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. 2026 Jan 4;26:59. doi: 10.1186/s12935-025-04089-y

Silencing Uracil-DNA glycosylase inhibits colorectal cancer progression

Jing-jing Yang 1,#, Li-zhou Shi 2,#, Yong-wei Hu 2, Hua Chen 3, Qing-hua Wang 1,✉, Wei Han 2,✉
PMCID: PMC12866510  PMID: 41486373

Abstract

Dysregulation of repair pathways, specifically the base excision repair (BER) axis, is frequently observed in cancer. This dysfunction is associated with accelerated tumor growth and reduced patient survival, suggesting that BER components may serve as potential targets for cancer therapy. This study focused on the expression of Uracil-DNA glycosylase (UNG), a key enzyme in BER, in colorectal tumor tissues and adjacent normal tissues. We systematically explored the clinical significance, biological functions, and molecular mechanisms of UNG in colorectal cancer (CRC). Our findings revealed that UNG was significantly upregulated in CRC, and its higher expression correlated with poorer patient outcomes. Functional assays demonstrated that silencing UNG inhibited cell proliferation, reduced migration and invasion capabilities, and induced apoptosis along with S/G2 phase cell cycle arrest. Mechanistically, UNG appears to interact with the AMPK, AKT/mTOR, and ERK signaling pathways. These results suggested that UNG exhibited oncogenic potential in CRC by regulating malignant behaviors and oncogenic signaling cascades, positioned it as a potential prognostic biomarker and therapeutic target for CRC treatment.

Keywords: Uracil-DNA glycosylase, Colorectal cancer, Proliferation, Apoptosis, AKT/mTOR signaling pathway

Introduction

Colorectal cancer (CRC) is the most common malignant tumor of the digestive tract, with high morbidity and mortality [1]. Despite advancements in diagnosis and treatment, postoperative recurrence and metastasis still lead to poor prognosis [2]. These clinical challenges underscore the urgent need to clarify the molecular mechanisms that drive CRC progression and to identify new therapeutic targets.

Genomic instability, a recognized hallmark of cancer [3], primarily arises from DNA damage and replication errors [4, 5], thereby driving tumorigenesis across various malignancies [6]. The maintenance of genomic integrity is facilitated by the Base Excision Repair (BER) pathway, which addresses most endogenous DNA damage and eliminates exogenous DNA base lesions. Additionally, the BER pathway plays a significant role in therapeutic strategies and cancer prognosis [7]. Uracil DNA glycosylase (UNG), a pivotal enzyme in the BER pathway, catalyzes the recognition and excision of uracil from DNA, thereby preventing detrimental mutations [8, 9]. Early studies have reported aberrant UNG expression in prostate cancer [10], lung cancer [11, 12], and colorectal cancer [13]. Specifically, the inhibition of UNG has been shown to impair the survival of prostate cancer cells by inducing DNA damage and apoptosis, while also increasing sensitivity to genotoxic stress [10]. Furthermore, UNG inhibition enhances the sensitivity of lung cancer cells to pemetrexed [11], while elevated UNG levels in non-small cell lung cancer (NSCLC) are significantly associated with poorer patient prognosis and a more aggressive phenotype [12]. Recent investigations have also indicated that UNG is upregulated in prostate cancer, potentially contributing to the maintenance of genomic stability and the promotion of tumor cell survival [14]. Given its critical role in maintaining BER-mediated genomic stability and its involvement in tumorigenesis and chemoresistance, UNG has attracted considerable attention in the field of oncology. Moreover, elevated UNG activity has been observed in human colorectal tumors [13], and germline alterations in UNG have been linked to susceptibility to colorectal cancer [15]. However, research on UNG in colorectal cancer remains preliminary. The precise biological role of UNG in CRC progression, along with its underlying molecular mechanisms, is not yet fully understood and warrants further investigation.

This study aimed to elucidate the role and underlying regulatory mechanisms of UNG in the progression of colorectal cancer. Initially, the prognostic value of UNG in CRC was assessed. Following this, the effects of UNG on cellular and biological behaviors in CRC were investigated, culminating in an exploration of its underlying regulatory mechanisms.

Materials and methods

Bioinformatic analysis

UNG expression in CRC and adjacent tissues was analyzed using GEPIA [16] (http://gepia.cancer-pku.cn/) and TIMER [17, 18] (http://timer.cistrome.org/). Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, along with Reactome and WikiPathways enrichment analyses, were employed to analyze the transcriptomes of shUNG and control colorectal cancer cells.

Tissue specimens and cell lines

Colorectal cancer tissues and matched adjacent normal mucosa (≥ 5 cm from the tumor margin) were collected from 185 primary colorectal cancer patients at the First People’s Hospital of Kunshan (Ethics No. 2025-03-063-H00-K01). Snap-frozen specimens, stored at −80 °C within 15 min post-resection, were utilized for qPCR and Western blot analyses.

Human colorectal cell lines NCM-460, SW480, RKO, HT29, and HCT116 were obtained from the Chinese Academy of Medical Sciences. The cells were cultured in DMEM or McCoy’s 5 A medium supplemented with 10% FBS at 37 °C in a 5% CO₂ atmosphere.

Immunohistochemistry (IHC) analysis

A tissue microarray containing 185 paired colorectal cancer and normal mucosa samples from the First People’s Hospital of Kunshan was utilized. After deparaffinization and antigen retrieval, sections were stained overnight at 4 °C with an anti-UNG antibody (1:200, Bioworld), followed by HRP-secondary antibody and DAB. Two blinded pathologists scored each core by multiplying the staining intensity (0–3) and the percentage of positive cells (0–4). A final score of < 6 indicated low UNG expression, while a score of 6 or greater indicated high expression.

Lentivirus constructs and cell transfection

CRC cells were infected with lentiviral particles containing either psi-LVE003-UNG-shRNA or the empty psi-LVE003 control (GeneCopoeia, China). Stable UNG knockdown cells were selected using puromycin, and the transduction efficiency was confirmed through qRT-PCR and western blotting.

RNA isolation and RT-qPCR assay

Total RNA was extracted using TRIzol™ (Thermo Fisher Scientific). cDNA was synthesized from 1 µg of RNA using the PrimeScript™ RT Reagent Kit (CWBIO), which included genomic DNA removal (42°C for 5 minutes and 37 °C for 60 minutes). qPCR was conducted with SYBR® Green (CWBIO) on an iQ5 real-time PCR detection system (Bio-Rad, USA). The primers used were as follows: UNG, Forward: 5’-CCCCACACCAAGTCTTCACC-3’, Reverse: 5’-TTGAACACTAAAGCAGAGCCC-3’; GAPDH, Forward: 5’-CAT GAG AAG TAT GAC AAC AGCCT-3’, Reverse: 5’-AGT CCT TCC ACG ATA CCA AAGT-3’. Samples were analyzed in triplicate, and relative expression was calculated using the 2−ΔΔCt method with efficiency correction.

Protein extraction and western blot analysis

Protein lysates were prepared from cells and tissues using RIPA buffer containing 1% PMSF. A total of 30 µg of protein per sample was separated by 10% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% skim milk. The membranes were incubated overnight at 4 °C with the following primary antibodies: UNG, P70 S6K, p-P70 S6K(S424) (1:1000, Bioworld), β-actin (1:1000, Abcam), cleaved caspase-3, AMPKα1/AMPKα2, p-AMPKα1(Thr183)/AMPKα2(Thr172) (1:1000, Beyotime), BAX, BCL2, AKT1/2/3, mTOR, and p-mTOR (Ser2448) (1:1000; BOSTER), cleaved caspase-9, p-AKT(Ser473), ERK1/2, and p-ERK1/2(Thr202/Tyr204)/(Thr185/Tyr187) (1:1000; ZEN BIO), and GAPDH (1:1000; Proteintech). Following incubation with HRP-conjugated secondary antibodies at room temperature, immunoreactive bands were developed using ECL reagent (Beyotime) and quantified by densitometry in ImageJ.

Cell counting Kit-8 (CCK-8) assay

Transfected cells were seeded at a density of 2,000 cells per well in 96-well plates. At 0, 24, 48, 72, and 96 h, the media were replaced with fresh medium containing 10% CCK-8 reagent. After a 2-hour incubation at 37 °C, the absorbance at 450 nm was measured. Assays were performed in triplicate.

EdU assay

Colorectal cancer cells were seeded at a density of 10,000 cells per well in 96-well plates and treated with 10 µM EdU for 6 h at 37 °C. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and then reacted with the Click-iT reaction mixture for 30 min in the dark. Nuclei were stained with Hoechst for 30 min, and images were acquired using fluorescence microscopy.

Colony formation assay

Colorectal cancer cells were seeded at a density of 1,000 cells per well in 6-well plates and cultured for 14 days. Colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted if they contained more than 50 cells. Experiments were performed in triplicate.

Wound healing assay

Six-well plates were marked with parallel reference lines. Cells were seeded at a density of 5 × 10⁵ cells per well and cultured to 80% confluency (37 °C, 5% CO₂). Wounds were created using sterile 200 µL pipette tips perpendicular to the reference lines. After washing with PBS three times, fresh serum-free medium was added. Wound closure was monitored at 0, 24, and 48 h using phase-contrast microscopy.

Cell migration and invasion assays

Transwell inserts (24-well, 8-µm pore) were pre-equilibrated in serum-free medium. Cells (1 × 10⁵/mL in serum-free DMEM, 100 µL per insert) were seeded into the upper chambers, with the lower chambers containing 600 µL 20% FBS medium. For invasion assays, inserts were pre-coated with Matrigel for 45 min. After 48 h, membranes were fixed with 4% PFA, stained with 0.5% crystal violet, and imaged.

Flow cytometric apoptosis and cell cycle analysis

For apoptosis analysis, cells were collected via centrifugation, washed twice with phosphate-buffered saline (PBS), and stained with Annexin V-APC and propidium iodide (PI) (KeyGen Biotech, China) for 15 min at room temperature in the dark. Following staining, the cells were resuspended in PBS and analyzed immediately using flow cytometry (BD Biosciences, USA).

For cell cycle analysis, cells were washed twice with ice-cold PBS and fixed in 80% ethanol at 4 °C overnight. After two additional washes with PBS, the cells were incubated with PI and RNase A (KeyGen Biotech, China) at 37 °C for 30 min in the dark. The DNA content was analyzed using flow cytometry (BD Biosciences, USA).

Statistical analysis

Data were analyzed using GraphPad Prism 9.0 and presented as mean ± standard deviation (SD). All data were assessed for normality using the Shapiro-Wilk test before parametric testing (e.g., Student’s t-test, one-way ANOVA). Only normally distributed data were analyzed with parametric tests; non-normally distributed data were analyzed using the non-parametric Mann-Whitney U test. Statistical significance was set at *P < 0.05, **P < 0.01, ***P < 0.001; P < 0.05 was considered statistically significant.

Results

UNG expression is elevated in human CRC and predicts poor prognosis in CRC patients

Initial bioinformatics analysis of the TCGA-COAD dataset indicated upregulation of UNG across various tumor types, with a pronounced increase observed in CRC (Fig. 1A). Furthermore, publicly available CRC datasets (GEPIA) corroborated high mRNA expression levels of UNG in CRC (Fig. 1B). To further investigate UNG expression in CRC, we conducted quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses on paired CRC and adjacent non-tumorous tissues. Both UNG protein levels (Fig. 2A-B) and mRNA levels (Fig. 2C) were significantly higher in CRC tissues compared to controls. Consistent with these findings, increased UNG protein expression (Fig. 2D-E) and mRNA levels (Fig. 2F) were observed in CRC cell lines compared to NCM460. Immunohistochemical analysis revealed that UNG protein was predominantly localized in the nuclei of both benign and malignant epithelial cells (Fig. 2G). To elucidate the clinical relevance of UNG in colorectal cancer, we correlated its protein levels with the clinicopathological features of 185 consecutive CRC patients. Among the 185 paired colorectal samples, tumors displayed significantly higher UNG protein expression compared to matched normal mucosae (P < 0.0001) (Fig. 2H). High UNG expression was significantly associated with positive lymphatic metastasis (P = 0.003) and advanced TNM stage (P = 0.011), while no significant correlations were found with age, sex, tumor location, histologic differentiation, T stage, or distant metastasis (all P > 0.05; Table 1). Collectively, these data suggest that UNG is markedly elevated in CRC and is associated with poor prognosis.

Fig. 1.

Fig. 1

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UNG expression is high in human CRC cancers. (A), Human UNG expression levels across various cancer types from TCGA data in the TIMER database (TCGA: The Cancer Genome Atlas; TIMER: Tumor Immune Estimation Resource). (B), Human UNG expression in Rectal adenocarcinoma (READ) and Colon adenocarcinoma (COAD) TCGA datasets extracted from GEPIA (red bar indicates tumor samples, grey bar indicates normal samples, P < 0.01). The error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

Fig. 2.

Fig. 2

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UNG is highly expressed in CRC and indicates poor prognosis in CRC patients. (A-B), The expression of UNG protein in paired CRC and adjacent noncancerous tissues (n = 12) by western blot (N: Normal, T: Tumor). Fold changes (Fc) are shown below the bars. Exact P-values: Tumor vs. Normal, ***P = 0.0001. (C) The expression of UNG mRNA in paired CRC (n = 12) and adjacent noncancerous tissues (n = 12) by qRT-PCR. Exact P-values: Tumor vs. Normal, ****P<0.0001. (D-F), The relative expression of UNG mRNA and UNG protein in colon mucosa epithelial cell lines (NCM460) and four CRC cell lines (SW480, RKO, HT29, and HCT116) by qRT-PCR and western blot, n = 3 biologically independent experiments (Mean ± SD). Fold changes (Fc) are shown below the bars. (G), UNG protein expression in paired CRC and adjacent noncancerous tissues by IHC (n = 185). Scale bars: 50 μm (×200) or 20 μm (×400). (H) Distribution of UNG expression in IHC-stained paired normal and cancerous tissues (n = 185). Exact P-values: Cancer vs. Normal, ****P < 0.0001

Table 1.

Relationship between UNG expression and the clinicopathological parameters of CRC patients

Characteristics Number of cases UNG expression χ² P
High Low
n = 185 72 113
Gender Male 95 40 55 0.834 0.361
Female 90 32 58
Age ≤ 65 68 28 40 0.231 0.631
>65 117 44 73
Tumor location Proximal colon 66 20 46 4.112 0.128
Distal colon 63 25 38
Rectum 56 27 29
Tumor size(cm) <5 116 46 70 0.071 0.79
≥ 5 69 26 43
Histologic differentiation Well or moderate 120 46 74 0.049 0.824
Poor 65 26 39
T stage T1-T2 27 10 17 0.047 0.828
T3-T4 158 62 96
Lymphatic metastasis Absent 95 27 68 9.053 0.003
Present 90 45 45
Distant metastasis Absent 175 67 108 0.546 0.46
Present 10 5 5
TNM stage I 15 6 9 11.057 0.011
II 76 19 57
III 84 42 42
IV 10 5 5
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Statistical analysis: χ² test was used for subgroups with n ≥ 5; Fisher’s exact test was used for small subgroups (n < 5)

UNG knockdown suppresses CRC cell proliferation and clonogenicity

To investigate the oncogenic role of UNG in CRC, we established two stable UNG-knockdown cell lines, SW480 and HCT116 (Fig. 3A). The efficient silencing was verified through qPCR and western blot analyses, which demonstrated a significant reduction in both UNG mRNA and protein levels compared to controls (Fig. 3B-D). Subsequent CCK-8 assays, EdU incorporation assays, and colony formation assays indicated that UNG depletion markedly attenuated the proliferative capacity and clonogenic capacities of CRC cells compared to control (Fig. 4A-C). Collectively, these findings suggest that UNG is essential for CRC cell growth and survival.

Fig. 3.

Fig. 3

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UNG Expression is Specifically Reduced in CRC Cell Lines via Lentivirus-Mediated Knockdown. (A) Representative images of SW480 and HCT116 transfection efficiency: bright-field (left) and green-fluorescence (right) microscopy (×200); n = 3 biologically independent experiments. Scale bar = 100 μm. (B-D) The efficiency of UNG knockdown in SW480 and HCT116 cells was confirmed by qRT-PCR and Western blot, n = 3 biologically independent experiments (Mean ± SD). Fold changes (Fc) are shown below the bars. Exact P-values: qRT-PCR verification: SW480 **P = 0.009, HCT116 *P = 0.0158; Western-blot quantification: SW480 ***P = 0.0008, HCT116 **P = 0.0048

Fig. 4.

Fig. 4

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Silencing UNG inhibited CRC cell proliferation. (A-C) UNG knockdown inhibited the proliferation ability of SW480 and HCT116 cells as determined by CCK8 assays, colony formation, and EDU assays, n = 3 biologically independent experiments (Mean ± SD). Exact P-values: Colony formation: SW480 **P = 0.0011, HCT116 ***P = 0.0006; EdU: SW480 ***P = 0.0005, HCT116 *P = 0.0105. Scale bar = 100 μm

Silencing UNG inhibits CRC cell motility, migration, and invasion

Next, we assessed whether UNG expression affects the metastatic potential of CRC cells. Matrigel transwell invasion assays, matrigel-free transwell migration assays, and wound healing assays consistently demonstrated that UNG knockdown significantly impaired CRC cell motility compared to the control group (Figs. 5A, B). Specifically, both the number of cells that traversed the Matrigel and the distance migrated by UNG-silenced cells were markedly reduced. This collective evidence establishes UNG as a novel driver of CRC cell migration and invasion, implicating its role in early metastatic progression.

Fig. 5.

Fig. 5

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Silencing UNG inhibited CRC cell migration and invasion. (A-B), Matrigel transwell assays, Matrigel-free transwell assays, and wound healing assays were used to determine the migration and invasion ability of UNG-downregulated CRC cells, n = 3 biologically independent experiments (Mean ± SD). Exact P-values: Migration: SW480 ***P = 0.0007, HCT116 ****P < 0.0001; Invasion: SW480 ****P < 0.0001, HCT116 ****P < 0.0001; Wound healing: 24 h-SW480 ***P = 0.001, 24 h-HCT116 *P = 0.022, 48 h-SW480 **P = 0.001, 48 h-HCT116 **P = 0.0083. Scale bar = 100/400 µm

UNG modulates cell-cycle progression and apoptosis in CRC

To elucidate the cellular mechanisms underlying UNG-driven tumorigenesis, we performed flow cytometric analysis on stable UNG-knockdown SW480 and HCT116 lines. UNG depletion significantly increased the early apoptotic rates (Figs. 6A-B), while inducing S/G2-phase arrest (Figs. 6C-D). These data indicate that UNG sustains colorectal cancer cell survival by simultaneously suppressing apoptosis and accelerating cell-cycle progression, positioning UNG as a pivotal regulator of the proliferation-apoptosis balance in CRC.

Fig. 6.

Fig. 6

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UNG regulates CRC cell cycle and apoptosis. (A-B) The effects of UNG downregulation on cell apoptosis by flow cytometry, n = 3 biologically independent experiments (Mean ± SD). Exact P-values: SW480 ***P = 0.0004, HCT116 **P = 0.0061. (C-D) The effects of UNG downregulation on cell apoptosis and cycle progression by flow cytometry

UNG regulates AKT/mTOR and MAPK/ERK signaling in CRC

Western blot profiling revealed that the knockdown of UNG significantly attenuated the phosphorylation of key effectors in the AKT/mTOR pathway (p-AKT, p-mTOR, and p-P70 S6K) and constituents of the MAPK/ERK pathway (p-ERK) (Fig. 7). Concurrently, the expression of the anti-apoptotic protein BCL2 was down-regulated, while the energy sensor p-AMPK and pro-apoptotic executors (BAX, cleaved caspase-9, and cleaved caspase-3) exhibited significant up-regulation (Fig. 7). These findings establish UNG as an upstream regulator that integrates metabolic and mitogenic signals to maintain a balance between proliferation and survival in CRC cells.

Fig. 7.

Fig. 7

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UNG activated several signaling pathways in CRC cells. Western blot was performed to detect the levels of mTOR, p-mTOR, p70 S6K, p-P70 S6K, AKT, p-AKT, AMPK, p-AMPK, ERK, p-ERK, Bax, Bcl2, cleavages of caspase-9, and caspase-3 in UNG-knockdown CRC cells. Fold changes (Fc) are shown below the bars

Transcriptomic landscape of UNG-depleted CRC cells

RNA sequencing of stable UNG-knockdown SW480 and HCT116 cells identified 2,634 differentially expressed genes (DEGs), with 1,267 genes up-regulated and 1,367 genes down-regulated (Fig. 8B). Unsupervised hierarchical clustering of the top 20 DEGs effectively segregated UNG-silenced replicates from control replicates (Fig. 8A). Among the most prominently down-regulated transcripts were CALB1, VAMP7, MMP1, DAPP1, PLA2G4F, LY6D, NCF2, LCN2, TBC1D3H, and H4C15—genes previously implicated in tumor invasion, neutrophil-mediated inflammation, and the formation of metastatic niches (Table 2). Conversely, the loss of UNG resulted in a significant upregulation of MAG, CLDN2, ZCCHC12, A2M, ATOH8, EFNA5, SNCA, ARMH4, SPARC, and MYBPH, several of which are known to exert tumor-suppressive or epithelial-integrity functions (Table 2). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and WikiPathways analyses collectively indicated that UNG silencing suppresses transcriptional programs associated with cancer progression. GO enrichment analysis identified significant terms related to epithelial-mesenchymal transition (EMT) and cell adhesion, with cellular component mapping indicating involvement of the extracellular matrix (ECM). Molecular function shifts were observed in DNA-binding transcription factor activity and signaling receptor binding (Fig. 9A). KEGG analysis highlighted alterations in pathways such as PI3K-Akt signaling, ECM-receptor interaction, focal adhesion, and pathways in cancer (Fig. 9B). Reactome analysis underscored changes related to ECM organization, developmental biology, and collagen metabolism and degradation (Fig. 9C), while WikiPathways also emphasized EMT, cell motility, PI3K-Akt signaling, and cancer-related pathways (Fig. 9D). Collectively, these transcriptomic findings demonstrate that UNG depletion suppresses the expression of pro-metastatic genes and modifies transcriptional networks, with a notable impact on the PI3K-Akt pathway. This provides a genome-wide mechanistic account for why UNG-deficient colorectal cancer cells exhibit reduced aggressiveness (Fig. 10).

Fig. 8.

Fig. 8

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Global gene expression profiling following UNG depletion in CRC. (A) Heatmap of hierarchical clustering for the most deregulated genes across biological replicates. (B) Volcano plot visualizing significantly dysregulated genes (red: upregulated; blue: downregulated)

Table 2.

Genes, expression, and functions related to UNG

Gene Names Chromosomal location Regulation (UNG silencing) Expression in CRC (GEPIA) Biological Function
MYBPH 1q32.1 ↑ ↑ MYBPH binds to myosin and possesses the ability to suppress cancer cell motility, invasion, and metastasis [19]
CLDN2 Xq22.3–q23 ↑ ↑ The level of CLDN2 can affect tumor proliferation, migration, and EMT [20]
SPARC 5q33.1 ↑ ↑ SPARC, a cysteine-rich acidic matricellular protein, modulates extracellular matrix remodeling and cell adhesion, exerting context-dependent dual roles in tumor suppression and metastasis [21, 22]
ARMH4 14q23.1 ↑ NR ARMH4 acts as a negative regulator of pro-inflammatory signaling, primarily through the inhibition of STAT3 and AKT pathways [23]
SNCA 4q22.1 ↑ ↓ SNCA inhibits tumor cell EMT and metastasis, while also reducing chemoresistance and promoting immune cell infiltration [24]
ZCCHC12 Xq24 ↑ ↓ ZCCHC12, a transcriptional coactivator for the BMP pathway, facilitates cancer cell proliferation and EMT [25, 26]
A2M 12p13.31 ↑ ↓ A2M, a potent protease inhibitor, regulates cancer growth, cell death, and anti-inflammatory cytokine responses [27]
EFNA5 5q21.3 ↑ ↓ EphrinA5 suppressed cancer cell proliferation, migration, and chemoresistance [28]
ATOH8 2p11.2 ↑ ↓ ATOH8 is an established tumor suppressor gene, with functional roles spanning embryonic development and carcinogenesis [29]
MAG 19q13.1 ↑ ↓ Myelin-associated glycoprotein (MAG) suppresses Schwann cell migration and induces their apoptosis[30]
TBC1D3H 17q12 ↓ NR TBC1D3H regulates GTPase activation and is implicated in tumorigenesis [31]
H4C15 1q21.2 ↓ NR H4C15, a core component of nucleosome, emerges as a novel potential therapeutic target for NETosis [32]
CALB1 8q21.3 ↓ ↑ CALB1 promotes tumor proliferation by inhibiting cellular senescence through the degradation of p53 [33]
MMP1 11q22.2 ↓ ↑ MMP1, as a matrix metalloproteinase, promotes tumor proliferation, invasion, and metastasis [34]
LCN2 9q34.11 ↓ ↑ LCN2, an oncogenic driver, orchestrates EMT, angiogenesis, and invasion to fuel tumor progression and poor prognosis [35]
LY6D 8q24.3 ↓ ↑ Lymphocyte antigen 6 family member D (LY6D) enhances colon cancer malignant phenotypes and tumorigenesis by activating the MAPK pathway [36]
DAPP1 4q23 ↓ ↑ DAPP1, a protein-dephosphorylation signal transducer, drives EGFR-mutant lung adenocarcinoma growth [37]
PLA2G4F 15q15.1 ↓ ↑ PLA2G4F, a key orchestrator of cancer metabolic reprogramming, sculpts an immunosuppressive niche that propels tumor growth [38]
NCF2 1q25.3 ↓ ↑ NCF2 directly interacts with NR2F2 to promote tumorigenesis, lymphangiogenesis, and metastasis [39]
VAMP7 Xq28 and Yq12 ↓ ↑ VAMP7, a vesicle-associated membrane protein, degrades the extracellular matrix to orchestrate immune-cell infiltration and foster cancer-cell dissemination and metastasis [40]
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Expression status (↑ or ↓) in the “Expression in CRC” column is based on data from the GEPIA database

NR: not reported; GTP: Guanosine triphosphate; EMT: Epithelial-mesenchymal transition; NETosis: Neutrophil extracellular traps

Fig. 9.

Fig. 9

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Transcriptomic analysis of UNG knockdown in CRC. (A) Gene Ontology (GO) enrichment analysis for biological processes, cellular components, and molecular functions. (B-D) Top associated pathways identified through KEGG, Reactome, and WikiPathways enrichment analyses

Fig. 10.

Fig. 10

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Schematic model of the carcinogenic mechanism of UNG in CRC. Created in https://BioRender.com

Discussion

Eukaryotic cells employ multifaceted mechanisms to protect genomic integrity from genotoxic stress, with the base excision repair (BER) pathway serving as a critical defense system. UNG is a key enzyme within this pathway, specifically responsible for removing uracil misincorporated into DNA, thereby preventing mutations and preserving genomic stability. The biological functions of UNG in relation to CRC remain to be elucidated. We observed elevated UNG expression in both CRC tissues and cell lines, consistent with previously published data [15]. Notably, increased levels of UNG were significantly associated with aggressive clinicopathological features and poor patient outcomes. These findings suggest that UNG contributes to malignant progression in CRC and may serve as a prognostic marker. Loss-of-function studies revealed that UNG knockdown significantly reduced the proliferation, migration, and invasion of CRC cells, accompanied by S/G2-phase cell-cycle arrest and increased early apoptosis. These findings strongly support the oncogenic role of UNG in CRC development and progression, highlighting its potential as a novel therapeutic target.

As the initiating enzyme in the BER pathway, UNG specifically recognizes and hydrolyzes uracil bases in DNA, which can arise from cytosine deamination or the misincorporation of dUTP. This process generates apurinic/apyrimidinic (AP) sites, thereby initiating the repair process [41]. The loss of UNG function leads to the persistent accumulation of uracil within the genome, resulting in the stalling of replication forks and the potential conversion of these lesions into DNA double-strand breaks (DSBs) [42]. These DNA lesions activate the DNA damage response (DDR) pathway. In this study, we observed that UNG silencing induces a robust S/G2 phase cell cycle arrest (Fig. 6). Such arrest is a well-recognized marker of genomic instability and functions as a key cellular checkpoint to prevent the division of damaged cells [43]. Our data thus provide functional evidence that UNG deficiency leads to DNA damage, which in turn activates the cell cycle checkpoint system. Building on this functional evidence, future studies will employ direct biochemical measurements, including uracil quantification and AP site assays, to precisely determine the extent of uracil accumulation and BER impairment. This DDR mechanism triggers a p53-p21-mediated S/G2 arrest through the ATM/ATR kinase cascade, providing a window for DNA repair [44, 45]. This interpretation is further supported by earlier research demonstrating that UNG dysfunction results in DNA double-strand breaks, as directly evidenced by increased γ-H2AX foci [46]. However, when the extent of DNA damage surpasses the repair capacity, the DDR signaling transitions towards a pro-apoptotic program. The mitochondrial apoptotic pathway is regulated by the Bcl-2 family of proteins, which comprises anti-apoptotic proteins (such as Bcl-2 and Bcl-xL) and pro-apoptotic proteins (such as Bax and Bak) [47]. Following DNA damage, activated p53 induces the transcription of Bax, promoting an increase in mitochondrial outer membrane permeability and the subsequent release of pro-apoptotic factors like cytochrome c from the mitochondria into the cytoplasm. The released pro-apoptotic substances then activate downstream caspase-9 and caspase-3, initiating the apoptotic cascade that culminates in cell death [48, 49]. Research has shown that the reduction of UNG expression triggers DNA damage and apoptosis in prostate cancer cells by altering key genes involved in cell survival and death, underscoring the critical role of UNG activity in maintaining the viability of malignant cells [10]. In this study, we observed that UNG knockdown significantly enhanced apoptosis in colorectal cancer cells, as evidenced by the upregulation of cleaved caspase-3/9 and an elevated Bax/Bcl-2 ratio. We propose that UNG knockdown leads to the accumulation of endogenous DNA damage and induces apoptosis via the mitochondrial pathway. Although direct detection of γ-H2AX remains a goal for future studies, our overall findings suggest that UNG loss suppresses tumors partly by inducing DNA damage and activating subsequent cell cycle checkpoints and apoptotic programs.

The cellular energy sensor AMP-activated protein kinase (AMPK) is activated by various physiological stimuli, including DNA damage, to modulate signal transduction and metabolic pathways [50]. Loss of UNG causes persistent repair stress, lowering ATP and raising the AMP/ATP ratio, which in turn triggers AMPK phosphorylation and activation [51]. Concurrently, the mammalian target of rapamycin complex 1 (mTORC1), a crucial regulator of cellular metabolism, survival, proliferation, and growth, is negatively regulated by AMPK. Activated AMPK inhibits mTORC1 activity through the phosphorylation of TSC2 or Raptor, leading to a substantial reduction in the phosphorylation of its downstream target, P70 S6K, ultimately suppressing cellular anabolism and proliferation [52]. In our study, we observed that p-mTOR and p-P70 S6K were downregulated while p-AMPK was upregulated in UNG-silenced CRC cells.

The PI3K/AKT/mTOR and MAPK/ERK pathways are critically involved in regulating cell proliferation, differentiation, apoptosis, and metabolism [53]. Serine/threonine kinase Akt is a central signaling hub for various growth factors and cytokines, mediating cancer and tumor progression primarily by promoting cell survival and inhibiting apoptosis [54]. Extracellular signal-regulated kinase (ERK), as the terminal activated kinase in the mitogen-activated protein kinase (MAPK) pathway, acts as a specific substrate and downstream effector of mitogen-activated protein/extracellular signal-regulated kinase (MEK). The core components of these pathways, specifically AKT and ERK, are frequently overactivated in most cancers [55], with their elevated activity also contributing to increased mTOR phosphorylation [54]. However, persistent DNA damage activates the ATM/ATR-mediated DDR pathways, which negatively regulate the PI3K/AKT signaling axis [56]. Additionally, the metabolic suppression induced by AMPK activation systematically inhibits crucial pro-growth signaling pathways, including PI3K/AKT and MAPK/ERK, which are essential for cell proliferation, survival, and migration. The energy-deprived environment resulting from UNG deficiency may further contribute to reduced ERK phosphorylation in the MAPK/ERK pathway, potentially by activating DNA damage-induced DUSP phosphatases or attenuating growth factor receptor signaling. Similarly, p-AKT and p-ERK levels were significantly reduced in UNG-silenced CRC cells. Transcriptome sequencing analysis also indicated that the PI3K/AKT/mTOR signaling pathway was enriched in CRC cells with UNG knockdown. These results suggest that UNG-induced proliferation and tumorigenesis may be mediated by the activation of the AMPK, AKT/mTOR, and MAPK/ERK pathways.

Traditionally, UNG has been regarded primarily as a guardian of genomic integrity through its canonical role in base-excision repair. This study provides the first comprehensive evidence that UNG also functions as a potent oncogenic driver in CRC by simultaneously fueling proliferative, anti-apoptotic, invasive, and metabolic programs. However, several important questions remain for future investigation. First, the molecular mechanisms underlying UNG-mediated regulation, whether through direct protein–protein interactions, ROS-mediated DNA damage signaling, or transcriptional control of receptor tyrosine kinases, warrant further elucidation. Second, while UNG knockdown significantly suppressed cancer cell migration and invasion, direct profiling of classical EMT markers (e.g., E-cadherin, N-cadherin) is needed to confirm its role in this process. Third, future studies should directly assess the metabolic consequences of UNG loss through ATP/ADP/AMP measurements and determine the hierarchical relationship between UNG and the observed signaling changes using constitutive activators and specific inhibitors of AKT/ERK/AMPK pathways. Fourth, the functional evidence of replication stress and DNA damage presented here should be extended through direct biochemical measurements, such as uracil quantification, AP site assays, and DNA fiber analysis, to precisely quantify BER impairment and replication fork dynamics. Most importantly, definitive confirmation of UNG as an oncogenic driver and a therapeutic target will require in vivo validation combined with rescue experiments. We plan to employ subcutaneous and orthotopic xenograft models incorporating UNG knockdown alongside rescue controls using an shRNA-resistant UNG cDNA. These studies will assess tumor growth, metastasis, and key molecular endpoints, including Ki-67, TUNEL, γH2AX, and p-AKT/p-ERK by IHC. Complementing these approaches, the development of highly selective UNG inhibitors will be essential for future therapeutic development. Finally, potential risks of targeting UNG in normal tissues, given its crucial role in preventing AP site toxicity, must be carefully evaluated in future translational studies.

In summary, the expression of UNG is elevated in colorectal cancer. The downregulation of UNG expression has been shown to inhibit the proliferation, invasion, migration, and tumorigenicity of CRC cells via the AMPK, AKT/mTOR, and MAPK/ERK signaling pathways. These findings indicate that UNG may serve as a promising therapeutic target for the treatment of colorectal cancer.

Acknowledgements

Not applicable.

Author contributions

Conceptualization, Wei Han and Qinghua Wang; formal analysis, investigation, and writing—original draft prepara-tion, Jingjing Yang; methodology and software, Li-zhou Shi; validation, Yong-wei Hu; writing—review and editing, Wei Han and Hua Chen; supervision, Wei Han. All authors reviewed the results and approved the final version of the manuscript.

Funding 

This research was funded by National Natural Science Foundation of China (No. 82503541); Kunshan Major Project of Social Research and Development (No. KS2309; No. KS2308; No. KS2516); Suzhou Municipal Bureau on Science and Technology (No. SYW2025030; No. SYW2025169; No. KJXW2023074; No. SKY2023029); and Jiangsu Provincial Maternal and Child Health Care Association (No. FYX202436).

Data availability

The RNA-seq data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1348306. The data will become publicly available on 2025-11-01 and can be accessed via https://www.ncbi.nlm.nih.gov/sra/PRJNA1348306.

Declarations

Ethics approval

The Ethics Committee of Kunshan First People’s Hospital approved this study and granted a waiver of informed consent due to its retrospective nature and the use of fully de-identified data (Ethics No. 2025-03-063-H00-K01).

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jing-jing Yang and Li-zhou Shi contributed equally to this work.

Contributor Information

Qing-hua Wang, Email: ksph_wqh@sina.com.

Wei Han, Email: hans64@126.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The RNA-seq data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1348306. The data will become publicly available on 2025-11-01 and can be accessed via https://www.ncbi.nlm.nih.gov/sra/PRJNA1348306.

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