003eUHRF1\u003c/em\u003e-overexpressing cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). This downregulation was further corroborated in HepG2 cells at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). IHC staining of clinical HCC tissues revealed an inverse correlation between UHRF1 and SFMBT2 protein expression: HCC tissues exhibited strong nuclear immunoreactivity for UHRF1, whereas adjacent non-cancerous tissues displayed much less staining. Conversely, SFMBT2 protein expression was markedly suppressed in HCC tissues compared with non-cancerous counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ).\u003c/p\u003e\u003cp\u003e\u003cb\u003eUHRF1 binds to the\u003c/b\u003e \u003cb\u003eSFMBT2\u003c/b\u003e \u003cb\u003epromoter, and SFMBT2 overexpression inhibits cell migration, invasion, and proliferation and the ERK/AKT/NF-κB signalling pathway\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe identified three CpG islands within the promoter of \u003cem\u003eSFMBT2\u003c/em\u003e using the MethPrimer database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.methprimer.com/index.html\u003c/span\u003e\u003cspan address=\"https://www.methprimer.com/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). ChIP (chromatin immunoprecipitation) assays demonstrated that UHRF1 protein binds to all three CpG islands in HepG2 cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for island 1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for islands 2 and 3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These results indicate that UHRF1 directly interacts with the \u003cem\u003eSFMBT2\u003c/em\u003e promoter, likely mediating transcriptional repression through CpG island targeting.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo validate these findings, \u003cem\u003eSFMBT2\u003c/em\u003e-overexpressing HepG2 and Huh7 cell lines were established. WB confirmed successful overexpression at the protein level, with distinct electrophoretic bands corresponding to SFMBT2 detected (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for both cell lines) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). SFMBT2 overexpression significantly reduced migration and invasion in both HepG2 and Huh7 cells, supporting its tumour-suppressive role (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and E). In addition, SFMBT2 overexpression significantly inhibited cell proliferation in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF and G).\u003c/p\u003e\u003cp\u003eWe next performed WB to examine the effects of SFMBT2 on the ERK/AKT/NF-κB signalling pathway. SFMBT2 overexpression significantly decreased phosphorylation levels of ERK and NF-κB in both HepG2 and Huh7 cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while total ERK and NF-κB protein levels remained unchanged, indicating selective inhibition of pathway activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH and I).\u003c/p\u003e\u003cp\u003e\u003cb\u003eOverexpression of SFMBT2 restores the tumour-promoting effect of UHRF1 both in vitro and in xenograft models\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the functional interplay between UHRF1 and SFMBT2 in HCC, Huh7 cells were co-transfected with UHRF1 and SFMBT2 overexpression plasmids. WB showed that UHRF1 protein overexpression significantly increased the phosphorylation levels of ERK and NF-κB. Strikingly, SFMBT2 protein overexpression reversed these effects, restoring p-ERK (phospho-extracellular signal-regulated kinase) and p-NF-κB (phospho-nuclear factor kappa-B) levels to near baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMTT assays demonstrated that UHRF1 protein overexpression significantly enhanced cell proliferation from 48 to 72 hours post-transfection, whereas co-expression of SFMBT2 protein abolished these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Transwell assays revealed that UHRF1 protein overexpression increased cell migration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while SFMBT2 protein co-expression restored migration to control levels. Similar effects were consistently observed in the corresponding invasion assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC\u0026ndash;E).\u003c/p\u003e\u003cp\u003eA subcutaneous xenograft model was established by inoculating HepG2 cells into nude mice. Quantitative analysis of tumour growth showed that UHRF1 protein overexpression significantly increased tumour volume (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and weight (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with vector controls at endpoint. Conversely, concomitant SFMBT2 protein overexpression reversed these oncogenic effects, reducing tumour volume and weight to levels comparable to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF\u0026ndash;H). Further IHC profiling of xenograft tissues confirmed that UHRF1 protein overexpression suppressed SFMBT2 protein expression while concomitantly increasing p-ERK and p-NF-κB expression. Consistent with previous experiments, SFMBT2 protein co-overexpression reversed these changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eUHRF1 downregulates SFMBT2 to promote HOXB13-dependent LTK transcription\u003c/h2\u003e\u003cp\u003ePrevious studies have shown that SFMBT2 protein can epigenetically repress \u003cem\u003eHOXB13\u003c/em\u003e (homeobox B13) transcription by binding methylated histones, thereby downregulating oncogenic pathways such as \u003cem\u003eLTK\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. To investigate the functional interplay among UHRF1, SFMBT2, and HOXB13 in HCC pathogenesis, HepG2 cells were transfected with UHRF1 and SFMBT2 overexpression plasmids, and WB was performed to assess HOXB13 and LTK protein levels. UHRF1 protein overexpression significantly increased HOXB13 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and LTK (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA), whereas SFMBT2 protein overexpression suppressed both (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn HepG2 cells with concomitant UHRF1 and SFMBT2 protein overexpression, UHRF1 significantly increased HOXB13 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and LTK (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) protein levels compared with empty vector controls. However, co-overexpression of SFMBT2 reversed these effects, reducing HOXB13 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and LTK (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). IHC analysis of HCC tissues confirmed that UHRF1 protein overexpression increased HOXB13 and LTK protein levels, whereas SFMBT2 protein co-expression reversed these oncogenic trends (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD and E).\u003c/p\u003e\u003cp\u003eChIP assays were then performed to examine enrichment of the \u003cem\u003eLTK\u003c/em\u003e promoter in HepG2 cells and to investigate the regulatory interplay between UHRF1 and HOXB13 in \u003cem\u003eLTK\u003c/em\u003e transcription. Following UHRF1 protein overexpression, chromatin fragments immunoprecipitated with a HOXB13-specific antibody showed significantly increased binding to \u003cem\u003eLTK\u003c/em\u003e promoter DNA compared with the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that UHRF1 enhances HOXB13-mediated transcriptional regulation of \u003cem\u003eLTK\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eTo further dissect HOXB13\u0026rsquo;s role in \u003cem\u003eLTK\u003c/em\u003e regulation, a HOXB13 overexpression plasmid was constructed and transfected into HepG2 cells. WB confirmed increased HOXB13 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG). Based on JASPAR database predictions, the highest-scoring HOXB13 binding site within the \u003cem\u003eLTK\u003c/em\u003e core promoter was subjected to site-directed mutagenesis. Wild-type and mutant-type \u003cem\u003eLTK\u003c/em\u003e promoter sequences were cloned into dual-luciferase reporter plasmids. Co-transfection of the HOXB13 overexpression plasmid with the wild-type reporter into HepG2 cells increased luciferase activity compared with the pcDNA3.1 empty vector control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas co-transfection with the mutant-type reporter abrogated this effect, demonstrating that HOXB13 regulates \u003cem\u003eLTK\u003c/em\u003e promoter activity via the predicted binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003eFinally, to validate the regulatory role of SFMBT2 in \u003cem\u003eLTK\u003c/em\u003e expression, wild-type and mutant-type \u003cem\u003eLTK\u003c/em\u003e promoter reporter plasmids were co-transfected with SFMBT2 overexpression plasmids into HepG2 cells. SFMBT2 protein overexpression significantly suppressed wild-type promoter activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but had no significant effect on the mutant-type construct, indicating that SFMBT2 modulates \u003cem\u003eLTK\u003c/em\u003e expression primarily through HOXB13-dependent chromatin interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHCC development is driven by complex epigenetic abnormalities. In the context of early diagnosis, DNA methylation biomarkers show significant clinical potential, with combined detection of promoter hypermethylation in specific genes markedly improving HCC identification\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. During tumour invasion and metastasis, histone modifications play a central regulatory role in modulating tumour cell invasion and in vivo dissemination\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. In addition, metabolite\u0026ndash;epigenetic interactions act as critical drivers: for example, acetyl-CoA accumulation induces epithelial\u0026ndash;mesenchymal transition, thereby promoting metastasis\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, while succinyl transferases accelerate tumour progression by enhancing glycolytic flux\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Environmental factors, such as alcohol, also contribute to carcinogenesis through mechanisms including acetaldehyde-mediated DNA adduct formation, oxidative stress, and alterations in DNA methylation patterns\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Together, these findings highlight the pivotal role of the epigenetic regulatory network in HCC and provide a theoretical basis for the development of early diagnostic biomarkers and targeted therapeutic strategies.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that suppression of the AMP-activated protein kinase signalling pathway\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, hypermethylation at the thioredoxin-interacting protein (\u003cem\u003eTXNIP\u003c/em\u003e) promoter\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, and p53 pathway inactivation\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e are involved in the tumour-suppressive effects of UHRF1 loss. In our study, we delineate a previously uncharacterised epigenetic mechanism by which UHRF1 contributes to HCC progression. Consistent with its established role as an epigenetic integrator, UHRF1 was markedly upregulated in HCC tissues and cell lines. These observations align with evidence from other malignancies, in which UHRF1 overexpression has been consistently linked to aggressive tumour phenotypes\u003csup\u003e[\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Mechanistically, we demonstrate that UHRF1 exerts transcriptional repression of the tumour suppressor gene \u003cem\u003eSFMBT2\u003c/em\u003e by directly binding to CpG-rich islands within its promoter region. This epigenetic silencing is consistent with UHRF1\u0026rsquo;s known ability to recruit DNA methyltransferases and modify chromatin structure, as reported in other cancers\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Importantly, our findings show that UHRF1-mediated repression of \u003cem\u003eSFMBT2\u003c/em\u003e facilitates the activation of downstream oncogenic signalling in HCC. These results position UHRF1 not only as a prognostic biomarker but also as a promising therapeutic target. Bioinformatics analyses further reinforced the clinical relevance of UHRF1 in HCC, revealing a strong correlation between UHRF1 overexpression and advanced tumour grade, as well as poorer overall survival. This suggests that UHRF1 is a robust prognostic biomarker for HCC \u0026mdash; a role similarly observed in other malignancies, where high UHRF1 expression predicts unfavourable clinical outcomes, including reduced overall survival and increased recurrence rates\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAccumulating evidence shows that SFMBT2 protein participates in the regulation of tumour suppressor genes through pathways such as miR-885-3p/\u003cem\u003eCHD7\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, miR-107/\u003cem\u003eSLC1A5\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e, and miR-182-5p/\u003cem\u003eCREB1\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. This regulatory activity modulates multiple signalling pathways, including Wnt/β-catenin and glutamine metabolism, ultimately playing a critical role in tumorigenesis, invasion, and metabolic adaptation. These findings not only highlight SFMBT2 as a pan-cancer regulatory factor but also underscore its potential as a novel diagnostic biomarker and therapeutic target. Our study, for the first time, reveals the collaborative role of SFMBT2 and UHRF1 proteins in promoting tumour progression. We establish SFMBT2 as a pivotal epigenetic regulator downstream of UHRF1, with substantial tumour-suppressive activity in HCC. SFMBT2 protein overexpression attenuated malignant phenotypes, including proliferation, migration, and invasion, while concurrently restoring ERK/AKT/NF-κB signalling pathway homeostasis. Notably, SFMBT2 protein was able to reverse the oncogenic effects induced by UHRF1 protein overexpression, both in vitro and in xenograft models. These findings underscore the functional importance of SFMBT2 in modulating UHRF1-driven transcriptional programmes.\u003c/p\u003e\u003cp\u003eIn addition to confirming UHRF1 and SFMBT2 proteins as key regulators of tumour aggressiveness, our study identifies the HOXB13/\u003cem\u003eLTK\u003c/em\u003e signalling cascade as a novel downstream effector axis. We show that UHRF1-mediated repression of SFMBT2 leads to upregulation of HOXB13 protein, which in turn transcriptionally activates \u003cem\u003eLTK\u003c/em\u003e. ChIP and luciferase reporter assays confirmed direct binding of HOXB13 to the \u003cem\u003eLTK\u003c/em\u003e promoter and subsequent transcriptional activation. Importantly, SFMBT2 overexpression significantly disrupted this interaction, resulting in diminished \u003cem\u003eLTK\u003c/em\u003e expression. These data suggest that HOXB13 serves as a critical transcriptional mediator bridging UHRF1/SFMBT2 dysregulation and \u003cem\u003eLTK\u003c/em\u003e-driven oncogenic signalling. The delineation of this regulatory axis offers a novel mechanistic framework for understanding HCC progression and highlights the HOXB13/\u003cem\u003eLTK\u003c/em\u003e pathway as a potential therapeutic target in UHRF1-dependent tumours.\u003c/p\u003e\u003cp\u003eHowever, this study is not without limitations. The relatively small sample size may limit the generalisability of our findings and necessitates caution when extrapolating results to a broader HCC population. Furthermore, the lack of longitudinal clinical validation restricts our ability to assess the temporal dynamics of UHRF1 expression and its prognostic significance over time. Additionally, while bioinformatics data provide valuable insights, they may introduce inherent biases and variability related to batch effects across datasets. These limitations underscore the importance of validating our findings in larger, more diverse cohorts and suggest that further investigations are warranted to confirm the role of UHRF1 as a prognostic biomarker and therapeutic target in HCC.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our findings suggest a novel oncogenic circuit in which UHRF1 promotes HCC progression by epigenetically repressing SFMBT2, thereby enabling activation of the ERK/AKT/NF-κB and HOXB13/\u003cem\u003eLTK\u003c/em\u003e pathways. The tumour-suppressive function of SFMBT2 serves as a critical counterbalance to UHRF1 activity, and its restoration significantly attenuates tumourigenic signalling. Moreover, our identification of the HOXB13/\u003cem\u003eLTK\u003c/em\u003e axis as a downstream effector of this epigenetic programme introduces new potential targets for therapeutic intervention. These findings provide a comprehensive framework for future studies aimed at disrupting UHRF1-mediated epigenetic repression and reactivating tumour suppressor pathways in HCC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHCC, hepatocellular carcinoma\u003c/p\u003e\u003cp\u003eLTK, leukocyte tyrosine kinase\u003c/p\u003e\u003cp\u003eUHRF1, ubiquitin-like containing PHD and RING finger domain 1\u003c/p\u003e\u003cp\u003eMEG3, maternally expressed gene 3\u003c/p\u003e\u003cp\u003eCSF1, colony-stimulating factor 1\u003c/p\u003e\u003cp\u003eSFMBT2, Scm-like with four mbt domains 2\u003c/p\u003e\u003cp\u003emiRNA, microRNA\u003c/p\u003e\u003cp\u003eTCGA, The Cancer Genome Atlas\u003c/p\u003e\u003cp\u003eUALCAN, Birmingham cancer data analysis portal\u003c/p\u003e\u003cp\u003eDMEM, Dulbecco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e\u003cp\u003eqRT-PCR, quantitative reverse transcription polymerase chain reaction\u003c/p\u003e\u003cp\u003eWB, western blotting\u003c/p\u003e\u003cp\u003eChIP, chromatin immunoprecipitation\u003c/p\u003e\u003cp\u003ecDNA, complementary DNA\u003c/p\u003e\u003cp\u003esiRNA, small interfering RNA\u003c/p\u003e\u003cp\u003eHOXB13, homeobox B13\u003c/p\u003e\u003cp\u003eIHC, immunohistochemistry\u003c/p\u003e\u003cp\u003ep-ERK, phospho-extracellular signal-regulated kinase\u003c/p\u003e\u003cp\u003ep-NF-κB, phospho-nuclear factor kappa-B\u003c/p\u003e\u003cp\u003eMTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\u003c/p\u003e\u003cp\u003eGEPIA, Gene Expression Profiling Interactive Analysis\u003c/p\u003e\u003cp\u003eAKT, serine/threonine kinase\u003c/p\u003e\u003cp\u003eTXNIP, thioredoxin-interacting protein\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University, School of Medicine ([2025B] IIT Ethics Approval No. 0483). All patients provided written informed consent.\u0026nbsp;All procedures were conducted in accordance with the ARRIVE guidelines, the AVMA Guidelines for the Euthanasia of Animals (2020), as well as the ethical guidelines of the\u0026nbsp;Declaration of Helsinki and the relevant local legislation and institutional requirements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data analysed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (No. 81970552).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZekai Hu and Qi Sun contributed equally to this work as co-first authors. Zekai Hu and Qi Sun performed the experiments and analysed. Zekai Hu drafted the manuscript. Weiliang Xia obtained funding, Weiliang Xia and Zenglei He reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA Cancer J. Clin.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e (3), 229\u0026ndash;263 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamaguchi, K. et al. Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (1), 2960 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu, Q. et al. 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Circ-SFMBT2 promotes the proliferation of gastric cancer cells through sponging miR-182-5p to enhance CREB1 expression. \u003cem\u003eCancer Manag Res.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 5725\u0026ndash;5734 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"UHRF1, SFMBT2, hepatocellular carcinoma, epigenetics, HOXB13","lastPublishedDoi":"10.21203/rs.3.rs-7981198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7981198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eHepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality, necessitating the identification of novel therapeutic targets. UHRF1 is an epigenetic regulator implicated in various cancers, but its precise mechanistic role in HCC progression remains incompletely understood. This study investigates the epigenetic mechanisms by which UHRF1 promotes HCC pathogenesis, with a focus on its interaction with SFMBT2.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA multifaceted approach integrating clinical sample analysis, bioinformatics, in vitro cell culture experiments, and in vivo xenograft models was employed. UHRF1 expression was assessed in HCC tissues and cell lines. Functional roles were investigated through overexpression and knockdown models, evaluated by MTT, Transwell, and Western blot assays. ChIP and luciferase reporter assays were used to examine promoter binding and transcriptional regulation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eUHRF1 was significantly upregulated in HCC tissues and correlated with advanced tumor grade and poor survival. UHRF1 promoted HCC cell proliferation, migration, and invasion by activating the ERK/AKT/NF-κB signaling pathways. Mechanistically, UHRF1 bound to CpG islands in the SFMBT2 promoter, epigenetically repressing its expression. SFMBT2 overexpression reversed UHRF1-driven oncogenic effects in vitro and in vivo. Furthermore, UHRF1-mediated SFMBT2 downregulation led to increased HOXB13 expression, which transcriptionally activated LTK.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study identifies a novel UHRF1/SFMBT2 epigenetic axis critical for HCC progression. UHRF1 represses SFMBT2 to activate ERK/AKT/NF-κB and HOXB13/LTK pathways, driving tumor aggressiveness. Restoration of SFMBT2 counteracts these effects, highlighting its tumor-suppressive role. These findings position UHRF1 as a promising prognostic biomarker and therapeutic target in HCC.\u003c/p\u003e","manuscriptTitle":"UHRF1 Drives Hepatocellular Carcinoma Progression via Epigenetic Repression of SFMBT2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 09:17:39","doi":"10.21203/rs.3.rs-7981198/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T08:41:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T03:58:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-07T10:37:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2491314230098690261617161647632458263","date":"2025-12-06T02:48:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"227399795792028354484741028651974134060","date":"2025-12-03T15:06:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T14:27:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-02T14:08:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-02T11:12:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-29T04:30:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-29T04:23:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"46560c98-87d5-4d22-8010-9a6344843c8c","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":59039386,"name":"Biological sciences/Cancer"},{"id":59039387,"name":"Biological sciences/Cell biology"},{"id":59039388,"name":"Biological sciences/Molecular biology"},{"id":59039389,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-04-20T16:09:37+00:00","versionOfRecord":{"articleIdentity":"rs-7981198","link":"https://doi.org/10.1038/s41598-026-45705-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-14 15:57:02","publishedOnDateReadable":"April 14th, 2026"},"versionCreatedAt":"2025-12-04 09:17:39","video":"","vorDoi":"10.1038/s41598-026-45705-9","vorDoiUrl":"https://doi.org/10.1038/s41598-026-45705-9","workflowStages":[]},"version":"v1","identity":"rs-7981198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7981198","identity":"rs-7981198","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}