Bioinformatics Analysis Revealsthe role of DLGAP4 in the development and progression of hepatocellular carcinoma

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This study aims to characterize DLGAP4 expression patterns, prognostic significance, and association with immune infiltration in the HCC tumor microenvironment, to assess its potential as a biomarker or therapeutic target. Methods We analyzed DLGAP4 expression in HCC and its prognostic significance using the TCGA database, performing survival analysis with the Kaplan-Meier method. Functional pathways linked to DLGAP4 were identified via GO and KEGG enrichment analyses, and further explored by gene set variation analysis (GSVA). Immune cell infiltration correlations were assessed using TIMER2.0. Clinical samples were immunohistochemically stained to validate bioinformatic results. Results DLGAP4 expression was significantly upregulated in HCC tissues and associated with poor patient prognosis. Enrichment analysis implicated DLGAP4 in biological processes including chromatin modification and protein translation. Single-cell data analysis revealed that high DLGAP4 expression correlated with features of the tumor microenvironment, such as tumor-associated macrophages, cancer-associated fibroblasts, stemness, angiogenesis, and metabolic reprogramming. Immunohistochemical results further confirmed a significant correlation between DLGAP4 expression and immune cell infiltration. Conclusion DLGAP4 is upregulated in HCC and associated with poor prognosis and immune infiltration in the tumor microenvironment, suggesting its potential as a prognostic biomarker and therapeutic target for HCC. Hepatocellular carcinoma DLGAP4 Chromatin remodeling Protein translation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Liver cancer is one of the most common malignant tumors worldwide, with hepatocellular carcinoma (HCC) accounting for more than 90% of cases [ 1 ] . Over recent decades, both the incidence and mortality rates of liver cancer have increased significantly. Current primary treatment options include surgical resection, liver transplantation, and targeted therapy; however, the prognosis remains poor, with a five-year survival rate of only 10–20% [2 , 3] . Due to the lack of specific early-stage biomarkers, most patients are diagnosed at an advanced stage. Therefore, identifying novel therapeutic targets is essential to improve the prognosis of liver cancer patients [ 4 ] .The DLGAP family comprises five members (DLGAP1-5), each encoded by distinct chromosomes. These genes are transcribed and translated into multiple protein isoforms, which perform diverse biological functions and represent key components of the DLGAP family [ 5 ] . Among them, DLGAP4 has been extensively studied and is well-established as being associated with several neurological disorders [ 6 ] , including bipolar disorder [ 7 ] , cerebellar ataxia [ 8 ] , motor clumsiness [ 9 ] , hand coordination deficits [ 10 ] , and tremors [ 11 ] . However, the role of DLGAP4 in hepatocellular carcinoma (HCC) remains poorly understood. Therefore, this study aims to investigate the biological functions and underlying mechanisms of DLGAP4 in HCC. In conclusion, bioinformatics integration offers a systematic and comprehensive approach for identifying potential therapeutic targets and developing innovative treatment strategies in hepatocellular carcinoma (HCC) [ 12 – 14 ] . To elucidate the biological function of DLGAP4 in HCC, this study analyzed clinical characteristics and survival data from The Cancer Genome Atlas (TCGA), revealing the significance of its expression in hepatocellular carcinoma. Furthermore, we investigated the correlation between DLGAP4 expression and immune cell infiltration using clinical samples. Our findings pave the way for improved clinical management of HCC and underscore the potential of DLGAP4 as a novel therapeutic target. Meterials and methods Patients and samples The collection of samples and clinical data in this study was approved by the Ethics Committee of Gansu Provincial People's Hospital. Written informed consent was obtained from all participating patients. Each sample was diagnosed by at least two experienced senior pathologists. Datasets Clinical data and RNA-seq results from 374 hepatocellular carcinoma (HCC) cases within the TCGA-Liver Hepatocellular Carcinoma (LIHC) project, including 50 matched pairs of tumor and adjacent normal tissues, were retrieved from the TCGA database. DLGAP4 mRNA expression was further examined using the GEO dataset GSE14520. All data supporting this study are publicly accessible through the TCGA data portal ( https://portal.gdc.cancer.gov/projects/TCGA-LIHC ). Single-cell RNA sequencing data were analyzed using datasets GSE151530 and GSE189903, available from the GEO repository ( http://www.ncbi.nlm.nih.gov/geo/ ). Functional enrichment analysis The genes most strongly associated with DLGAP4, along with a characteristic gene set of the cell cluster, were submitted to the Database for Annotation, Visualization, and Integrated Discovery (DAVID, version 6.8). Analysis was performed using official gene symbols as identifiers and Homo sapiens as the background species. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were subsequently conducted. The top six significantly enriched terms (P < 0.05), ranked in ascending order of P-value, are presented in this study. Gene set variation analysis (GSVA) The gene list associated with chromatin remodeling was obtained from the AmiGO2 portal ( http://amigo.geneontology.org/amigo ). The functional enrichment score for each hepatocellular carcinoma (HCC) sample was computed using the specified package in the R environment under default parameters. A heatmap visualizing the enrichment results was generated with the pheatmap package in R. The correlation between DLGAP4 expression and chromatin remodeling was assessed using Pearson correlation analysis. Single-cell sequencing The Seurat package (version 4.5.1) was used to perform cell clustering in both HCC patients and co-culture models. The standard preprocessing workflow for single-cell sequencing data was conducted as described in previous studies. The resulting cell clustering outcomes have been presented in an earlier section of this article. TIMER2.0 analysis The TIMER2.0 database enables the analysis of differential gene expression between tumor and adjacent normal tissues based on TCGA data. We utilized TIMER2.0 to examine DLGAP4 expression levels across various cancer types and to evaluate the correlation between DLGAP4 and immune cell infiltration. Statistical significance of differential expression was assessed using the Wilcoxon test along with the TIMER deconvolution method. HE staining and Immunohistochemistry (IHC) All tissue specimens were fixed in 4% neutral buffered formalin, embedded in paraffin, and sectioned continuously at a thickness of 4 µm. Sections were then subjected to HE staining and EnVision two-step immunohistochemical staining. For IHC, HCC tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Endogenous peroxidase activity was blocked with 3% H₂O₂, and non-specific binding sites were blocked with 5% goat serum. The sections were incubated overnight at 4 ℃ with a rabbit anti-human DLGAP4 antibody (1:500, Bioss, China). After washing three times with phosphate-buffered saline (PBS), the sections were incubated for 30 minutes with a goat anti-rabbit IgG secondary antibody (1:500, Bioss, China). Detection was performed using 3,3′-diaminobenzidine (DAB), followed by counterstaining, dehydration, and mounting. DLGAP4-positive cells were observed under an inverted fluorescence microscope. Statistical analyses Statistical analyses and data visualization were conducted using R (version 4.5.1) and IBM SPSS Statistics (version 25.0). Differences between two groups were assessed using an unpaired t-test, while comparisons among more than two groups were evaluated by one-way ANOVA. Correlations between variables were examined using Pearson correlation analysis. Survival analysis was performed with Kaplan-Meier curves, and the log-rank test was applied to determine the statistical significance of survival differences. A P-value of less than 0.05 was considered statistically significant. Results DLGAP4 is highly expressed in HCC First, we investigated the expression of DLGAP4 across multiple cancer types using the TIMER database. Differential expression analysis revealed that DLGAP4 was significantly up-regulated in various tumor tissues compared to normal controls (Fig. 1 a). This up-regulation was further confirmed by volcano plot analysis, which identified DLGAP4 as one of the prominently upregulated genes (Fig. 1 b). Subsequent analyses in the TCGA database, including both non-matched and matched comparisons of tumor versus normal tissues, consistently showed elevated DLGAP4 expression in cancerous tissues (Fig. 1 c-d). Moreover, receiver operating characteristic (ROC) analysis indicated that DLGAP4 expression exhibited high specificity in distinguishing cancer from non-cancer samples (Fig. 1 e).We also assessed the association between DLGAP4 expression levels and clinicopathological characteristics. In the TCGA cohort, higher DLGAP4 expression was asymmetrically distributed across several clinical parameters, including recurrence, AFP levels, tumor grade, presence of liver cirrhosis, HBV status, age, and gender (Fig. 1 f). Comparative analyses among different subgroups showed that DLGAP4 expression was significantly elevated in samples with high AFP levels (> 300 ng/mL; Fig. 1 g), older age (> 60 years; Fig. 1 h), female gender (Fig. 1 i), and HBV-positive status (Fig. 1 j). Tumor cell DLGAP4 regulates chromatin remodeling To investigate the biological functions associated with DLGAP4, genes most correlated with DLGAP4 (|R| >0.5, P < 0.05) were identified using Pearson correlation analysis in both the TCGA and GSE14520 datasets. Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), were performed based on these gene sets.In the TCGA database, the biological processes most significantly associated with DLGAP4 were positive regulation of transcription by RNA polymerase II, regulation of DNA-templated transcription, and neuron projection development (Fig. 2 a). The primary cellular components linked to DLGAP4 were the nucleoplasm, nucleus, and cytosol (Fig. 2 b). For molecular function, DLGAP4 was predominantly associated with protein binding and cadherin binding (Fig. 2 c). The most relevant signaling pathways included endocytosis, bacterial invasion of epithelial cells, and Shigellosis (Fig. 2 d). Similar enrichment profiles for biological processes, cellular components, molecular functions, and signaling pathways were observed in the GSE14520 dataset (Fig. 2 e-h).These findings suggest that DLGAP4 may play an important role in chromatin remodeling and the regulation of protein translation in hepatocellular carcinoma cells. Tumor cell CD161 is positively correlated with transcription During cancer cell proliferation, chromatin remodeling and protein translation play critical roles. To investigate the potential influence of DLGAP4 on these processes, we assessed its association with transcriptional and translational activity. Using gene set variation analysis (GSVA) on the TCGA and GSE14520 datasets, enrichment scores for chromatin remodeling-related pathways were evaluated. Correlation analysis revealed that DLGAP4 expression was positively associated with the majority of chromatin remodeling and protein translation functions (Fig. 3 a). These findings were further validated in the TCGA cohort (Fig. 3 b). Collectively, these results suggest that DLGAP4 is involved in chromatin reorganization and the regulation of transcription and translation in HCC cells. Protein translation regulation and chromatin remodeling are positively correlated with high CD161 expression DLGAP4 has been implicated in tumor-related transcription and translation processes. To further explore this, we examined the relationship between DLGAP4 and several known protein translation genes,including EIF4G1, RPS6, KRAS, PTEN, EIF4A1, CTNNB1, TP53, and PDCD4,using data from the GSE14520 and TCGA databases (Fig. 4 a). DLGAP4 expression showed a significant positive correlation with these genes.Additionally, we assessed seven transcription-related gene clusters as markers of transcriptional activity: activated-induced cytidine deaminase (AICDA), β-tubulin (ACTB), apoptosis-inducing factor (AIF), cell division cycle protein1(CDT1), cyclin-dependent kinase 9 (CDK9), bromo-adjacent homology domain-containing protein 1 (BAHD1), and histone acetyl-lysine reader (CECR2). Correlation analyses revealed that DLGAP4 was significantly positively correlated with each of these meta-gene clusters in both the TCGA and GSE14520 datasets (Fig. 4 b-c).These results support the hypothesis that DLGAP4 interacts with and upregulates key genes involved in tumor transcription and translation, thereby potentially promoting tumor cell growth. The expression characteristics of DLGAP4 in different cell types of HCC tissues This study analyzed hepatocellular carcinoma (HCC) patient samples and classified the cells into 25 distinct clusters based on their specific gene expression profiles (Fig. 5 a). Among these, clusters 12 and 21 were identified as tumor cells. Notably, stem cell markers (PROM1), tumor-associated macrophage markers (APOE and SLC40A1), metabolic reprogramming markers (SQLE and ACSL4), and cell cycle regulators (CDKN3 and PRC1) all exhibited high expression levels within these clusters (Fig. 5 b).The enrichment of DLGAP4 in these cell clusters was further validated using a public single-cell sequencing dataset (GSE189903), in which cells from liver cancer samples were categorized into 32 clusters (Fig. 5 c). Consistent with our initial findings, the same markers showed pronounced enrichment features in tumor cells (Fig. 5 d). The association between DLGAP4 expression and the characteristics of immune cell infiltration Furthermore, using the TIMER2.0 database, we analyzed the correlation between DLGAP4 expression and immune cell infiltration in liver cancer. The analysis revealed positive correlations between DLGAP4 expression and several immune cell types, with correlation coefficients greater than 0.3 for CD4⁺ T cells, B cells, dendritic cells, macrophages, and neutrophils(Fig. 6 a-f). In contrast, the correlation with CD8⁺ T cells was weak (r = 0.086). These results suggest that DLGAP4 expression in liver cancer is significantly associated with increased infiltration of multiple immune cell types. DLGAP4 is an independent prognostic factor for patients with HCC To evaluate the prognostic value of DLGAP4 in hepatocellular carcinoma (HCC), we performed Kaplan–Meier survival analysis using data from the TCGA and GSE14520 databases. In the TCGA cohort, patients with high DLGAP4 expression had a significantly shorter overall survival (median survival: 697 days) compared to those with low expression (median survival: 870 days; Fig. 7 a). The prognostic significance of DLGAP4 was further confirmed in the GSE14520 dataset (Fig. 7 b). Immunohistochemical detection of the expression of DLGAP4 in liver cancer tissues We collected 40 tissue specimens from patients with hepatocellular carcinoma and performed immunohistochemical staining to evaluate the expression of DLGAP4, CD3, CD8, CD45RO, Foxp3, and Granzyme B (Fig. 8 a). The results demonstrated that DLGAP4 expression was significantly higher in tumor tissues compared to normal tissues. Furthermore, tumor tissues exhibited high expression of CD3 and Foxp3, but low expression of CD8, CD45RO, and Granzyme B (Fig. 8 b–g). These findings suggest that DLGAP4 may suppress the immune response within the tumor microenvironment by modulating regulatory T cell activity in hepatocellular carcinoma. Discussion The DLGAP protein family comprises five members, among which DLGAP5 has been the primary focus in cancer research. Overexpression of DLGAP5 has been shown to promote malignant progression in several cancers, including lung adenocarcinoma [ 15 ] , bladder cancer [ 16 ] , gallbladder cancer [ 17 ] , endometrial cancer [ 18 ] ,breast cancer [ 19 ] , colorectal cancer [ 20 ] , gastric cancer [ 21 ] , and glioma [ 22 ] . Currently, DLGAP4 expression is primarily and closely associated with neurological and psychiatric disorders such as cerebellar movement disorders [ 23 ] , congenital cerebellar ataxia [ 24 ] , and schizophrenia [ 25 ] .Due to high conservation in molecular structure and function across the DLGAP family, Liu et al. proposed that DLGAP4 may exhibit oncogenic roles similar to those of DLGAP5 [ 26 ] . Their study demonstrated that DLGAP4 expression is significantly elevated in gastric cancer tissues compared to normal gastric tissues, and high DLGAP4 expression was correlated with poor prognosis in gastric cancer patients, suggesting its potential as a prognostic biomarker. However, research on DLGAP4 in hepatocellular carcinoma (HCC) remains limited.The present study revealed that DLGAP4 expression is significantly upregulated in HCC tissues compared to adjacent normal liver tissues and is closely associated with clinicopathological features of HCC patients. Furthermore, high DLGAP4 expression was identified as an independent risk factor for poor prognosis in HCC. These findings imply that DLGAP4 may play a critical role in the malignant progression of hepatocellular carcinoma. Chen H et al. reported that SOX11 may serve as a potential prognostic biomarker in hepatocellular carcinoma, associated with immune infiltration and ferroptosis [ 27 ] .Analysis using the TIMER2.0 online database revealed that DLGAP4 is highly expressed in multiple cancer types, including breast cancer, cholangiocarcinoma, colorectal cancer, and hepatocellular carcinoma. In a related context, Jia Hui et al. evaluated the diagnostic and prognostic value of GPI in lung adenocarcinoma using ROC curve analysis [ 28 ] . Similarly, the current study demonstrated that DLGAP4 expression in tumor tissues exhibits high specificity. Wang Di et al. analyzed the clinicopathological characteristics associated with KLRB1 through heatmap visualization [ 29 ] .Our results indicate that DLGAP4 expression correlates with clinical features such as AFP levels and HBV status. Furthermore, Cairong Dong et al. reported that high DLGAP4 expression promotes proliferation and metastasis in HCC by upregulating PPARβ/δ protein [ 30 ] .In this study, analyses of the TCGA and GSE14520 databases indicated that DLGAP4 plays a significant role in biological functions related to chromatin remodeling and protein translation. Subsequent GSVA further confirmed that DLGAP4 is critically involved in transcriptional and translational regulation. Moreover, Pearson correlation analysis identified a positive association between DLGAP4 expression and genes regulating both protein translation and chromatin remodeling. The findings presented above underscore the significant role of DLGAP4 in chromatin remodeling and other cellular processes within hepatocellular carcinoma. In a related study, Jiajing Li et al. identified CDK1 and DLGAP5 as key regulators of tumor immune cell infiltration in HCC [ 31 ] . Analysis of single-cell sequencing data from our work further suggests that DLGAP4-expressing tumor cells exhibit stem-like properties, metabolic reprogramming, and pro-angiogenic activity. Moreover, DLGAP4 expression shows a correlation with both tumor-associated macrophages and cancer-associated fibroblasts, indicating its potential importance in maintaining a tumor-promoting microenvironment conducive to malignant progression.Zhou Hongrui et al. demonstrated that POC1A plays a critical role in immune cell infiltration in hepatocellular carcinoma (HCC) [ 32 ] . In line with this, our analysis using the TIMER2.0 database revealed a positive correlation between DLGAP4 expression and the infiltration levels of multiple immune cells, including CD4⁺ T cells and B cells. These findings imply that DLGAP4 may facilitate the recruitment or activation of immune cells, potentially shifting the tumor microenvironment toward an immunoreactive(“hot tumor”)phenotype, which is generally associated with improved prognosis and enhanced responsiveness to immunotherapy [33] .However, Kaplan–Meier survival analysis indicated that high DLGAP4 expression was correlated with poorer overall survival, identifying DLGAP4 as an independent risk factor for unfavorable prognosis in HCC patients. This apparent contradiction may reflect complex roles of DLGAP4 in both immune activation and tumor progression. Further validation using immunohistochemical staining of clinical tissue specimens confirmed a positive correlation between DLGAP4 expression and immune markers, supporting the notion that DLGAP4 contributes critically to the immune microenvironment in HCC.Collectively, these results underscore the potential of DLGAP4 as a predictive biomarker and a future target for immunotherapy in hepatocellular carcinoma.Although we performed a systematic analysis of DLGAP4 and validated our findings across multiple independent databases, this study has several limitations. First, the publicly available data may be subject to inherent biases or incompleteness due to the retrospective nature of the datasets. Second, the precise molecular mechanisms through which DLGAP4 influences chromatin remodeling and protein translation in hepatocellular carcinoma remain unclear. Further experimental studies are needed to elucidate these mechanisms and confirm our bioinformatic observations. Conclusion This study establishes DLGAP4 as a potential prognostic biomarker and therapeutic target in hepatocellular carcinoma (HCC), showing significant upregulation correlated with poor survival. DLGAP4 is involved in chromatin remodeling and protein translation, and is associated with an immunosuppressive tumor microenvironment characterized by infiltration of tumor-associated macrophages and cancer-associated fibroblasts, along with stemness, angiogenesis, and metabolic reprogramming. Despite validation across databases, the mechanistic role of DLGAP4 requires further experimental investigation. Declarations Competing interests The authors declare no conflicts of interest. Ethics approval The collection of samples and clinical data in this study was approved by the Ethics Committee of Gansu Provincial People's Hospital. Consent for publication All authors agree to publication. Funding This work was supported by the Science and Technology Department of Gansu Province, Projects Unit (Grant No: 22JR5RA671). Author Contribution Feng Yang designed research; Shoufeng Chang and Ruxia Li wrote the manuscript with contributions from all authors. All authors read and approved the initial manuscript. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Hwang SY, Danpanichkul P, Agopian V, Mehta N, et al. Hepatocellular carcinoma: updates on epidemiology, surveillance, diagnosis and treatment. Clin Mol Hepatol. 2025;31(Suppl):S228–54. https://doi.org/10.3350/cmh.2024.0824 . Yeo YH, Abdelmalek M, Khan S, Moylan CA, et al. Current and emerging strategies for the prevention of hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol. 2025;22(3):173–90. https://doi.org/10.1038/s41575-024-01021-z . Chan YT, Zhang C, Wu J, Lu P, et al. 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Cite Share Download PDF Status: Published Journal Publication published 03 Dec, 2025 Read the published version in Journal of Gastrointestinal Cancer → Version 1 posted Editorial decision: Revision requested 14 Oct, 2025 Reviews received at journal 13 Oct, 2025 Reviewers agreed at journal 12 Oct, 2025 Reviewers invited by journal 23 Sep, 2025 Editor assigned by journal 23 Sep, 2025 Submission checks completed at journal 15 Sep, 2025 First submitted to journal 14 Sep, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Dang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDCCA2DShoefmfnAgQ8/iNeSJifZzpZ4cGYP8VoOGxuc5zE+zMFGhA6+42cPv+bNYU5sOMzz4TADD4M8v9gB/Fokz+SlWfNuY0tsbObdcLjAgsFw5uwE/FoMDuSYGfNu40lsZgZqmcHDkGBwm5CW829AWiQS25h5HhzmYSNGy40c48e82wyMeZh5GIjTInnjjRnj3G0JchLMbAbAQJYg7Be+8znGH95u+89jf/7w4w8fftjI80sT0AIEbFI8CI4EQeUgwPyRqGQyCkbBKBgFIxcAABWVR/hMJI+HAAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"yamei","middleName":"","lastName":"Dang","suffix":""},{"id":524258853,"identity":"b0540b62-acf1-4e93-a893-e64663af5672","order_by":1,"name":"feng yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"feng","middleName":"","lastName":"yang","suffix":""},{"id":524258854,"identity":"8aa3876e-6c26-47c1-8999-274fc5790843","order_by":2,"name":"shoufeng chang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"shoufeng","middleName":"","lastName":"chang","suffix":""},{"id":524258855,"identity":"7922661f-22a6-48c0-b2aa-305f582820fa","order_by":3,"name":"ruxia li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"ruxia","middleName":"","lastName":"li","suffix":""},{"id":524258856,"identity":"9539c923-92f8-48d9-9139-e63bc1bb9be0","order_by":4,"name":"jing wei","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"jing","middleName":"","lastName":"wei","suffix":""},{"id":524258857,"identity":"3b4ba9a1-56f9-4b08-bcf7-1edf66373c74","order_by":5,"name":"hua zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"hua","middleName":"","lastName":"zhang","suffix":""},{"id":524258858,"identity":"4281084a-0d28-432e-b94a-b5e0c6a11c94","order_by":6,"name":"qiqi wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"qiqi","middleName":"","lastName":"wang","suffix":""},{"id":524258859,"identity":"893e12fb-c3e5-413b-b5f1-0eead75ac02e","order_by":7,"name":"zhenjun li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"zhenjun","middleName":"","lastName":"li","suffix":""}],"badges":[],"createdAt":"2025-09-14 15:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7613609/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7613609/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12029-025-01359-w","type":"published","date":"2025-12-03T15:57:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93006939,"identity":"2af935c2-9666-4aee-bfb1-92a035c656f8","added_by":"auto","created_at":"2025-10-08 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06:48:30","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":101529,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7613609/v1/484ea1d76a1e883576aa5a62.html"},{"id":93007983,"identity":"d7754309-ac90-4074-9110-4f0921bcc7b7","added_by":"auto","created_at":"2025-10-08 07:04:30","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":300027,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and clinicopathological characteristics of DLGAP4 in HCC. (a) The expression of DLGAP4 in pan-cancer.(b)Volcano plot of DGEs in TCGA datasets, the red dots represent up-regulated genes, the blue dots represent down-regulated genes, and the grey dots represent genes with no significant difference in expression. The screening criteria are set to P\u0026lt;0.0001, |log2FC|≥2.(c)DLGAP4 levels across tumor/normal specimens from TCGA;(d)DLGAP4 levels in paired tumor and normal samples (TCGA);(e) ROC curve analysis for DLGAP4 expression in tumor and normal tissue used by TCGA;(f)The landscape of DLGAP4-related clinicopathological features of HCC in the TCGA database.(g-j)DLGAP4 was significantly increased in higher AFP,higher-age,Female and HBV(+) HCC in theTCGA databases. The significance of the difference was tested by one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7613609/v1/48d4f70cb9fe5e0a10b77a00.jpeg"},{"id":93006934,"identity":"8923cf3c-dc6d-49e4-a478-e4d879e0bd8e","added_by":"auto","created_at":"2025-10-08 06:48:29","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":106293,"visible":true,"origin":"","legend":"\u003cp\u003eDLGAP4 is closely associated with transcription and translationimmune process regulation in HCC.(a-g), Biological processes (BP), cellular components (CC), and molecular functions(MF) are mostly related to DLGAP4 in the TCGA database and GSE14520.(d-h)Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DLGAP4 in the TCGA database and GSE14520.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7613609/v1/9218c457cb7ce2f8ae5ffec3.jpeg"},{"id":93007832,"identity":"0eb111c9-0c34-4a43-a17b-72ce971e40d8","added_by":"auto","created_at":"2025-10-08 06:56:30","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":223583,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis between DLGAP4 expression and chromatin remodeling enrichment scores. (a-b)The heatmap showed the expression of DLGAP4 and the enrichment scores of chromatin remodeling functions of each patient in the GSE14520 and The Cancer Genome Atlas (TCGA) databases. The samples were arranged in ascending order of the expression of DLGAP4. The column graph and line graph on the right showed the R-value and P-value of the correlation analysis.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7613609/v1/f529e9eabc8c61e69b398b6c.jpeg"},{"id":93007981,"identity":"c495f45f-1a7e-43a7-8792-5d0eacfdd883","added_by":"auto","created_at":"2025-10-08 07:04:30","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":89133,"visible":true,"origin":"","legend":"\u003cp\u003eThe correlation between DLGAP4 expression and protein translation and chromatin remodeling in the GSE14520 and The Cancer Genome Atlas(TCGA)databases.(a)Pearson correlation between DLGAP4 and protein translation.The width of the band represented the R-value.The color of the band represented the P-value.The correlation was tested by Pearson correlation analysis.(b-c)Correlation matrix of DLGAP4 and chromatin remodeling.The bottom left showed the correlation coefficient.The correlation coefficients were demonstrated as the proportion of the pie charts.The red parts represented a positive correlation,while the green parts represented a negative correlation.The correlation was tested by Pearson correlation analysis.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7613609/v1/8550a6c00b942c4c0eba048f.jpeg"},{"id":93006943,"identity":"46f0131e-740d-4e90-b92e-50e74ac4af9a","added_by":"auto","created_at":"2025-10-08 06:48:30","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107143,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression pattern of DLGAP4 in HCC.(A)Subtypes of lymphocytes from the surgical samples of HCC patients.(a and c)Cell clustering from the surgical samples of HCC patients.(b and d ) Association between DLGAP4 and Cancer cell markers(AFP,ALB,HNF4A and GPC3),stem cell markers(PROM1),tumor-associated macrophage markers(APOE and SLC40A1),Cancer-associated fibroblasts markers(ACTA2),angiogenesis markers(PECAM1),metabolic reprogramming(SQLE and ACSL4),cell cycle(CDKN3 and PRC1) of HCC.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7613609/v1/c406ae2bbe9aed8de5335d04.jpeg"},{"id":93007835,"identity":"db77c38a-0162-414e-82b2-b27d4791b8a5","added_by":"auto","created_at":"2025-10-08 06:56:30","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116735,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis of DLGAP4 in tumor immune cells.(a-f)Correlation analysis of DLGAP4 in CD4\u003csup\u003e+\u003c/sup\u003e T cells,CD8\u003csup\u003e+\u003c/sup\u003e T cells,B cells,dendritic cell,macrophage and neutrophil.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7613609/v1/d5784a34dfe28b77b677486a.jpeg"},{"id":93006935,"identity":"6d39f5f6-2dc5-4d0d-829c-cdd5a14e2504","added_by":"auto","created_at":"2025-10-08 06:48:30","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":56575,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier analysis of DLGAP4 expression in the GSE14520 and The Cancer Genome Atlas (TCGA) databases. (a) and (b) The cutoff of the group is the median expression of DLGAP4. The significance of the prognostic value was tested by a log-rank test.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7613609/v1/1a1d2384a4e5cdd1f8f81fba.jpeg"},{"id":93007837,"identity":"1c2d3e50-9914-4ce9-841c-762c19629bf8","added_by":"auto","created_at":"2025-10-08 06:56:30","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":135252,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical detection of the expression of DLGAP4 and other proteins in liver cancer tissues. \u0026nbsp;(a) Immunohistochemical detection of the expression levels of DLGAP4, CD3, CD8, CD45RP, Foxp3 and Granzyme B. (b-g) Bar charts showing the results of various indicator tests.The significance of the difference was tested by one-way ANOVA\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7613609/v1/a130a343dd48f8ffe94ffbab.jpeg"},{"id":97724004,"identity":"c2817023-96b3-4e68-8cbf-7539c86d57a4","added_by":"auto","created_at":"2025-12-08 16:10:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1944148,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7613609/v1/0ae0fa52-628f-4aaf-85fa-e20d912ce24a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bioinformatics Analysis Revealsthe role of DLGAP4 in the development and progression of hepatocellular carcinoma","fulltext":[{"header":"Background","content":"\u003cp\u003eLiver cancer is one of the most common malignant tumors worldwide, with hepatocellular carcinoma (HCC) accounting for more than 90% of cases\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Over recent decades, both the incidence and mortality rates of liver cancer have increased significantly. Current primary treatment options include surgical resection, liver transplantation, and targeted therapy; however, the prognosis remains poor, with a five-year survival rate of only 10\u0026ndash;20%\u003csup\u003e[2\u003c/sup\u003e, \u003csup\u003e3]\u003c/sup\u003e. Due to the lack of specific early-stage biomarkers, most patients are diagnosed at an advanced stage. Therefore, identifying novel therapeutic targets is essential to improve the prognosis of liver cancer patients\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.The DLGAP family comprises five members (DLGAP1-5), each encoded by distinct chromosomes. These genes are transcribed and translated into multiple protein isoforms, which perform diverse biological functions and represent key components of the DLGAP family\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Among them, DLGAP4 has been extensively studied and is well-established as being associated with several neurological disorders\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, including bipolar disorder\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, cerebellar ataxia\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, motor clumsiness\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, hand coordination deficits\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, and tremors\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, the role of DLGAP4 in hepatocellular carcinoma (HCC) remains poorly understood. Therefore, this study aims to investigate the biological functions and underlying mechanisms of DLGAP4 in HCC.\u003c/p\u003e\u003cp\u003eIn conclusion, bioinformatics integration offers a systematic and comprehensive approach for identifying potential therapeutic targets and developing innovative treatment strategies in hepatocellular carcinoma (HCC)\u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. To elucidate the biological function of DLGAP4 in HCC, this study analyzed clinical characteristics and survival data from The Cancer Genome Atlas (TCGA), revealing the significance of its expression in hepatocellular carcinoma. Furthermore, we investigated the correlation between DLGAP4 expression and immune cell infiltration using clinical samples. Our findings pave the way for improved clinical management of HCC and underscore the potential of DLGAP4 as a novel therapeutic target.\u003c/p\u003e"},{"header":"Meterials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatients and samples\u003c/h2\u003e\u003cp\u003e The collection of samples and clinical data in this study was approved by the Ethics Committee of Gansu Provincial People's Hospital. Written informed consent was obtained from all participating patients. Each sample was diagnosed by at least two experienced senior pathologists.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDatasets\u003c/h3\u003e\n\u003cp\u003eClinical data and RNA-seq results from 374 hepatocellular carcinoma (HCC) cases within the TCGA-Liver Hepatocellular Carcinoma (LIHC) project, including 50 matched pairs of tumor and adjacent normal tissues, were retrieved from the TCGA database. DLGAP4 mRNA expression was further examined using the GEO dataset GSE14520. All data supporting this study are publicly accessible through the TCGA data portal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/projects/TCGA-LIHC\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/projects/TCGA-LIHC\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Single-cell RNA sequencing data were analyzed using datasets GSE151530 and GSE189903, available from the GEO repository (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eFunctional enrichment analysis\u003c/h3\u003e\n\u003cp\u003eThe genes most strongly associated with DLGAP4, along with a characteristic gene set of the cell cluster, were submitted to the Database for Annotation, Visualization, and Integrated Discovery (DAVID, version 6.8). Analysis was performed using official gene symbols as identifiers and Homo sapiens as the background species. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were subsequently conducted. The top six significantly enriched terms (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), ranked in ascending order of P-value, are presented in this study.\u003c/p\u003e\n\u003ch3\u003eGene set variation analysis (GSVA)\u003c/h3\u003e\n\u003cp\u003eThe gene list associated with chromatin remodeling was obtained from the AmiGO2 portal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://amigo.geneontology.org/amigo\u003c/span\u003e\u003cspan address=\"http://amigo.geneontology.org/amigo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The functional enrichment score for each hepatocellular carcinoma (HCC) sample was computed using the specified package in the R environment under default parameters. A heatmap visualizing the enrichment results was generated with the pheatmap package in R. The correlation between DLGAP4 expression and chromatin remodeling was assessed using Pearson correlation analysis.\u003c/p\u003e\n\u003ch3\u003eSingle-cell sequencing\u003c/h3\u003e\n\u003cp\u003eThe Seurat package (version 4.5.1) was used to perform cell clustering in both HCC patients and co-culture models. The standard preprocessing workflow for single-cell sequencing data was conducted as described in previous studies. The resulting cell clustering outcomes have been presented in an earlier section of this article.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eTIMER2.0 analysis\u003c/h2\u003e\u003cp\u003eThe TIMER2.0 database enables the analysis of differential gene expression between tumor and adjacent normal tissues based on TCGA data. We utilized TIMER2.0 to examine DLGAP4 expression levels across various cancer types and to evaluate the correlation between DLGAP4 and immune cell infiltration. Statistical significance of differential expression was assessed using the Wilcoxon test along with the TIMER deconvolution method.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHE staining and Immunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eAll tissue specimens were fixed in 4% neutral buffered formalin, embedded in paraffin, and sectioned continuously at a thickness of 4 \u0026micro;m. Sections were then subjected to HE staining and EnVision two-step immunohistochemical staining. For IHC, HCC tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Endogenous peroxidase activity was blocked with 3% H₂O₂, and non-specific binding sites were blocked with 5% goat serum. The sections were incubated overnight at 4 ℃ with a rabbit anti-human DLGAP4 antibody (1:500, Bioss, China). After washing three times with phosphate-buffered saline (PBS), the sections were incubated for 30 minutes with a goat anti-rabbit IgG secondary antibody (1:500, Bioss, China). Detection was performed using 3,3\u0026prime;-diaminobenzidine (DAB), followed by counterstaining, dehydration, and mounting. DLGAP4-positive cells were observed under an inverted fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eStatistical analyses and data visualization were conducted using R (version 4.5.1) and IBM SPSS Statistics (version 25.0). Differences between two groups were assessed using an unpaired t-test, while comparisons among more than two groups were evaluated by one-way ANOVA. Correlations between variables were examined using Pearson correlation analysis. Survival analysis was performed with Kaplan-Meier curves, and the log-rank test was applied to determine the statistical significance of survival differences. A P-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDLGAP4 is highly expressed in HCC\u003c/h2\u003e\u003cp\u003eFirst, we investigated the expression of DLGAP4 across multiple cancer types using the TIMER database. Differential expression analysis revealed that DLGAP4 was significantly up-regulated in various tumor tissues compared to normal controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This up-regulation was further confirmed by volcano plot analysis, which identified DLGAP4 as one of the prominently upregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Subsequent analyses in the TCGA database, including both non-matched and matched comparisons of tumor versus normal tissues, consistently showed elevated DLGAP4 expression in cancerous tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d). Moreover, receiver operating characteristic (ROC) analysis indicated that DLGAP4 expression exhibited high specificity in distinguishing cancer from non-cancer samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).We also assessed the association between DLGAP4 expression levels and clinicopathological characteristics. In the TCGA cohort, higher DLGAP4 expression was asymmetrically distributed across several clinical parameters, including recurrence, AFP levels, tumor grade, presence of liver cirrhosis, HBV status, age, and gender (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Comparative analyses among different subgroups showed that DLGAP4 expression was significantly elevated in samples with high AFP levels (\u0026gt;\u0026thinsp;300 ng/mL; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), older age (\u0026gt;\u0026thinsp;60 years; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh), female gender (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei), and HBV-positive status (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTumor cell DLGAP4 regulates chromatin remodeling\u003c/h2\u003e\u003cp\u003eTo investigate the biological functions associated with DLGAP4, genes most correlated with DLGAP4 (|R| \u0026gt;0.5, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were identified using Pearson correlation analysis in both the TCGA and GSE14520 datasets. Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), were performed based on these gene sets.In the TCGA database, the biological processes most significantly associated with DLGAP4 were positive regulation of transcription by RNA polymerase II, regulation of DNA-templated transcription, and neuron projection development (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The primary cellular components linked to DLGAP4 were the nucleoplasm, nucleus, and cytosol (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). For molecular function, DLGAP4 was predominantly associated with protein binding and cadherin binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The most relevant signaling pathways included endocytosis, bacterial invasion of epithelial cells, and Shigellosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Similar enrichment profiles for biological processes, cellular components, molecular functions, and signaling pathways were observed in the GSE14520 dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-h).These findings suggest that DLGAP4 may play an important role in chromatin remodeling and the regulation of protein translation in hepatocellular carcinoma cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eTumor cell CD161 is positively correlated with transcription\u003c/h2\u003e\u003cp\u003eDuring cancer cell proliferation, chromatin remodeling and protein translation play critical roles. To investigate the potential influence of DLGAP4 on these processes, we assessed its association with transcriptional and translational activity. Using gene set variation analysis (GSVA) on the TCGA and GSE14520 datasets, enrichment scores for chromatin remodeling-related pathways were evaluated. Correlation analysis revealed that DLGAP4 expression was positively associated with the majority of chromatin remodeling and protein translation functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). These findings were further validated in the TCGA cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Collectively, these results suggest that DLGAP4 is involved in chromatin reorganization and the regulation of transcription and translation in HCC cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eProtein translation regulation and chromatin remodeling are positively correlated with high CD161 expression\u003c/h2\u003e\u003cp\u003eDLGAP4 has been implicated in tumor-related transcription and translation processes. To further explore this, we examined the relationship between DLGAP4 and several known protein translation genes,including EIF4G1, RPS6, KRAS, PTEN, EIF4A1, CTNNB1, TP53, and PDCD4,using data from the GSE14520 and TCGA databases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). DLGAP4 expression showed a significant positive correlation with these genes.Additionally, we assessed seven transcription-related gene clusters as markers of transcriptional activity: activated-induced cytidine deaminase (AICDA), β-tubulin (ACTB), apoptosis-inducing factor (AIF), cell division cycle protein1(CDT1), cyclin-dependent kinase 9 (CDK9), bromo-adjacent homology domain-containing protein 1 (BAHD1), and histone acetyl-lysine reader (CECR2). Correlation analyses revealed that DLGAP4 was significantly positively correlated with each of these meta-gene clusters in both the TCGA and GSE14520 datasets (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-c).These results support the hypothesis that DLGAP4 interacts with and upregulates key genes involved in tumor transcription and translation, thereby potentially promoting tumor cell growth.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eThe expression characteristics of DLGAP4 in different cell types of HCC tissues\u003c/h2\u003e\u003cp\u003eThis study analyzed hepatocellular carcinoma (HCC) patient samples and classified the cells into 25 distinct clusters based on their specific gene expression profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Among these, clusters 12 and 21 were identified as tumor cells. Notably, stem cell markers (PROM1), tumor-associated macrophage markers (APOE and SLC40A1), metabolic reprogramming markers (SQLE and ACSL4), and cell cycle regulators (CDKN3 and PRC1) all exhibited high expression levels within these clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).The enrichment of DLGAP4 in these cell clusters was further validated using a public single-cell sequencing dataset (GSE189903), in which cells from liver cancer samples were categorized into 32 clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Consistent with our initial findings, the same markers showed pronounced enrichment features in tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eThe association between DLGAP4 expression and the characteristics of immune cell infiltration\u003c/h2\u003e\u003cp\u003eFurthermore, using the TIMER2.0 database, we analyzed the correlation between DLGAP4 expression and immune cell infiltration in liver cancer. The analysis revealed positive correlations between DLGAP4 expression and several immune cell types, with correlation coefficients greater than 0.3 for CD4⁺ T cells, B cells, dendritic cells, macrophages, and neutrophils(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-f). In contrast, the correlation with CD8⁺ T cells was weak (r\u0026thinsp;=\u0026thinsp;0.086). These results suggest that DLGAP4 expression in liver cancer is significantly associated with increased infiltration of multiple immune cell types.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eDLGAP4 is an independent prognostic factor for patients with HCC\u003c/h2\u003e\u003cp\u003eTo evaluate the prognostic value of DLGAP4 in hepatocellular carcinoma (HCC), we performed Kaplan\u0026ndash;Meier survival analysis using data from the TCGA and GSE14520 databases. In the TCGA cohort, patients with high DLGAP4 expression had a significantly shorter overall survival (median survival: 697 days) compared to those with low expression (median survival: 870 days; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The prognostic significance of DLGAP4 was further confirmed in the GSE14520 dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemical detection of the expression of DLGAP4 in liver cancer tissues\u003c/h2\u003e\u003cp\u003eWe collected 40 tissue specimens from patients with hepatocellular carcinoma and performed immunohistochemical staining to evaluate the expression of DLGAP4, CD3, CD8, CD45RO, Foxp3, and Granzyme B (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The results demonstrated that DLGAP4 expression was significantly higher in tumor tissues compared to normal tissues. Furthermore, tumor tissues exhibited high expression of CD3 and Foxp3, but low expression of CD8, CD45RO, and Granzyme B (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb\u0026ndash;g). These findings suggest that DLGAP4 may suppress the immune response within the tumor microenvironment by modulating regulatory T cell activity in hepatocellular carcinoma.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe DLGAP protein family comprises five members, among which DLGAP5 has been the primary focus in cancer research. Overexpression of DLGAP5 has been shown to promote malignant progression in several cancers, including lung adenocarcinoma\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, bladder cancer\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, gallbladder cancer\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, endometrial cancer\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e,breast cancer\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, colorectal cancer\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, gastric cancer\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, and glioma\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Currently, DLGAP4 expression is primarily and closely associated with neurological and psychiatric disorders such as cerebellar movement disorders\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, congenital cerebellar ataxia\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, and schizophrenia\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.Due to high conservation in molecular structure and function across the DLGAP family, Liu et al. proposed that DLGAP4 may exhibit oncogenic roles similar to those of DLGAP5\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Their study demonstrated that DLGAP4 expression is significantly elevated in gastric cancer tissues compared to normal gastric tissues, and high DLGAP4 expression was correlated with poor prognosis in gastric cancer patients, suggesting its potential as a prognostic biomarker. However, research on DLGAP4 in hepatocellular carcinoma (HCC) remains limited.The present study revealed that DLGAP4 expression is significantly upregulated in HCC tissues compared to adjacent normal liver tissues and is closely associated with clinicopathological features of HCC patients. Furthermore, high DLGAP4 expression was identified as an independent risk factor for poor prognosis in HCC. These findings imply that DLGAP4 may play a critical role in the malignant progression of hepatocellular carcinoma.\u003c/p\u003e\u003cp\u003eChen H et al. reported that SOX11 may serve as a potential prognostic biomarker in hepatocellular carcinoma, associated with immune infiltration and ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.Analysis using the TIMER2.0 online database revealed that DLGAP4 is highly expressed in multiple cancer types, including breast cancer, cholangiocarcinoma, colorectal cancer, and hepatocellular carcinoma. In a related context, Jia Hui et al. evaluated the diagnostic and prognostic value of GPI in lung adenocarcinoma using ROC curve analysis\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Similarly, the current study demonstrated that DLGAP4 expression in tumor tissues exhibits high specificity. Wang Di et al. analyzed the clinicopathological characteristics associated with KLRB1 through heatmap visualization\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.Our results indicate that DLGAP4 expression correlates with clinical features such as AFP levels and HBV status. Furthermore, Cairong Dong et al. reported that high DLGAP4 expression promotes proliferation and metastasis in HCC by upregulating PPARβ/δ protein\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.In this study, analyses of the TCGA and GSE14520 databases indicated that DLGAP4 plays a significant role in biological functions related to chromatin remodeling and protein translation. Subsequent GSVA further confirmed that DLGAP4 is critically involved in transcriptional and translational regulation. Moreover, Pearson correlation analysis identified a positive association between DLGAP4 expression and genes regulating both protein translation and chromatin remodeling.\u003c/p\u003e\u003cp\u003eThe findings presented above underscore the significant role of DLGAP4 in chromatin remodeling and other cellular processes within hepatocellular carcinoma. In a related study, Jiajing Li et al. identified CDK1 and DLGAP5 as key regulators of tumor immune cell infiltration in HCC\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Analysis of single-cell sequencing data from our work further suggests that DLGAP4-expressing tumor cells exhibit stem-like properties, metabolic reprogramming, and pro-angiogenic activity. Moreover, DLGAP4 expression shows a correlation with both tumor-associated macrophages and cancer-associated fibroblasts, indicating its potential importance in maintaining a tumor-promoting microenvironment conducive to malignant progression.Zhou Hongrui et al. demonstrated that POC1A plays a critical role in immune cell infiltration in hepatocellular carcinoma (HCC)\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. In line with this, our analysis using the TIMER2.0 database revealed a positive correlation between DLGAP4 expression and the infiltration levels of multiple immune cells, including CD4⁺ T cells and B cells. These findings imply that DLGAP4 may facilitate the recruitment or activation of immune cells, potentially shifting the tumor microenvironment toward an immunoreactive(\u0026ldquo;hot tumor\u0026rdquo;)phenotype, which is generally associated with improved prognosis and enhanced responsiveness to immunotherapy\u003csup\u003e[33]\u003c/sup\u003e.However, Kaplan\u0026ndash;Meier survival analysis indicated that high DLGAP4 expression was correlated with poorer overall survival, identifying DLGAP4 as an independent risk factor for unfavorable prognosis in HCC patients. This apparent contradiction may reflect complex roles of DLGAP4 in both immune activation and tumor progression. Further validation using immunohistochemical staining of clinical tissue specimens confirmed a positive correlation between DLGAP4 expression and immune markers, supporting the notion that DLGAP4 contributes critically to the immune microenvironment in HCC.Collectively, these results underscore the potential of DLGAP4 as a predictive biomarker and a future target for immunotherapy in hepatocellular carcinoma.Although we performed a systematic analysis of DLGAP4 and validated our findings across multiple independent databases, this study has several limitations. First, the publicly available data may be subject to inherent biases or incompleteness due to the retrospective nature of the datasets. Second, the precise molecular mechanisms through which DLGAP4 influences chromatin remodeling and protein translation in hepatocellular carcinoma remain unclear. Further experimental studies are needed to elucidate these mechanisms and confirm our bioinformatic observations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study establishes DLGAP4 as a potential prognostic biomarker and therapeutic target in hepatocellular carcinoma (HCC), showing significant upregulation correlated with poor survival. DLGAP4 is involved in chromatin remodeling and protein translation, and is associated with an immunosuppressive tumor microenvironment characterized by infiltration of tumor-associated macrophages and cancer-associated fibroblasts, along with stemness, angiogenesis, and metabolic reprogramming. Despite validation across databases, the mechanistic role of DLGAP4 requires further experimental investigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eThe collection of samples and clinical data in this study was approved by the Ethics Committee of Gansu Provincial People\u0026apos;s Hospital.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eAll authors agree to publication.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Department of Gansu Province, Projects Unit (Grant No: 22JR5RA671).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eFeng Yang designed research; Shoufeng Chang and Ruxia Li wrote the manuscript with contributions from all authors. All authors read and approved the initial manuscript.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHwang SY, Danpanichkul P, Agopian V, Mehta N, et al. Hepatocellular carcinoma: updates on epidemiology, surveillance, diagnosis and treatment. Clin Mol Hepatol. 2025;31(Suppl):S228\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3350/cmh.2024.0824\u003c/span\u003e\u003cspan address=\"10.3350/cmh.2024.0824\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYeo YH, Abdelmalek M, Khan S, Moylan CA, et al. Current and emerging strategies for the prevention of hepatocellular carcinoma. 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Signal Transduct Target Ther. 2024;9(1):274. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41392-024-01979-x\u003c/span\u003e\u003cspan address=\"10.1038/s41392-024-01979-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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":"journal-of-gastrointestinal-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijgc","sideBox":"Learn more about [Journal of Gastrointestinal Cancer](https://www.springer.com/journal/12029)","snPcode":"12029","submissionUrl":"https://submission.nature.com/new-submission/12029/3","title":"Journal of Gastrointestinal Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Hepatocellular carcinoma, DLGAP4, Chromatin remodeling, Protein translation","lastPublishedDoi":"10.21203/rs.3.rs-7613609/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7613609/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eAlthough the DLGAP4 gene is well-established in neurological disorders, its function in hepatocellular carcinoma (HCC) remains unclear. This study aims to characterize DLGAP4 expression patterns, prognostic significance, and association with immune infiltration in the HCC tumor microenvironment, to assess its potential as a biomarker or therapeutic target.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe analyzed DLGAP4 expression in HCC and its prognostic significance using the TCGA database, performing survival analysis with the Kaplan-Meier method. Functional pathways linked to DLGAP4 were identified via GO and KEGG enrichment analyses, and further explored by gene set variation analysis (GSVA). Immune cell infiltration correlations were assessed using TIMER2.0. Clinical samples were immunohistochemically stained to validate bioinformatic results.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eDLGAP4 expression was significantly upregulated in HCC tissues and associated with poor patient prognosis. Enrichment analysis implicated DLGAP4 in biological processes including chromatin modification and protein translation. Single-cell data analysis revealed that high DLGAP4 expression correlated with features of the tumor microenvironment, such as tumor-associated macrophages, cancer-associated fibroblasts, stemness, angiogenesis, and metabolic reprogramming. Immunohistochemical results further confirmed a significant correlation between DLGAP4 expression and immune cell infiltration.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eDLGAP4 is upregulated in HCC and associated with poor prognosis and immune infiltration in the tumor microenvironment, suggesting its potential as a prognostic biomarker and therapeutic target for HCC.\u003c/p\u003e","manuscriptTitle":"Bioinformatics Analysis Revealsthe role of DLGAP4 in the development and progression of hepatocellular carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 06:48:25","doi":"10.21203/rs.3.rs-7613609/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-15T01:45:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-13T08:32:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106957201624932275517767074334209414446","date":"2025-10-12T07:03:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-23T12:13:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-23T10:03:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-15T07:03:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Gastrointestinal Cancer","date":"2025-09-14T15:31:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-gastrointestinal-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijgc","sideBox":"Learn more about [Journal of Gastrointestinal Cancer](https://www.springer.com/journal/12029)","snPcode":"12029","submissionUrl":"https://submission.nature.com/new-submission/12029/3","title":"Journal of Gastrointestinal Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"00f900b5-1491-4f44-ba92-6a0a088a4c6b","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:05:15+00:00","versionOfRecord":{"articleIdentity":"rs-7613609","link":"https://doi.org/10.1007/s12029-025-01359-w","journal":{"identity":"journal-of-gastrointestinal-cancer","isVorOnly":false,"title":"Journal of Gastrointestinal Cancer"},"publishedOn":"2025-12-03 15:57:40","publishedOnDateReadable":"December 3rd, 2025"},"versionCreatedAt":"2025-10-08 06:48:25","video":"","vorDoi":"10.1007/s12029-025-01359-w","vorDoiUrl":"https://doi.org/10.1007/s12029-025-01359-w","workflowStages":[]},"version":"v1","identity":"rs-7613609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7613609","identity":"rs-7613609","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}