Comprehensive analysis of potential prognostic genes for the construction of a competing endogenous RNA regulatory network in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is an extremely common malignant tumor with worldwide prevalence. The aim of this study was to identify potential prognostic genes and construct a competing endogenous RNA (ceRNA) regulatory network to explore the mechanisms underlying the development of HCC.

Methods: Integrated analysis was used to identify potential prognostic genes in HCC with R software based on the GSE14520, GSE17548, GSE19665, GSE29721, GSE60502, and the Cancer Genome Atlas databases. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway-enrichment analyses were performed to explore the molecular mechanisms of potential prognostic genes. Differentially expressed miRNAs (DEMs) and lncRNAs (DELs) were screened based on the Cancer Genome Atlas database. An lncRNA-miRNA-mRNA ceRNA regulatory network was constructed based on information about interactions derived from the miRcode, TargetScan, miRTarBase, and miRDB databases.

Results: A total of 152 potential prognostic genes were screened that were differentially expressed in HCC tissue and significantly associated with overall survival of HCC patients. There were 13 key potential prognostic genes in the ceRNA regulatory network: eleven upregulated genes (CCNB1, CEP55, CHEK1, EZH2, KPNA2, LRRC1, PBK, RRM2, SLC7A11, SUCO, and ZWINT) and two downregulated genes (ACSL1 and CDC37L1) whose expression might be regulated by eight DEMs and 61 DELs. Kaplan-Meier curve analysis showed that nine DELs (AL163952.1, AL359878.1, AP002478.1, C2orf48, C10orf91, CLLU1, CLRN1-AS1, ERVMER61-1, and WARS2-IT1) in the ceRNA regulatory network were significantly associated with HCC-patient prognoses.

Conclusion: This study identified potential prognostic genes and constructed an lncRNA- miRNA-mRNA ceRNA regulatory network of HCC, which not only has important clinical significance for early diagnoses but also provides effective targets for HCC treatments and could provide new insights for HCC-interventional strategies.

Keywords: ceRNA; hepatocellular carcinoma; prognostic gene.

Figure 1

Log₂FC heat map of the top 20 upregulated and downregulated genes of the five expression-microarray groups downloaded from the GEO data set. Notes: The differentially expressed gene (DEG) list of each expression microarray from the GEO data was integrated using R software with the RRA package. The abscissa is the GEO ID, and the ordinate is the gene name. Red represents upregulated gene expression, and green represents downregulated gene expression in hepatocellular carcinoma tissues compared with adjacent normal liver tissues. Numbers in the box represent log₂FC values that resulted from the integrated analysis. Abbreviations: FC, fold change; GEO, Gene Expression Omnibus; RRA, robust rank aggregation.

Figure 2

Hierarchical clustering heat map (A) and volcano plot (B) of differentially expressed genes screened based on TCGA HCC WTS data. Notes: Protein-encoding mRNA-expression data were screened and analyzed using R software and the Limma package. Log₂FC >1 and P<0.05 were used as cutoff criteria. Abbreviations: FC, fold change; TCGA, the Cancer Genome Atlas; HCC, hepatocellular carcinoma; WTS, whole-transcriptome sequencing; FDR, false discovery rate.

Figure 3

Hierarchical clustering heat map and volcano plot of screened DEMs (A, B) and DELs (C, D) based on TCGA HCC WTS data. Notes: miRNA- and lncRNA-expression data were downloaded and analyzed using R software and the Limma package. Log₂FC >2 and P<0.01 were used as cutoff criteria. Abbreviations: DEMs, differentially expressed miRNAs; DELs, differentially expressed lncRNAs; FC, fold change; TCGA, the Cancer Genome Atlas; HCC, hepatocellular carcinoma; WTS, whole-transcriptome sequencing.

Figure 4

The ceRNA regulatory network in HCC. Notes: The oval represents mRNA, the diamond represents lncRNA, and the square represents miRNA. Red indicates the gene was upregulated and green indicates the gene downregulated in HCC tissues compared with adjacent normal liver tissues. The line between the genes indicates that there could be regulatory relationships between the two genes. Abbreviations: ceRNA, the competing endogenous RNA; HCC, hepatocellular carcinoma.

Figure 5

Kaplan–Meier curve analysis was performed on the DELs in the ceRNA regulatory network. P<0.05 was used as the cutoff criterion. Abbreviations: DELs, differentially expressed lncRNAs; ceRNA, competing endogenous RNA.

Figure 6

mRNA-expression levels in the ceRNA regulatory network of the 20 pairs of HCC tissue and adjacent normal liver tissue. Notes: Experiments were repeated three times. *P<0.05. Abbreviations: ceRNA, competing endogenous RNA; HCC, hepatocellular carcinoma.