Biliary atresia (BA) is a severe neonatal cholangiopathy characterized by progressive inflammation and fibrosis of both intrahepatic and extrahepatic bile ducts (1). If left untreated, it can lead to bile duct obstruction, liver cirrhosis, and ultimately liver failure (2). Early diagnosis and prompt surgical intervention, particularly the Kasai portoenterostomy, are critical for improving outcomes in infants with BA (3, 4). Despite surgical treatment, BA remains the leading cause of end-stage liver disease and the primary indication for pediatric liver transplantation (1, 3, 5). While substantial research has been conducted, the etiology of BA remains poorly understood, with genetic variants, environmental factors, toxins, viral infections, and immune dysfunction all thought to contribute to its pathogenesis (6–8). Thus, further investigation into the disease mechanisms and potential therapeutic targets for BA is crucial to improving patient outcomes.

Epigenetics refers to heritable changes in gene function that do not involve alterations in the DNA sequence, thereby influencing phenotype (9). Epigenetic mechanisms, including DNA methylation, histone modifications, and chromatin remodeling, play critical roles in regulating gene expression and are implicated in the development of various liver diseases (10–12). Epigenetic factors serve as key regulators of gene expression, bridging genetic and environmental influences (13). Although several studies have examined genetic alterations in BA, the findings remain inconclusive, highlighting the need to explore non-coding and epigenetic factors that may contribute to disease onset (6). Notably, the interaction between genetic and epigenetic predispositions, combined with environmental exposures during pregnancy, may act as triggers for BA (8, 10). Approximately 20% of BA cases are associated with embryonic development, with epigenetic factors playing a pivotal role in disease pathogenesis (14). Epigenetic mechanisms may lead to abnormal expression of proliferation-related genes in BA; however, there is currently no research exploring the specific molecular mechanisms involved. Therefore, further identification of key genes associated with these epigenetic mechanisms is crucial for understanding the specific mechanisms linking BA.

Although research on BA has advanced, the specific epigenetic modifications and their functional implications remain poorly understood. To address this, the present study leverages transcriptomic data from public BA databases to identify key genes associated with epigenetic factors through protein-protein interaction (PPI) analysis. Various bioinformatics techniques will be employed to identify biological pathways related to these key genes. Additionally, molecular regulatory networks will be constructed, and potential therapeutic drugs will be predicted, providing theoretical support for BA treatment. To validate the bioinformatics findings, RT-qPCR experiments will be conducted to assess the mRNA expression levels of key genes, confirming their alignment with computational analyses. These results will offer a deeper understanding of the molecular mechanisms linking BA with epigenetic factors and may contribute to the development of new therapeutic approaches.

BA is not a singular disease with a single cause, but rather a phenotype resulting from multiple pathogenic factors and mechanisms (31). Increasing evidence suggests that patients with BA exhibit a genetic predisposition to heightened biliary injury and pathological repair (1). Recent studies have identified various genetic loci and potential epigenetic factors that may contribute to its pathogenesis, highlighting the complexity of the disease (6). The inconsistent presentation of BA in twins, particularly monozygotic twins, underscores the significant role of epigenetic factors in its development (32). This study identified five key genes—AURKA, BUB1, CDK1, RAD51, and TOP2A—that are linked to epigenetic factors in BA through comprehensive bioinformatics analyses. The functions of these genes were explored using GESA and molecular regulatory networks. Additionally, thirteen potential therapeutic drugs were screened through drug prediction, identifying the strongest binding interactions. Finally, the expression of these key genes was experimentally validated, offering new perspectives for the research and treatment of BA.

AURKA (Aurora Kinase A) is a protein kinase critical for cell mitosis, regulating various aspects of cell division, including centrosome maturation and spindle assembly (33, 34). Dysregulated expression or activity of AURKA is strongly associated with the development of various cancers (35, 36). AURKA is also significantly involved in epigenetic regulation and liver diseases. In cholangiocarcinoma, the TF FOSL1 modulates AURKA expression, influencing cell proliferation and tumor growth (37). Furthermore, AURKA is implicated in cell cycle regulation and epigenetic modifications (38), illustrating the complex regulatory roles of AURKA in liver disease development (39). The upregulation of AURKA can drive the abnormal proliferation of biliary epithelial cells, potentially disrupting bile duct development and function (40). Consequently, AURKA represents a promising target for therapeutic intervention, as modulating its activity could restore normal cell cycle regulation and mitigate the pathological processes underlying BA.

BUB1 (Budding uninhibited by benzimidazoles 1) is integral in monitoring the connection between spindle microtubules and chromosome kinetochores during mitosis (41, 42). Elevated BUB1 levels may serve as a compensatory response to errors in cell division or genomic instability (43). Previous studies have linked high BUB1 expression to aneuploidy (44), a condition that exacerbates the development of various diseases, including human sarcoma (45). Additionally, BUB1 expression in human lymphocytes suggests its involvement in immune responses (46), which may play a significant role in BA, where immune-mediated damage to biliary epithelial cells contributes to disease progression (47). Targeting BUB1 could provide therapeutic benefits by addressing both cell cycle dysregulation and immune-related factors in BA.

CDK1 (Cyclin-dependent kinase 1) is a key regulatory factor in the cell cycle (48), closely related to the transition of cells from the G2 phase to the M phase (49). Dysregulation of CDK1 may lead to uncontrolled cell proliferation (50), which subsequently impairs normal cell differentiation and function. Elevated levels of CDK1 typically indicate abnormal activation of cell cycle regulatory pathways (51). This study found that CDK1 is significantly upregulated in BA liver tissue through transcriptome analysis, suggesting that it may accelerate disease progression by driving cholangiocyte cell cycle dysregulation. Notably, CDK1 is also involved in multiple signaling pathways related to epigenetic regulation, including pathways such as the Wnt/β-catenin signaling cascades. Its activity may be influenced by epigenetic modifications, such as histone acetylation and methylation (52), which further affect gene expression and cell function. However, RT-qPCR results showed no significant differences in CDK1 expression between BA and control samples. This discrepancy may be due to heterogeneity among RT-qPCR samples, including differences in sample source and quality. Additionally, the small sample size may have limited the detection of significant differences. Therefore, future research should explore the molecular interaction mechanisms between CDK1 and epigenetic factors in BA to better understand the epigenetic mechanisms underlying cell cycle dysre.

RAD51, a recombinase essential for homologous recombination, is a core factor in the homologous recombination repair of DNA. It is highly expressed in rapidly proliferating tissues such as the testes, involved in germ cell development, and the colon, responsible for intestinal epithelial renewal, consistent with its role in maintaining genomic stability. RAD51 plays a key role in repairing DNA double-strand breaks and maintaining genomic stability (53, 54). Notably, RAD51 can also influence the activity of DNA methyltransferase DNMT1 by regulating the ubiquitin ligase UHRF1 through two distinct mechanisms, thereby participating in the maintenance of genomic DNA methylation (55). Moreover, in cancer cell lines, RAD51 is associated with the regulation of the autophagy pathway and works together with E-box proteins such as USF1, USF2, and MITF to regulate the expression of autophagy-related genes (56). Autophagy itself is crucial in cellular stress responses and cell death processes (57). In this study, the upregulation of RAD51 in BA suggests that it may indirectly affect the differentiation or function of cholangiocytes by influencing DNA damage repair, modulating the autophagy pathway, and maintaining DNA methylation. Therefore, further research on the specific role of RAD51 in BA will help reveal the molecular mechanisms of this disease and provide a theoretical basis for developing new prevention and treatment strategies.

TOP2A is highly expressed in 721_B lymphoblasts (leukemia cells) and in the testis. It encodes DNA topoisomerase IIα, which is essential for DNA replication and chromosome segregation. Although its overexpression is associated with poor prognosis in various cancers (58–61), the expression of TOP2A is tightly regulated by epigenetic mechanisms, particularly DNA methylation (62). High methylation of its promoter typically suppresses its expression, while low methylation promotes its expression. We found that TOP2A is upregulated in BA, suggesting that there is likely low methylation of the promoter. This finding is consistent with previous observations that low DNA methylation in zebrafish plays a key role in biliary defects (63, 64). Therefore, this epigenetic dysregulation disrupts the tissue-specific expression of TOP2A, leading to its aberrant activation in cholangiocytes and resulting in genomic instability and abnormal cell proliferation in BA cholangiocytes.

GSEA analysis indicates that the pathogenesis of BA is closely linked to the abnormal activation of the cell cycle and DNA replication pathways. Abnormal regulation of the cell cycle pathway strongly correlates with the five key genes, including CDK1, suggesting that these genes work synergistically to promote cell cycle dysregulation. Dysregulation of cell cycle control has been shown to cause excessive proliferation or prevent differentiation in cholangiocytes, leading to disruption of the normal bile duct structure (65). As a key regulator of the cell cycle, CDK1 overexpression accelerates the G2/M phase transition by phosphorylating downstream target proteins, such as retinoblastoma (Rb) protein, which drives uncontrolled cholangiocyte proliferation (66). Moreover, epigenetic modifications, including long non-coding RNAs, may exacerbate cell cycle dysregulation by modulating CDK1 expression (67). This abnormal proliferative state contributes to bile duct fibrosis, which is consistent with the progressive bile duct injury and liver fibrosis seen in patients with BA (65). The significant activation of the DNA replication pathway suggests DNA replication stress and cholangiocyte damage, pointing to potential genomic instability in BA. DNA replication stress can result in replication fork stalling and DNA double-strand breaks (68). This activates the ATR/CHK1 signaling pathway, which may induce apoptosis or cellular senescence (69). The positive correlation between RAD51 and primary immunodeficiency observed in this study may reflect a compensatory enhancement of DNA repair mechanisms in response to sustained replication stress. However, when DNA repair capacity is overwhelmed, accumulated DNA damage may trigger apoptosis through the p53 pathway (70). The negative correlation between the ascorbic acid metabolism pathway and RAD51 suggests that oxidative stress may exacerbate DNA damage. An imbalance in ascorbic acid, a key antioxidant, could reduce cellular defenses against oxidative injury (71). The interaction between these pathways and disease progression indicates that abnormalities in the cell cycle and DNA replication create a vicious cycle. Specifically, an accelerated cell cycle increases the demand for DNA replication, resulting in replication stress (72), while DNA damage can suppress the cell cycle through CDK inhibitors like p21 (73). This imbalance likely explains the simultaneous occurrence of abnormal proliferation and increased apoptosis in cholangiocytes in BA (74). Furthermore, epigenetic modifications, such as irregularities in DNA methyltransferases, may amplify the pathological effects of these pathways by regulating multiple key genes at once (75). In summary, the results from GSEA emphasize the critical role of the cell cycle and DNA replication pathways in the pathogenesis of BA. Disruptions in these pathways may drive dysregulated cholangiocyte proliferation, genomic instability, and metabolic disorders through an epigenetic regulatory network, ultimately leading to the progressive destruction of the bile ducts.

The constructed molecular regulatory network reveals that TFs like CEBPB and miRNAs such as hsa-miR-98-5p regulate key genes. CEBPB is a multifunctional TF involved in regulating inflammation, cell growth, and differentiation (76). CEBPB may serve as a critical link between inflammatory responses and abnormal cell proliferation (76). The presented study identified CEBPB as a regulator of RAD51 and AURKA, suggesting its involvement in the pathological processes of BA by influencing cell proliferation and genomic stability. miR-98-5p inhibits hepatic stellate cell (HSC) activation by directly targeting key factors in the TGF-β signaling pathway, thereby slowing liver fibrosis progression (77). In the present study, hsa-miR-98-5p targeted both TOP2A and AURKA, indicating its potential to protect against BA by inhibiting fibrosis and maintaining genomic stability. However, further experiments are needed to validate the specific regulatory mechanisms of TFs like CEBPB and miRNAs such as hsa-miR-98-5p in the context of BA.

Identifying therapeutic drugs that target key genes associated with BA marks a significant advancement in developing effective treatment strategies. Among the predicted drugs interacting with key genes, Amuvatinib stands out for its strong binding affinity to RAD51, with a docking score of −9.9 kcal/mol. Amuvatinib selectively inhibits receptor tyrosine kinases and has been primarily studied for its efficacy against various cancers, where it impedes tumor growth and induces cancer cell apoptosis (78). Studies suggest that Amuvatinib can reduce tumor cell drug resistance by inhibiting RAD51 protein expression (79, 80). Although the role of RAD51 in BA remains unexplored, it may contribute to cholangiocyte function by regulating DNA repair. Additionally, Protuboxepin A, which targets CDK1, exhibits a docking score of −9.7 kcal/mol. CDK1 is a key regulator of the cell cycle, and its dysfunction is associated with various cancers (81). Protuboxepin A binds directly to α,β-tubulin, stabilizing tubulin polymerization and disrupting microtubule dynamics. This disruption leads to chromosome misalignment and metaphase arrest, ultimately causing apoptosis in cancer cells (81). While the mechanisms of action of Protuboxepin A and CDK1 in BA have not been reported, Protuboxepin A presents a theoretical foundation for potential therapeutic intervention. These findings are crucial not only for identifying individual drug-gene interactions but also for exploring the possibility of combination therapies that target multiple pathways in BA's pathogenesis. However, the aforementioned drugs are currently in the experimental research stage as anticancer agents, and there is still a lack of clinical data supporting their safety, optimal dosage, and potential toxicity in the pediatric population, especially in BA infants. In light of this, future research should prioritize conducting in vivo experiments using a BA mouse model induced by rhesus monkey rotavirus. These studies should systematically evaluate the efficacy and safety of the drugs, while optimizing dosing strategies based on the pharmacokinetic characteristics of neonates.

However, this study has several limitations. First, the analysis results depend on data quality, algorithm accuracy, and methodological assumptions. These factors may lead to data noise and difficulties in interpretation due to algorithm complexity; they can also cause issues such as false positives and false negatives. Moreover, the imbalance between the samples of the disease group and the control group may increase the risk of false positives and false negatives. Additionally, although validation was performed through RT-qPCR, the small sample size and differences in experimental methods limited the statistical power to significantly detect the expression pattern of CDK1. To address these issues, future plans include expanding the sample sizes of both the disease group and the control group, as well as optimizing the sample selection strategy to improve the stability and reliability of the research findings. At the same time, we plan to further validate the research findings through experimental techniques such as immunohistochemistry and Western blot. These will be combined with animal models and functional experiments like overexpression and deletion to enhance the credibility of the research conclusions.

This study identified five key genes (AURKA, BUB1, CDK1, RAD51, TOP2A) related to epigenetic regulation in BA through transcriptomic analysis, whose abnormal expression may promote the progression of BA by interfering with the cell cycle and DNA repair pathways. These genes may serve as potential therapeutic targets for BA, and small molecule inhibitors or antibody drugs targeting them could be developed in the future, which could be further explored for their therapeutic potential. Additionally, this study discovered potential drugs such as Amuvatinib and Protuboxepin A, which may exert therapeutic effects by targeting key genes, providing new hope for the treatment of BA. Future research will focus on investigating the specific regulatory mechanisms of epigenetic modifications (such as DNA hypomethylation) on key genes in animal models, and further experiments will validate the feasibility of these genes as therapeutic targets.