Identification of potential pseudogenes for predicting the prognosis of hepatocellular carcinoma

Abstract

Purpose: Hepatocellular carcinoma (HCC) remains a highly deadly malignant tumor with high recurrence and metastasis rates. Cancer stem cells (CSCs) are involved in tumor metastasis, recurrence, and resistance to drugs, which have attracted widespread attention in recent years. Research has shown that pseudogenes may regulate stemness to promote the progression of HCC, but its specific mechanisms and impact on prognosis remain unclear.

Methods: In this study, clinical prognosis information of HCC was first downloaded from The Cancer Genome Atlas (TCGA) database. Then we calculated the mRNA expression-based stemness index (mRNAsi) of HCC. We also screened the differentially expressed pseudogene (DEPs) and conducted univariate Cox regression analysis to investigate their effect on the prognosis of HCC. Further, genomic mutation frequency analysis and weighted gene co-expression network analysis (WGCNA) were performed to compare the role of pseudogenes and stemness in promoting the progression of HCC. Finally, we conducted the correlation analysis to examine the potential mechanism of pseudogenes regulating stemness to promote the progression of HCC and detected the possible pathways through the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.

Results: Herein, we revealed that the high stemness of HCC correlated with an unfavorable prognosis. We obtained 31 up-regulated and 8 down-regulated DEPs in HCC and screened CTB-63M22.1, a poor prognostic indicator of HCC. In addition, CTB-63M22.1 had a mutation frequency similar to mRNAsi and acted in a module similar to that of mRNAsi on HCC. We then screened two RNA-binding proteins (RBPs) LIN28B and NOP56 with the highest correlation with stemness. We also discovered that they were primarily enriched in the biological process as examples of mitotic nuclear division and cell cycle.

Conclusions: Collectively, these results revealed that pseudogenes CTB-63M22.1 may regulate cancer stemness by regulating RBPs, suggesting that CTB-63M22.1 may serve as an innovative therapeutic target and a reliable prognostic marker for HCC.

Keywords: CTB-63M22.1; Differential pseudogenes; Hepatocellular carcinoma (HCC); Prognosis; Stemness; TCGA.

Fig. 1

Flowchart of our research

Fig. 2

The stemness of HCC and its clinical relevance. A Stemness index of HCC in normal and tumor groups, different stages (B), and different grades (C). Kaplan–Meier survival curves for D OS, E DSS, F DFI, and G PFI analyses in the high- and low-stemness index groups

Fig. 3

Heatmap of the DEPs in HCC

Fig. 4

Univariate Cox regression analysis of the 16 DEPs. The forest plots for univariate Cox regression analysis of OS (A), DSS (B), DFI (C), and PFI (D) in HCC. E Venn diagram of DEPs selected from the above Univariate Cox analysis results for PFI, DFI, DSS, and OS in HCC, respectively (P < 0.05 as a filter)

Fig. 5

Waterfall plots of mutation frequency for CTB-63M22.1 and mRNAsi. Waterfall plots reflecting the frequency of gene mutations in HCC for A tumor group, B high-mRNAsi group, C low-mRNAsi group, D high-CTB-63M22.1 group, and E low-CTB-63M22.1 group

Fig. 6

WGCNA analysis for identification of co-expression modules of mRNAsi and CTB-63M22.1. A Cluster dendrogram of the co-expression network modules based on topological overlap. B Heatmap depicting the correlation between module eigengenes and mRNAsi and CTB-63M22.1. C Co-expression network of the black module genes. The enrichment analysis of BP (D), CC (E), and MF (F) by GO analysis in the black module is displayed in a bar chart

Fig. 7

The potential mechanism of CTB-63M22.1 promoting HCC progression through stemness. A Scatter plot of the correlation between mRNAsi and RBPs of CTB-63M22.1. B The regulatory networks of the RBPs (LIN28B and NOP56) corresponding to the DEGs between the high-/low-mRNAsi groups. C–E The enrichment analysis of BP (C), CC (D), and MF (E) by GO analysis of genes co-regulated by RBPs of CTB-63M22.1 and the stemness. For each GO term in the outer circle, the logFC was assigned to each gene and shown as a scatter plot, the green dots indicating downregulated genes and the red dots indicating upregulated genes. F KEGG pathway enrichment analysis of genes co-regulated by RBPs of CTB-63M22.1 and the stemness. For each KEGG term in the outer circle, the logFC was assigned to each gene and shown as a scatter plot, the green dots indicating downregulated genes and the red dots indicating upregulated genes