CDT1 Is a Novel Prognostic and Predictive Biomarkers for Hepatocellular Carcinoma

Abstract

Objective: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors endangering human health and life in the 21st century. Chromatin licensing and DNA replication factor 1 (CDT1) is an important regulator of DNA replication licensing, which is essential for initiation of DNA replication. CDT1 overexpression in several human cancers reportedly leads to abnormal cell replication, activates DNA damage checkpoints, and predisposes malignant transformation. However, the abnormal expression of CDT1 in HCC and its diagnostic and prognostic value remains to be elucidated.

Methods: TCGA, ONCOMINE, UALCAN, HCCDB, HPA, Kaplan-Meier plotter, STRING, GEPIA, GeneMANIA, and TIMER were conducted for bioinformatics analysis. CDT1 protein expression was evaluated by immunohistochemistry in HCC tissues through a tissue microarray. qRT-PCR, western blot and a cohort of functional experiments were performed for in vitro validation.

Results: In this study, we discovered remarkably upregulated transcription of CDT1 in HCC samples relative to normal liver samples through bioinformatic analysis, which was further verified in clinical tissue microarray samples and in vitro experiments. Moreover, the transcriptional level of CDT1 in HCC samples was positively associated with clinical parameters such as clinical tumor stage. Survival, logistic regression, and Cox regression analyses revealed the significant clinical prognostic value of CDT1 expression in HCC. The receiver operating characteristic curve and nomogram analysis results demonstrated the strong predictive ability of CDT1 in HCC. Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analyses indicated that CDT1 was mainly associated with the cell cycle, DNA repair, and DNA replication. We further demonstrated the significant correlation between CDT1 and minichromosome maintenance (MCM) family genes, revealing abnormal expression and prognostic significance of MCMs in HCC. Immune infiltration analysis indicated that CDT1 was significantly associated with immune cell subsets and affected the survival of HCC patients. Finally, knockdown of CDT1 decreased, whereas overexpression of CDT1 promoted the proliferation, migration, invasion of HCC cells in vitro.

Conclusions: Our study findings demonstrate the potential diagnostic and prognostic significance of CDT1 expression in HCC, and elucidate the potential molecular mechanism underlying its role in promoting the occurrence and development of liver cancer. These results may provide new opportunities and research paths for targeted therapies in HCC.

Keywords: CDT1; bioinformatics analysis; immune infiltration; liver hepatocellular carcinoma; prognostic value.

Figure 1

The workflow of the study.

Figure 2

CDT1 expression levels in different types of human cancers. (A) Transcription expression of CDT1 in 33 distinct cancer types (TCGA and GTEx). (B) CDT1 expression in tumor and paired tissues (TCGA and GTEx). (C) Transcription expression of CDT1 in 20 distinct cancer types (ONCOMINE). (D) Expression of CDT1 in various tumor cell lines (CCLE). *p < 0.05, **p < 0.01, ***p < 0.001. The “ns” stands for not significant.

Figure 3

The relative expression of CDT1 in HCC at the cell and tissue levels. (A) CDT1 expression in normal and tumor tissues (TCGA and GTEx). (B) CDT1 expression in paired tissues (TCGA and GTEx). (C) The mRNA level of CDT1 in different HCC datasets (HCCDB). (D, E) CDT1 mRNA expression in normal and tumor tissues (ONCOMINE). (F) The protein expression level of CDT1 in HCC and normal liver tissues (HPA). (G) The IHC staining results of CDT1 level in HCC and adjacent non-tumor tissue (clinical tissue microarray of HCC) and the corresponding scatter diagram. Statistical significance was determined by paired t-test. (H, I) Western blot and qRT-PCR results of CDT1 expression in normal human liver cell line and HCC cell lines. Data are expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Correlation between CDT1 expression and the clinical parameters of HCC patients and its prognostic significance. (A–D) Relationship of CDT1 mRNA levels with individual cancer stages, tumor grade, age, and TP53 mutation status of HCC patients. (E–H) Relationship of CDT1 expression with OS, PFS, RFS, and DSS in TCGA database. (I, J) Relationship of CDT1 expression with OS of HCC patients in GEO and ICGC databases. (K) Forest plot showing the impact of CDT1 on OS at different TNM stages and ages. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Diagnostic value of CDT1 mRNA level in HCC. (A) ROC curve for CDT1 in HCC and normal liver tissue. (B–H) Subgroup analysis for stage I/II, stage III/IV, T1/T2, T3/T4, N0, M0, and G1/G2. (I) Nomogram predicting the probability of patients with 1-, 3- and 5-year overall survival.

Figure 6

Volcano plots and heatmap plots of DEGs between the expression of CDT1^high and CDT1^low in HCC samples. (A) The volcano plot described 3755 DEGs (|log2fold change| > 1 and adjusted p-value < 0.05). (B) The histogram showing the number of up-regulated or down-regulated genes. (C, D) The heatmaps depicted the expression of 15 significant upregulated and downregulated genes in HCC samples with CDT1^high and CDT1^low expression. *p < 0.05, ***p < 0.001.

Figure 7

Results of enrichment analysis. (A, B) GO and KEGG enrichment results of CDT1-related DEGs (15 most significant upregulated and 15 most significant downregulated genes in HCC samples with CDT1^high and CDT1^low expression). (C–H) Gene set enrichment plots of (C) DNA replication, (D) cell cycle checkpoints, (E) mitotic prometaphase, (F) integrated cancer pathway, (G) DNA repair, and (H) cellular senescence with high CDT1 expression from GSEA.

Figure 8

(A, B) Interaction network of CDT1 and their most similar genes on GeneMANIA (A) and STRING (B) datasets. (C) Heatmap of the expression of MCM family genes in HCC samples with CDT1^high and CDT1^low expression. (D–G) Scatter plot results using Spearman correlation analysis between MCMs and CDT1 at the transcriptional level. ***p < 0.001.

Figure 9

(A) mRNA expression levels of all MCM family genes in HCC tissues and normal tissues. (B) The expression of MCM family members in paired HCC tissues. (C–J) Survival analysis of MCMs in HCC. ***p < 0.001.

Figure 10

The CDT1 expression and immune cell infiltration using the TIMER database. (A–F) Different proportions of immune cell subtypes in HCC samples in CDT1^high and CDT1^low groups. (G) The correlation between CDT1 expression and 24 immune infiltrating cells. ***p < 0.001.

Figure 11

Silencing of CDT1 inhibited the proliferation, migration and invasion of HCC cells. (A) Western blot detection of CDT1 expression after knockdown of CDT1 in liver cancer cells. (B) The effect of CDT1 knockdown on cell viability at 24, 48, 72 and 96h after seeding in plates was measured by CCK-8 assay. (C–E) Transwell analysis and wound healing assay reflected the migration ability of LM3 and Hep3B cell lines. (F) Images of the colony formation assay after knockdown of CDT1 in HCC cells (G, H) Relative quantification of the colony areas is shown. Scale bars in (F) equal 5mm. Data are expressed as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 12

Overexpression of CDT1 promoted the proliferation, migration and invasion of HCC cells. (A) Western blotting detection of CDT1 expression after overexpression of CDT1 in the LM3 and Hep3B cell lines. (B) The effect of CDT1 overexpression on cell viability at 24, 48, 72 and 96h after seeding in plates was measured by CCK-8 assay. (C, D) Transwell analysis and wound healing assay reflected the migration ability of LM3 and Hep3B cell lines. (E) Images of the colony formation assay after overexpression of CDT1 in HCC cells. Scale bars in (E) equal 5mm. Data are expressed as means ± SD of three independent experiments.**p < 0.01.