Biology subjects

Wu, G.

Publications and source records attributed to Wu, G..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Exploring DNA methylation profiles in the pathogenesis of human osteoporosis via whole-genome bisulfite sequencing

BackgroundOsteoporosis is a prevalent bone metabolic disorder characterized by reduced bone mass, disruption of bone microarchitecture, and increased bone fragility, leading to a heightened risk of fracture. This condition significantly impairs patients quality of life and increases mortality risk. Emerging evidence suggests that DNA methylation may play a crucial role in regulating the expression of genes related to bone metabolism, thereby influencing the development of osteoporosis. However, the precise relationship between DNA methylation and osteoporosis remains unclear and warrants further investigation. ResultsOur study revealed significant differences in both the quantity and ratio of DNA methylation between individuals with osteoporosis and non-osteoporosis controls, with differences predominantly occurring in CpG islands. GO/KEGG enrichment analyses highlighted distinct osteoporosis-related gene pathways. Notably, we identified six genes, MSX1, HOXD4, AXIN2, WNT5A, TGFB1, and STAT3, respectively, that are potentially involved in the pathogenesis of osteoporosis and are broadly involved in various diseases and biological processes. ConclusionsThese findings indicate distinct methylation patterns between osteoporosis patients and healthy individuals, with differential methylation levels in genes associated with osteoporosis. This research offers new insights into the epigenetic mechanisms underlying osteoporosis.

genetics↗

bioRxiv2025

UC Irvine Brain Initiative Cell Atlas Network (BICAN) Brain Procurement Program for the Center for Multiomic Human Brain Cell Atlas Project

High-quality neurotypical postmortem human brain tissue is essential but difficult to obtain for constructing comprehensive human brain cell atlases. Here we describe the establishment of UC Irvines Brain Procurement Program, a coordinated initiative to collect and process neurotypical donor brains for multiomic mapping studies within the NIH BRAIN Initiative Cell Atlas Network (BICAN) consortium. Through partnerships with the Orange County Coroners Office, the UC Irvine Willed Body Program, UCI Medical Center, and the Childrens Hospital of Orange County, we have developed standardized workflows encompassing donor identification, postmortem brain recovery and processing, region-of-interest dissection, neurotypical donor selection, and data management. Our experience demonstrates the feasibility of a community-based, multi-institutional procurement framework while highlighting challenges in recruiting neurotypical donors and ensuring demographic representation reflective of Southern California. We further identify opportunities to strengthen outreach and donation pathways. This program provides a scalable model for advancing population-reflective, high-quality human brain cell atlas efforts. HighlightsO_LIEstablish a multi-site pipeline for procuring high-quality neurotypical human brains. C_LIO_LIDemonstrate feasibility of donor collection across childhood to adulthood. C_LIO_LIIdentify barriers and propose strategies to improve broad donor recruitment. C_LI

neuroscience↗

bioRxiv2025

Application of Large Language Models for Annotating Genes into Reactome Pathways

Reactome is the most comprehensive, open source, open access biological pathway knowledgebase, widely used in the research community. To ensure the highest quality of its content, human pathway data in Reactome is manually curated. However, manual curation is labor-intensive, time-consuming, and increasingly difficult to keep up with the ever-growing biomedical literature. Large language model (LLM)-driven artificial intelligence (AI) technologies are transforming many fields, including bioinformatics resource development. Applying LLM/AI technologies in Reactome may offer a powerful way to scale curation and consolidate pathway-related data into a single resource. This manuscript describes the first stage of our attempt to adopt LLM/AI technologies for Reactome manual curation. We developed an LLM workflow that can assist curators in adding new genes to existing pathways and refining the functional annotations of existing ones. The workflow predicts pathways in which genes are likely to function, identifies PubMed-indexed literature that may support these predictions, generates text summaries describing potential molecular mechanisms, and extracts functional relationships among biological entities from full-text PDF papers. To validate the workflow output, we used a computational approach based on semantic similarity between LLM workflow-generated summaries and Reactome manual annotations. The results show significant enrichment of high-similarity matches. Manual evaluation of 19 genes indicated that more than half of the outputs are useful for supporting curation. Based on these results, we developed an enhanced workflow that incorporates protein-protein interaction data, facilitating Reactomes reaction-based annotation. In summary, our initial adoption of LLM/AI technologies produced encouraging results and provides a practical framework for integrating AI-assisted methods into Reactomes curation pipeline. The strategies described here may be broadly applicable to community knowledgebases in general.

bioinformatics↗

bioRxiv2025

Multiscale Spatial Transcriptomic Atlas of Human Basal Ganglia Cell-Type and Cellular Community Organization

We generated a multi-region, subcellular-resolution spatial transcriptomic atlas of the human basal ganglia by integrating MERFISH+ and Stereo-seq across four neurotypical donors. These datasets profiled [~]7 million cells spanning the caudate, putamen, nucleus accumbens, and globus pallidus, resolving 60 transcriptionally distinct cell types. We show region-selective, molecular and spatial diversification of medium-spiny-neuron cell types and multiple non-neuronal populations with distinct molecular identities and spatial localizations. Subcellular RNA localization captures somatic size and projection-inferred signatures that reflect direct and indirect pathway topology. Cellular community analyses reveal the enrichment of sub-clusters of astrocytes and oligodendrocytes at striosome-matrix borders, while primate-expanded interneurons are confined to matrix territories. Cross-species mapping uncovers orthologous striosome-matrix organization and conserved dorsolateral-ventromedial gene expression gradients. This atlas provides a foundational molecular and spatial framework for studying human basal ganglia architecture, offering a multi-centimeter scale resource that links cell types, spatial architecture, and subcellular transcript topography across multiple nuclei. HighlightsO_LIOur multi-centimeter scale spatial taxonomy identifies the precise locations of 60 neuronal and glial cell types of human basal ganglia. C_LIO_LIMERFISH+ and Stereo-seq platforms map consistent spatial modules that align with classical neuroanatomical nuclei. C_LIO_LID1D2 hybrid MSNs and primate-expanded interneurons show regional and domain specific organization C_LIO_LISubcellular RNA localization reports soma morphology and projection-inferred signatures. C_LI

neuroscience↗

bioRxiv2025

The Genomic Architecture of Human DNA Replication Origins

The exact sites of DNA replication origins in human and other metazoans remain elusive. Examining human whole-genome sequencing data of 2,616 specimens, we observed conjoining reads at 2,025,756 non-random genomic positions, likely arising from nascent DNA and thus defining replication origins. These origins exhibited a 16 bp motif and periodic occurrence at the intervals of 10.5 bp and 200 bp. Genome-wide replication activity is related to the expression of DNA replication-related genes. Across the genome, DNA replication initiation is more active in early replicating regions, correlated with transcription activity in cis, enriched for de novo mutations and trait-associated polymorphisms. Our high-resolution mapping of human DNA replication origins points to molecular features that govern where and when replication begins in the genome.

cell biology↗

bioRxiv2025

RNA editing fine-tunes transcriptional plasticity and enhances the adaptability of Myzus persicae to diverse host plants

Parthenogenetic organisms can adapt to diverse and rapidly changing environments despite limited standing genetic variation, but the molecular mechanisms enabling such adaptability remain unclear. Here, we investigate this question in the green peach aphid (Myzus persicae, GPA), an asexual generalist herbivore capable of colonizing a wide range of host plants without underlying genetic change. By integrating RNA-seq and DNA-seq from genetically identical clones reared on different hosts, we identified 1,368 high-confidence RNA editing sites (RES), many of which exhibited strong host-dependent differences in editing levels. These RES encompassed all 12 possible nucleotide substitutions, with A-to-I, C-to-U, and U-to-C conversions being particularly prevalent. Editing at several sites shifted dynamically within 48 hours of host transfer and often coincided with changes in transcript abundance. Functional knockdown of the editing genes ADAR1 and ADAR2 reduced editing at specific sites, diminished fecundity, and abolished aphid survival on new hosts. Comparative analyses across aphid species reveal that although the biochemical signatures of A-to-I editing are deeply conserved, the genomic locations and functional roles of edited transcripts are species specific, reflecting lineage-specific adaptive strategies. Together, our findings demonstrate that RNA editing provides a rapid, reversible mechanism for environmental acclimation in GPA, enabling adaptive phenotypic plasticity even in the absence of genetic variation. This work establishes RNA editing as a key molecular basis for the remarkable ecological generalism of clonal organisms. Significance statementA major question in evolutionary biology is how clonal organisms with little genetic variation rapidly adapt to diverse, changing environments. Here we show that RNA editing provides a mechanism enabling the parthenogenetic green peach aphid (Myzus persicae) to colonize a wide range of host plants. RNA editing generates transcriptomic diversity, responds quickly to host shifts, and is essential for adaptation, as silencing ADAR genes disrupts A-to-I editing, reduces fitness, and prevents survival on new hosts. Comparative analyses across aphid species reveal that although the biochemical features of A-to-I editing are conserved, its genomic deployment and functional outcomes differ among species, reflecting lineage-specific strategies. These findings demonstrate that RNA editing creates a dynamic regulatory layer that offsets constraints of asexual reproduction.

molecular biology↗

bioRxiv2025

Mechanistic Investigation of LncRNA-ZMIZ1-AS1 in the Malignant Phenotype of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide, with high recurrence and mortality rates. LncRNA-ZMIZ1-AS1 has attracted considerable attention for its role in the tumor microenvironment, particularly in the regulation of HCC cell growth, invasion, and biological behavior. However, the specific mechanisms of LncRNA-ZMIZ1-AS1 in HCC remain unclear. MethodsWe analyzed the relationship between LncRNA-ZMIZ1-AS1 and HCC clinical prognosis using bioinformatics with GEO and TCGA datasets. We also conducted molecular biology experiments, including cell culture, real-time PCR, and Western blotting, as well as cell viability and flow cytometry analyses, to investigate the effects of LncRNA-ZMIZ1-AS1 knockdown on HCC cell proliferation, migration, invasion, and apoptosis. Furthermore, we used Gene Set Enrichment Analysis (GSEA) to identify potential signaling pathways involving LncRNA-ZMIZ1-AS1. ResultsLncRNA-ZMIZ1-AS1 was highly expressed in HCC patients and significantly associated with clinical characteristics, such as gender and clinical stage. Knockdown of LncRNA-ZMIZ1-AS1 notably inhibited HCC cell proliferation, migration, and invasion while increasing apoptosis rates. Additionally, GSEA suggested a potential association between high LncRNA-ZMIZ1-AS1 expression and the NOTCH signaling pathway. ConclusionThe high expression of LncRNA-ZMIZ1-AS1 in HCC likely promotes malignant phenotypes by regulating signaling pathways such as NOTCH. This finding underscores its potential as a biomarker for HCC, providing a theoretical basis for further research on its molecular mechanisms and clinical applications in liver cancer.

molecular biology↗

bioRxiv2025

De Novo Design of a Protein Binder to Probe Gas Channel and Enhance the Oxygen Tolerance of Hydrogenase

[NiFe]-hydrogenases exhibit outstanding catalytic performance for H2 production and oxidation but suffer from extreme O2 sensitivity, limiting their biotechnological potential. The identity and functional roles of gas channels that connect to the buried [NiFe] active site have remained unclear, impeding the rational engineering of O2-tolerant enzyme variants. In this study, we present an artificial intelligence-guided de novo protein binder design strategy, utilizing compact protein binders as selective molecular probes to map hydrophobic O2 diffusion pathways in Escherichia coli hydrogenase-2 (Hyd-2). By integrating RFdiffusion, ProteinMPNN, and AlphaFold, approximately 100,000 candidate binders were computationally screened, leading to the identification of two high-affinity binders, L1 and L2. Structural, biophysical, and electrochemical analyses reveal that L1 specifically occludes the primary O2 ingress channel, thereby enhancing the enzymes oxygen tolerance by more than threefold, whereas L2, which targets a putative secondary channel, exerts a negligible effect, demonstrating the nonfunctionality of this pathway. These results provide the first direct experimental evidence for a hierarchical organization of O2 diffusion channels in [NiFe]-hydrogenases and establish binder-mediated channel occlusion as a generalizable, mutation-free approach for probing gas transport mechanisms and selectivity in metalloenzymes.

biochemistry↗

bioRxiv2025

Mineral guided molecular enrichment: An interfacial driving force for protocell emergence on early Earth

The emergence of protocells from a dilute prebiotic environment is a fundamental challenge in origins-of-life research. which requires the simultaneous overcoming of molecular dilution, the establishment of metabolic cycles and the formation of selectively permeable compartments. But where and how such critical conditions are satisfied is very obscure and debated. We demonstrate, using contemporary model proteins and enzymatic systems, that mineral surfaces can facilitate the co-adsorption and spatial arrangement of nucleotides, proteins, and lipids into locally enriched, protocell-like assemblies under the examined laboratory conditions. We showed that geochemically relevant minerals efficiently co-adsorb and colocalize diverse biomolecules, creating crowded interfacial microenvironments that support enzyme cascades with substrate channeling-like behavior. Subsequently, these mineral-protein complexes template the assembly of lipid membranes, leading to the formation of discrete compartments that maintain metabolic activity while permitting molecular exchange. This mineral-guided surface enrichment (MSE) mechanism integrates key aspects of "metabolism-first" and "membrane-first" scenarios into a unified pathway, reconciling long-standing conceptual divides in prebiotic chemistry. Our findings establish mineral interfaces as active organizers of protocell emergence, offering a geochemically plausible framework for the origin of cellular life.

evolutionary biology↗

bioRxiv2025

R-loops orchestrate the maternal-to-zygotic transition by harnessing RNA polymerase II pause release

R-loops, ubiquitous three-stranded nucleic acid structures abundant in the mammalian genome, play pivotal roles in diverse biological processes1-3. However, the dynamics and functions of R-loops during mammalian preimplantation embryonic development remain poorly understood. Here we optimized an innovative, ultra-sensitive RIAN-seq method capable of mapping genome-wide R-loop landscape using ultra-low cell numbers to investigate R-loop dynamics in mouse gametes and early embryos. Our findings reveal the widespread presence of AT-rich R-loops during early mouse embryonic development and highlight their critical roles in orchestrating transcription machinery during zygotic genome activation (ZGA). We demonstrate that the stability and inheritance of R-loops through developmental stages are governed by their sequence composition, with GC-rich R-loops exhibiting greater stability. Notably, R-loops, particularly AT-rich R-loops, inhibit DDX21 helicase activity on the 7SK/HEXIM1 snRNP complex, thereby reducing CDK9 release. This, in turn, modulates the phosphorylation of Ser2 at C-terminal domain (CTD) of RNA polymerase II (RNAPIIS2p) at the promoters of major ZGA and maternal genes. Furthermore, R-loops promote RNAPII accumulation at major ZGA gene promoters, preventing RNAPII from premature release and ensuring timely activation of major ZGA genes. Conversely, R-loops enhance the retention of stalled RNAPII at maternal gene promoters, effectively repressing their expression and facilitating the maternal-to-zygotic transition. In summary, our study unveils a key role for R-loops in regulating the maternal-to-zygotic transcription transition and early embryonic development in mice.

developmental biology↗

bioRxiv2025

HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36

Human cytomegalovirus (HCMV) establishes latency in CD34+ hematopoietic progenitor cells (HPCs), where reactivation is intimately linked to cellular differentiation. We demonstrate that the Notch signaling pathway, a key regulator of stem cell maintenance and differentiation, functions as a barrier to HCMV reactivation. Two viral gene products, UL8 and miR-UL36, modulate this pathway during reactivation. UL8 promotes degradation of the Notch3 receptor via the endosomal/lysosomal pathway, dependent on two tyrosine-based motifs (Y305/314) in its cytoplasmic tail. A UL8 mutant lacking these motifs fails to degrade Notch3, resulting in sustained Notch signaling and impaired reactivation in vitro and in humanized mice. Similarly, miR-UL36 reduces expression of Notch3 and the Notch transcription factor Recombination Signal Binding Protein For Immunoglobulin Kappa J Region (RBPJ), suppressing Notch signaling. Deletion of miR-UL36 inhibits reactivation, but this defect, like that of the UL8 mutant, can be rescued by pharmacologic Notch inhibition. Thus, HCMV employs multiple gene products to suppress Notch signaling and promote conditions conducive to reactivation. These findings reveal how HCMV manipulates host differentiation pathways to control latency and suggest therapeutic strategies to prevent viral recurrence in immunocompromised patients. ImportanceHuman cytomegalovirus (HCMV) establishes lifelong latency, posing significant risks to transplant recipients and other immunocompromised individuals. Reactivation depends on progenitor cell differentiation, yet the viral mechanisms governing this process remain unclear. We identify Notch signaling as a major inhibitory pathway to reactivation and show that HCMV uses UL8 and miR-UL36 to suppress this pathway. UL8 degrades Notch3, while miR-UL36 downregulates Notch3 and RBPJ, together reducing Notch signaling and enabling reactivation. Mutant viruses lacking these regulators fail to reactivate efficiently, but this can be reversed by pharmacological inhibition of Notch. These findings establish Notch pathway suppression as a critical viral strategy for reactivation and highlight potential therapeutic targets for preventing HCMV disease.

microbiology↗

bioRxiv2025

Cross-species connectome comparisons reveal the network attributes of memory capacity and time series prediction

The brains connectome provides a powerful blueprint for designing efficient neural networks, yet the impact of incorporating its intricate, non-random architecture into machine learning models remains underexplored. Here, we integrate empirical structural connectomes from four model species--fruit fly, mouse, rat, and macaque--as the recurrent layer in echo state networks (ESNs). We demonstrate that biologically realized networks, particularly the macaque connectome, achieve superior performance in chaotic time series prediction and exhibit higher memory capacity compared to randomly shuffled controls. This computational advantage correlates with small-world topology, which scales with phylogenetic level. Crucially, we identify that weakly connected but highly central nodes are essential for optimal network dynamics; their targeted perturbation significantly degrades performance. Furthermore, functional connectomes from Alzheimers disease patients show computational deficits resembling those induced by weak-tie disruption in healthy networks. Our findings establish that evolved connectome topology is fundamental to efficient information processing, providing key principles for bio-inspired artificial intelligence.

neuroscience↗

bioRxiv2025

Aphid-derived cross-kingdom RNA dynamics underpin maize resistance

Aphids are major pests of maize (Zea mays), yet the molecular mechanisms underlying their interactions with host plants remain poorly understood. Here, we identified and functionally characterized long non-coding RNAs (lncRNAs) from the aphid-specific Ya gene family in the cereal-specialist aphids Rhopalosiphum maidis and R. padi. We showed that Ya genes formed lineage-specific clusters and were transcriptionally active across both species. Multiple Ya transcripts secreted into maize tissues exhibited remarkable stability compared with rapidly degraded aphid mRNAs and migrated systemically, as visualized in planta using an RNA switch-controlled RNA-triggered fluorescence system. RNA interference of Ya genes significantly reduced aphid fecundity, while ectopic expression of Ya lncRNAs in maize enhanced aphid colonization. Importantly, maize genotypes differing in aphid resistance selectively influenced the persistence of aphid mRNAs but not Ya lncRNAs, indicating a decoupled fate of distinct aphid RNA classes in planta. These findings establish Ya lncRNAs as cross-kingdom effectors critical for aphid virulence, and suggest new molecular markers for breeding aphid-resistant maize.

plant biology↗

bioRxiv2025

VO: The Vaccine Ontology

With the widespread use of vaccines in research and clinical settings, there is an urgent need to standardize vaccine representation, integrate information across diverse vaccine types, and support computer-assisted reasoning. Accordingly, we have since 2007 developed the community-based Vaccine Ontology (VO), which aligns with the Basic Formal Ontology and adheres to OBO Foundry principles. VO models ontologically vaccines, vaccine components, vaccine immune responses, vaccine investigation studies and other vaccine-related topics. VO represents more than 10,000 vaccines targeting 289 infectious pathogens and cancers in humans and over 30 nonhuman animal species. VO provides mappings to external resources such as RxNorm, CVX, FDA, and USDA. Various VO use cases exist. VO facilitates vaccine standardization in resources such as the VIOLIN vaccine database, ImmPort, and the Vaccine Adjuvant Compendium (VAC). Semantic queries can be made to query VO. VO has been shown to enhance experimental and clinical vaccine data analysis and vaccine literature mining. Overall, VO standardizes vaccine modeling and representation and greatly supports vaccine AI research in the Semantic Web era.

bioinformatics↗

bioRxiv2025

An improved SIFT algorithm based method for detecting of tumor tissue movement in respiratory motion

BackgroundThis paper intends to make use of the excellent performance of the SIFT algorithm to accurately calculate the displacement of tumor tissues during respiratory movement. Material and methodFirst, we moved phantoms to up,down,right,left respectively to simulate the different directions of movement during respiratory. And rotated phantom to left and right respectively by the angle of 5,10,30,45. Then, used SIFT and cross-correlation algorithms to search the phantoms. Last, use the t-test to check the differences between different directions. ResulThe t-test result proved that there was no significant difference in the detection of moving phantom in all directions. There was no statistical difference between the SIFT of the moving motif and the cross-correlation. The detection results of rotating phantom indicated that the SIFT test results could be used to detect distortion. ConclusionSIFT algorithm can be used to calculate the distortion caused by the thoracic tissue during respiratory movement.

biophysics↗

bioRxiv2025

MiroSCOPE: An AI-driven digital pathology platform for annotating functional tissue units

Cancer tissue analysis in digital pathology is typically conducted across different spatial scales, ranging from high-resolution cell-level modeling to lower-resolution tile-based assessments. However, these perspectives often overlook the structural organization of functional tissue units (FTUs), the small, repeating structures which are crucial to tissue function and key factors during pathological assessment. The incorporation of FTU information is hindered by the need for detailed manual annotations, which are costly and time-consuming to obtain. While artificial intelligence (AI)-based solutions hold great promise to accelerate this process, there is currently no comprehensive workflow for building the large, annotated cohorts required. To remove these roadblocks and advance the development of more interpretable approaches, we developed MiroSCOPE, an end-to-end AI-assisted platform for annotating FTUs at scale, built on QuPath. MiroSCOPE integrates a fine-tunable multiclass segmentation model and curation-specific usability features to enable a human-in-the-loop system that accelerates AI annotation by a pathologist. The system is used to efficiently annotate over 71,900 FTUs on 184 prostate cancer hematoxylin and eosin (H&E)-stained tissue samples and demonstrates ready translation to breast cancer. Furthermore, we publicly release a dataset named Miro-120, consisting of 120 prostate cancer H&E with 30,568 annotations, which can be used by the community as a high-quality resource for FTU-level machine learning aims. In summary, MiroSCOPE provides an adaptable AI-driven platform for annotating functional tissue units, facilitating the use of structural information in digital pathology analyses.

bioinformatics↗

bioRxiv2025

Granulin+ macrophages promote lineage plasticity in prostate cancer through paracrine signaling loops

The role of tumor-infiltrating immune cells in driving phenotype switching remains unclear, despite the well-established association between lineage transition and drug resistance in prostate cancer. This study employed an integrated analysis of single-cell multiomics to investigate the dynamics of immune infiltration, transcriptional programs, and cell-cell communication in prostate cancer. Our results demonstrated that granulin (GRN) positive macrophages facilitated the transition from adenocarcinoma to a multilineage state with mesenchymal and stem-like traits by activating intra-tumoral NF-{kappa}B signaling. Subsequently, the multilineage clones induced macrophages to highly express granulin through the secretion of CSF1, forming a positive feedback cell communication loop. Next, we validated the biological function of granulin in mediating epithelial-mesenchymal transition in vitro. Additionally, organoids drug resistance assay demonstrated that granulin drove resistance to androgen receptor (AR)-targeted drug (enzalutamide). Moreover, pharmacologic blockade of the CSF-1/CSF-1R axis in TRAMP mouse models reduced the expression of GRN in macrophages and suppressed the formation of multilineage subclones in prostate malignant cells. Furthermore, multiplex immunofluorescence staining of tumor samples from TRAMP mouse models revealed the VIM lineages were spatially in close contact with macrophages. Meanwhile, Cytometry by Time-Of-Flight (CyTOF) analysis validated our findings at single-cell protein level in patients with castration-resistant prostate cancer. Besides, three distinct tumor-infiltrating subsets associated with disease relapse were identified, including DCN+ endothelial cells, CCL7+ fibroblasts, and IFIT1+ neutrophils. These results offer potential therapeutic targets to address lineage plasticity-driven resistance to AR-targeted therapy.

cancer biology↗

bioRxiv2025

Targeted inhibition of colorectal carcinoma using a designed CEA-binding protein to deliver TCF/LEF transcription factor decoy DNA and p53 protein

Colorectal carcinoma (CRC) is characterized by mutations in Wnt signaling pathway components and the p53 protein, and anti{square}PD-1/anti{square}PD-L1 immunotherapy has shown limited efficacy in microsatellite stable CRC patients. In this work, CEABP1, a de novo-designed binding protein for the CRC marker carcinoembryonic antigen (CEA), and cell-penetrating peptide (CPP) were employed for targeted delivery in CRC cells. The consensus binding motifs for TCF/LEF and Max were concatenated, and Max DNA binding domain protein fused with CPP and CEABP1 was used to deliver this TCF/LEF transcription factor decoy (TFD) DNA specifically into CRC cells by recognizing CEA on the CRC cell surface to inhibit Wnt target gene transcription, leading to marked suppression of CRC cell proliferation and xenograft tumor growth. In addition, p28 was employed as a CPP to specifically deliver the p28-p53-CEABP1 protein into CRC cells, which significantly enhanced p53 inhibition of CRC cell proliferation and xenograft tumor growth. Furthermore, codelivery of the p14ARF protein together with p53 increased its antitumor activity by prolonging its effective duration. These findings paved the way for the development of related biomacromolecular anticancer therapeutics.

cancer biology↗