Bioinformatics analysis to reveal key biomarkers for early and late hepatocellular carcinoma

Abstract

Background: The aim of this study was to find the long non-coding RNAs (lncRNAs) and mRNAs acting as biomarker of early and late hepatocellular carcinoma (HCC).

Methods: The Cancer Genome Atlas (TCGA) dataset was used to identify shared differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) between early and late HCC and normal tissue. Functional annotation and protein-protein interaction network of shared DEmRNAs were performed. Furthermore, DElncRNAs-DEmRNAs co-expression network of early and late HCC were also performed. The expression of selected candidate genes were validated by the quantitative real time polymerase chain reaction (qRT-PCR).

Results: A total of 1,201 shared DEmRNAs and 162 shared DElncRNAs were identified in both early and late HCC compared with normal controls. Cell cycle, p53 signaling pathway, retinol metabolism and metabolism of xenobiotics by cytochrome P450 were four significantly enriched pathways. Base on the protein-protein interaction network, CDK1, AURKA, CDC20, PLK1, AURKB, HIST1H2BG, BUB1B, CCNA2, CCNB1 and CDT1 were key protein. CTD-2510F5.4 and HAND2-AS1 were hub lncRNAs in both early and late HCC. Overall, the confirmation results of qRT-PCR were generally consistent with our integrated analysis.

Conclusions: A total of 4 DEmRNAs (CDK1, KIFC1, CENPF and ECM1) and 2 DElncRNAs (CTD-2510F5.4 and HAND2-AS1) were identified as key biomarkers for HCC. This study may contribute to reveal the pathogenesis of early and late HCC and provide new and accurate therapeutic targets for HCC.

Keywords: DElncRNA-DEmRNA co-expression; Hepatocellular carcinoma (HCC); differentially expressed lncRNAs (DElncRNAs); differentially expressed mRNAs (DEmRNAs).

Figure 1

Hierarchical clustering analysis of top 100 differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) between hepatocellular carcinoma (HCC) and normal tissues. (A) DEmRNAs in early HCC. (B) DElncRNAs in early HCC. (C) DEmRNAs in late HCC. (D) DElncRNAs in late HCC. Row and column represented DElncRNAs/DEmRNAs and tissue samples, respectively. Orange and light blue color mean the (early and late) HCC and normal control, respectively. The color scale represented the expression levels. Red color represents the relative expression level of genes was higher than mean, and green color represents the relative expression of genes was lower than mean.

Figure 2

Venny analysis of DEmRNAs and DEmRNAs in early HCC vs. normal control and late HCC vs. normal control. (A) DEmRNAs; (B) DElncRNAs. DEmRNAs, differentially expressed mRNAs; DElncRNAs, differentially expressed long non-coding RNAs; HCC, hepatocellular carcinoma.

Figure 3

Top 15 significantly enriched Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for shared differentially expressed mRNAs (DEmRNAs) in early and late hepatocellular carcinoma (HCC). The x-axis shows −log P value, and the y-axis shows GO terms or KEGG pathways. (A) Biological process; (B) cellular component; (C) molecular function; (D) KEGG pathways. The change color from red to green indicates that the significance is high to low. The larger the dot size, the greater the proportion of this enrichment item.

Figure 4

Protein-protein interaction network. Ellipses are used to represent nodes, and lines are used to represent edges. Red and green represent up- and down-ward adjustments, respectively. The blue border indicates the top 20 up- and down-regulated proteins.

Figure 5

DElncRNAs-DEmRNAs co-expression network of early HCC. Ellipses and rhombus represent DEmRNAs and DElncRNAs, respectively. Red and green colors represent up- and down-regulation, respectively. The blue border indicates the top 20 up- and down-regulated DElncRNAs and DEmRNAs. DEmRNAs, differentially expressed mRNAs; DElncRNAs, differentially expressed long non-coding RNAs; HCC, hepatocellular carcinoma.

Figure 6

DElncRNAs-DEmRNAs co-expression network of late HCC. Ellipses and rhombus represent DEmRNAs and DElncRNAs, respectively. Red and green colors represent up- and down-regulation, respectively. The blue border indicates the top 20 up- and down-regulated DElncRNAs and DEmRNAs. DEmRNAs, differentially expressed mRNAs; DElncRNAs, differentially expressed long non-coding RNAs; HCC, hepatocellular carcinoma.

Figure 7

Validation optimal DEmRNAs and DElncRNAs in HCC tissue by qRT-PCR. All of the assays were performed three times independently at least. The x-axis shows DEmRNAs or DElncRNAs and y-axis shows log2(Foldchange). The log2(Foldchange) >0 and log2(Foldchange) <0 indicates up- and down-regulation, respectively. *P<0.05, **P<0.01, ***P<0.001. DEmRNAs, differentially expressed mRNAs; DElncRNAs, differentially expressed long non-coding RNAs; HCC, hepatocellular carcinoma.