The mRNA-miRNA-lncRNA Regulatory Network and Factors Associated with Prognosis Prediction of Hepatocellular Carcinoma

Abstract

The aim of this study was to identify novel prognostic mRNA and microRNA (miRNA) biomarkers for hepatocellular carcinoma (HCC) using methods in systems biology. Differentially expressed mRNAs, miRNAs, and long non-coding RNAs (lncRNAs) were compared between HCC tumor tissues and normal liver tissues in The Cancer Genome Atlas (TCGA) database. Subsequently, a prognosis-associated mRNA co-expression network, an mRNA-miRNA regulatory network, and an mRNA-miRNA-lncRNA regulatory network were constructed to identify prognostic biomarkers for HCC through Cox survival analysis. Seven prognosis-associated mRNA co-expression modules were obtained by analyzing these differentially expressed mRNAs. An expression module including 120 mRNAs was significantly correlated with HCC patient survival. Combined with patient survival data, several mRNAs and miRNAs, including CHST4, SLC22A8, STC2, hsa-miR-326, and hsa-miR-21 were identified from the network to predict HCC patient prognosis. Clinical significance was investigated using tissue microarray analysis of samples from 258 patients with HCC. Functional annotation of hsa-miR-326 and hsa-miR-21-5p indicated specific associations with several cancer-related pathways. The present study provides a bioinformatics method for biomarker screening, leading to the identification of an integrated mRNA-miRNA-lncRNA regulatory network and their co-expression patterns in relation to predicting HCC patient survival.

Keywords: Hepatocellular carcinoma; Prognostic factor; Systems biology; TCGA database; mRNA–miRNA–lncRNA regulatory network.

Figure 1

KEGG pathway and GO biological process enrichment analyses of DEmRNAs in HCC tumors A. KEGG pathways enriched by DEmRNAs in HCC tumors. B. Top 30 GO biological processes enriched by DEmRNAs in HCC tumors. KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; DEmRNA, differentially expressed mRNA; HCC, hepatocellular carcinoma.

Figure 2

WGCNA of mRNA co-expression in HCC A. The soft threshold for the construction of the mRNA co-expression network. B. Enrichment of survival genes in different modules. Modules 1–7 indicate different co-expressed mRNA patterns identified by WGCNA. Module 0 labeled in gray corresponds to the set of mRNAs which have not been clustered in any module. C. mRNA relationships in modules. WGCNA, weighted correlation network analysis; PCC, Pearson correlation coefficient.

Figure 3

The mRNA–miRNA–lncRNA regulatory network and survival-associated subnetworks A. The constructed DEmiRNA–mRNA network. B. Functional enrichment analysis of DEmiRNAs. C. The constructed mRNA–miRNA–lncRNA complex regulatory network. D. mRNA–miRNA–lncRNA subnetworks associated with survival. DEmiRNA, differentially expressed miRNA; DElncRNA, differentially expressed lncRNA.

Figure 4

The potential biomarkers for the prognosis of HCC A. Kaplan-Meier survival curves for HCC patients with different expression levels of CHST4, SLC22A8, and STC2. B. ROC curves of CHST4, SLC22A8, and STC2 expression for predicting overall survival of HCC patients. C. Kaplan-Meier survival curves for HCC patients with different expression levels of hsa-miR-326 and hsa-miR-21. D. ROC curves of hsa-miR-326 and hsa-miR-21 expression for predicting overall survival of HCC patients. ROC, receiver operating characteristic; AUROC, area under the ROC curve.

Figure 5

Target mRNAs and enriched pathways of hsa-miR-326 and hsa-miR-21 miRNA and miRNA target are represented with square and diamond, respectively. Green diamond represents the target enriched in the pathway, and red diamond represents the target that is not enriched in the pathway; circle represents a significantly enriched pathway (the larger the node, the more significant the pathway; the darker the color, the greater the proportion of genes enriched in this pathway).

Figure 6

Clinical significance of CHST4, SLC22A8, STC2, hsa-miR-326, and hsa-miR-21 A. Typical images of IHC staining for SLC22A8, CHST4, and STC2 in HCC patients with distinct prognosis. Scale bar, 50 μm. B. Clinical validation of the prognostic significance of SLC22A8, CHST4, and STC2. Upper: the significance for predicting recurrence; lower: the significance for predicting overall survival. C. Clinical validation of the prognostic significance of has-miR-326 and has-miR-21. Upper: the significance for predicting recurrence; lower: the significance for predicting overall survival. D. Expression status of selected mRNAs/miRNAs between HCC and paired adjacent normal liver tissues. The average expression levels of indicated mRNAs/miRNAs in normal tissues were set as 1.0, and the expression levels of mRNAs/miRNAs in HCC tissues were calculated as HCC/normal to determine the FC in expression. IHC, immunohistochemistry; FC, fold change.