Transcriptomic Analysis of mRNA-lncRNA-miRNA Interactions in Hepatocellular Carcinoma

Abstract

Fully elucidating the molecular mechanisms of non-coding RNAs (ncRNAs), including micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs), underlying hepatocarcinogenesis is challenging. We characterized the expression profiles of ncRNAs and constructed a regulatory mRNA-lncRNA-miRNA (MLMI) network based on transcriptome sequencing (RNA-seq) of hepatocellular carcinoma (HCC, n = 9) patients. Of the identified miRNAs (n = 203) and lncRNAs (n = 1,090), we found 16 significantly differentially expressed (DE) miRNAs and three DE lncRNAs. The DE RNAs were highly enriched in 21 functional pathways implicated in HCC (p < 0.05), including p53, MAPK, and NAFLD signaling. Potential pairwise interactions between DE ncRNAs and mRNAs were fully characterized using in silico prediction and experimentally-validated evidence. We for the first time constructed a MLMI network of reciprocal interactions for 16 miRNAs, three lncRNAs, and 253 mRNAs in HCC. The predominant role of MEG3 in the MLMI network was validated by its overexpression in vitro that the expression levels of a proportion of MEG3-targeted miRNAs and mRNAs was changed significantly. Our results suggested that the comprehensive MLMI network synergistically modulated carcinogenesis, and the crosstalk of the network provides a new avenue to accurately describe the molecular mechanisms of hepatocarcinogenesis.

Figure 1

DE ncRNAs. A volcano plot of miRNAs (a) and lncRNAs (b). (c) Significantly DE miRNAs identified using two algorithms (edgeR and DESeq). (d) Filtration of lncRNAs using PLAR and slncky. (e) Heatmap of significantly DE ncRNAs. A boxplot showed the expression of the DE miRNAs (f) and lncRNAs (g). Each ncRNA contains two boxes with different colors (dark sky blue: normal and Indian red: tumor). (h) RT-qPCR results of selected DE ncRNAs (dark sky blue: normal and Indian red: tumor).

Figure 2

Interactions between miRNAs, lncRNAs, and mRNAs. (a) The number of miRNA-mRNA targets based on different sequence regions in both predicted and previously reported miRNA-targeted mRNAs. The 3′-UTR, 5′-UTR, and CDS were the regions of the targeted mRNAs that were used for the predictions. (b) A Venn plot of the miRNA-mRNA target predictions based on different regions. (c) A summary of the miRNA-lncRNA targets for both the predicted and previously reported interactions. (d) A summary of the lncRNA-mRNA targets for both the predicted and previously reported miRNA-targeted lncRNAs. Venn plots for the interactions of significantly DE miRNAs and mRNAs (e), miRNAs and lncRNAs (f), lncRNAs and mRNAs (h). and (g). A co-expression network of the MLMI based on weighted Pearson’s correlations (GS1: average gene significance; cyan: module 1; blue: module 2; and gray: non-modules).

Figure 3

A MLMI network of significantly DE RNAs. Different colors indicate different RNA molecules (gray: mRNAs and colorful dots: ncRNAs). The outer circle includes mRNAs targeted by a single ncRNA, the middle circle is miRNAs and lncRNAs (three dots in khaki), and the inner circle is mRNAs targeted by multiple ncRNAs.

Figure 4

Analyses of functional pathway for ncRNAs. (a) Gene ontology (GO) analysis of the cellular localization, biological processes and molecular functions of the miRNAs and lncRNAs. Each point represents a GO term that is highly enriched for the non-coding RNAs, and the total number of each GO class is listed in the legend. The most significant terms (p < 10⁻¹⁵) are presented. Points in gray indicate significant enrichment of GO terms (p < 0.05). (b) Functional pathway analyses of miRNAs and lncRNAs using KEGG, Panther, and Wiki. Each point represents a pathway, and points with different colors represent the HCC-associated pathways listed in the legend. The numbers in round brackets represent the number of genes that operate in that pathway predominantly from our input gene list. The total number of significantly enriched pathways obtained from all three databases is shown in the legend.

Figure 5

The role of MEG3 validated by its overexpression in vitro. (a) The intersection of DE miRNAs identified in vitro (HepG₂ and LO₂ cell lines) and the sequenced HCC cohorts. (b) DE miRNAs identified in the sequenced HCC that was validated in vitro. (c) DE miRNAs identified in LO₂ with overexpression of MEG3.

Figure 6

Survival analysis of TCGA HCC cohort based on the DE ncRNAs (red line: patients with a higher expression levels (upper 25%, n = 90); blue line: patients with a lower expression levels (lower 25%, n = 90).