1. Identification of key genes in Hepatocellular carcinoma
To more comprehensively determine the gene expression profile and identify key genes associated with the occurrence and development of Hepatocellular Carcinoma, we performed a comprehensive analysis of the publicly available genomic data generated from liver tissue from human with primary HCC, NASH or HBV driven HCC in the GEO database (Supporting Table S1). Both upregulated and downregulated genes were identified by differentially expressed gene (DEG) analysis in each comparative group (Figure 1 A-E). Total 100 genes with consistent pattern changes across five databases were discovered by intersection analysis (Figure 1F), detail information in Supporting data.
The underlying mechanisms of action of these common crossover genes GO enrichment analysis revealed that the main biological processes (BP) mainly include cell division, cell cycle, negative regulation of growth DNA repair signaling pathway, etc. Cellular components (CC) mainly involve nucleus, cytoplasm, cytosol, nucleoplasm, and centrosome. molecular functions (MF) involved in protein binding, DNA binding (Figure. 1G); KEGG pathway analysis showed that the cross-targets mainly involved mineral absorption, cell cycle, oocyte meiosis and p53 signaling pathway (Figure. 1H). Subsequently, we queried and screened the interaction relationships between indicated target proteins by the STRING online database to obtain a protein interaction network (Figure. 1). Besides, we visualized PPI analysis of the pivotal genes by the Cytoscape (Figure. 1J) and obtained a core target interaction network consisting of 12 core genes (Supporting Table S2): Aspm, Ccnb2, Cdk1, Cdkn3, Dlgap5, Dtl, Hmmr, Nusap1, Pbk, Racgap1, Top2a and Zwint. Taken together, our joint analysis reveals key genomic alterations in HCC.
2. Expression of key genes in HCC from TCGA
Through the analysis of the GSE dataset, we identified a set of DEGs that exhibited significant differential expression in HCC. To further confirm the importance of these indicated genes, total of 12 DEGs, were screened according to stromal scores in HCC from TCGA dataset, which show the same expression patterns as in the GSE database (Figure. 2). To substantiate the significance of the identified genes, we investigated their expression in Cholangiocarcinoma (CHOL) from TCGA. The study revealed a high expression level of these genes in CHOL (Supporting Figure. S1), providing additional evidence for the accuracy of our screening results and suggesting their crucial roles in liver tumorigenesis, which strengthen the reliability of GEO data.
3. CDK1and DLGAP5 is highly expressed and associated with poor prognosis in HCC
To further investigate the significance and identify the most crucial genes in HCC, we utilized RNA-Seq data from the TCGA database to analyze these DEGs expression levels in HCC samples along with survival analysis. Kaplan-Meier survival analysis demonstrated that high expression levels of Aspm, Ccnb2, Cdk1, Cdkn3, Dlgap5, Dtl, Hmmr, Nusap1, Pbk, Racgap1, Top2a and Zwint were significantly associated with poor overall survival in HCC patients, among them, CDK1 and DLGAP5 are the most significant (Figure. 3A). Therefore, we focused on CDK1 and DLGAP5 to reveal its important role in HCC.
Next, we validated the protein expression levels of CDK1 and DLGAP5 in HCC tissues using protein expression databases Human Protein Atlas. Our analysis revealed a significant upregulation of CDK1 and DLGAP5 expression in HCC tissues compared to normal liver tissues (Figure. 3B). Additionally, CDK1 and DLGAP5 expression were also significantly overexpressed in HCC mice model (Figure. 3C). More importantly, these two genes were also significantly changed under the treatment of sorafenib, suggesting that they also play an important role in patients with HCC and sorafenib tolerance (Figure. 3D). These findings suggested that CDK1 and DLGAP5 may play crucial roles in HCC progression and serve as potential prognostic indicators.
4. CDK1 and DLGAP5 are influenced by individual cancer types, patient characteristics, and TP53 mutation status
Subsequently, we conducted a correlation and expression patterns in different tumors analysis of these two genes, and the results suggest CDK1/DLGAP5 can be used as dual targets for treatment (Supporting Figure. S2A-B). The above results indicated the expression of CDK1 and DLGAP5 may play a crucial role in the development and progression of HCC. Understanding the factors that influence their expression, such as individual cancer type, patient characteristics (age, weight, gender, race), and TP53 mutation status, can provide valuable insights into disease mechanisms and potential therapeutic targets. Expression of CDK1 and DLGAP5 in LICH based on individual cancer, patient's age, patient's weight, patient's gender, patient's race, and TP53 mutation status were conducted. The expression patterns of CDK1 and DLGAP5 in LICH are influenced by individual cancer types, patient characteristics, and TP53 mutation status (Figure. 4A-B).
Additionally, investigating the RNA expression of CDK1 and DLGAP5 at different periods and times during the cell cycle can shed light on their regulatory roles in cell division and proliferation. RNA expression of CDK1 and DLGAP5 in different periods of the cell cycle (G1, S, G2/M). Furthermore, their expression levels vary across different periods of the cell cycle (Supporting Figure. S2C). These findings contribute to our understanding of HCC biology and may aid in the development of personalized treatment strategies targeting CDK1 and DLGAP5.
5. CDK1 and DLGAP5 is associated with tumor immune cell infiltration
Next, we aim to find out the mechanisms by which CDK1 and DLGAP5 potentially regulate the occurrence of HCC. Understanding the interaction between tumor immune cells and key genes involved in cancer progression is crucial for improving cancer treatment strategies. To explore whether these two genes are related to tumor immunity in the development of HCC. In this study, we conducted a correlation analysis to explore the relationship between CDK1 and DLGAP5 in tumor immune cells.
We utilized the GSE166635 dataset, which provides single-cell transcriptomic data, our analysis revealed the distribution of CDK1 and DLGAP5 in different TME cell types at single-cell resolution. The cells with significant expression of the prognostic genes, indicated by the red box, suggest the presence of a potential correlation between CDK1 and DLGAP5 in specific tumor immune cell populations (Figure. 5A). Conversely, the cells with trace expression of the prognostic genes, indicated by the green box, might represent cells where CDK1 and DLGAP5 are not significantly expressed. Specifically, we investigated the distribution of these prognostic genes across different types of tumor microenvironment (TME) cells, and found that CDK1 and DLGAP5 is associated with tumor immune cell infiltration (Figure. 5B). Taken together, these data indicated that CDK1 and DLGAP5 may promote HCC through tumor immune cell infiltration.