Transcriptomic Analysis of Long Non-coding RNA-MicroRNA-mRNA Interactions in the Nucleus Accumbens Related to Morphine Addiction in Mice

Abstract

Recent research suggest that some non-coding RNAs (ncRNAs) are important regulators of chromatin dynamics and gene expression in nervous system development and neurological diseases. Nevertheless, the molecular mechanisms of long non-coding RNAs (lncRNAs), acting as competing endogenous RNAs (ceRNAs), underlying morphine addiction are still unknown. In this research, RNA sequencing (RNA-seq) was used to examine the expression profiles of lncRNAs, miRNAs and mRNAs on the nucleus accumbens (NAc) tissues of mice trained with morphine or saline conditioned place preference (CPP), with differential expression of 31 lncRNAs, 393 miRNAs, and 371 mRNAs found. A ceRNA network was established for reciprocal interactions for 9 differentially expressed lncRNAs (DElncRNAs), 10 differentially expressed miRNAs (DEmiRNAs) and 12 differentially expressed mRNAs (DEmRNAs) based on predicted miRNAs shared by lncRNAs and mRNAs. KEGG pathway enrichment analyses were conducted to explore the potential functions of DEmRNAs interacting with lncRNAs in the ceRNA network. These DEmRNAs were enriched in synaptic plasticity-related pathways, including pyrimidine metabolism, ECM-receptor interaction, and focal adhesion. The correlation between the relative expression of lncRNAs, miRNAs and mRNAs was analyzed to further validate predicted ceRNA networks, and the Lnc15qD3-miR-139-3p-Lrp2 ceRNA regulatory interaction was determined. These results suggest that the comprehensive network represents a new insight into the lncRNA-mediated ceRNA regulatory mechanisms underlying morphine addiction and provide new potential diagnostic and prognostic biomarkers for morphine addiction.

Keywords: addiction; ceRNA network; lncRNA; morphine; nucleus accumbens.

FIGURE 1

Establishment of the morphine CPP model. (A) Chematic experimental design for the training and test of morphine-CPP. (B) CPP scores of saline- and morphine-treated mice on Days 0 and 7. The data are presented as the mean ± SEM, n = 12, ***p < 0.001, compared with the saline group. (C) Up: The position of the razor blades used to cut the nucleus accumbens (NAc) are shown in a sagittal section of the mouse brain; down: Schematic illustrations of NAc location in coronal slices (red area). (D) Flowchart for the identification and analysis of ncRNAs and mRNAs in the ceRNA network.

FIGURE 2

Heatmaps of differentially expressed lncRNA (A), miRNA (B), and mRNA (C) profiles. Unsupervised clustering analysis show the expression profiles of differentially expressed lncRNAs, miRNAs (top 40), and mRNAs (top 40) in the morphine group compared to saline group. MOR1, MOR2, and MOR3 represent the NAc tissue of three mice treated with morphine. NS1, NS2, and NS3 represent the NAc tissue of three mice treated with saline. Red indicates upregulation, and blue indicates downregulation.

FIGURE 3

Differential expression of distinct RNAs and characteristics of LncRNAs. The relative expression levels of eight lncRNAs (A), four miRNAs (B) and five mRNAs (C) are shown by comparing the morphine group with the saline group via qRT-PCR. The data are presented as the mean ± SEM, n = 4, *p < 0.05. Subclasses (D) and subcellular localization (E) of significantly dysregulated lncRNAs were analyzed.

FIGURE 4

The lncRNA-associated ceRNA networks in morphine/saline treated mice. The ceRNA network was constructed based on identified lncRNA–miRNA and miRNA–mRNA interactions. In the network, red indicates upregulated lncRNAs, blue indicates downregulated miRNAs, and green indicates upregulated mRNAs.

FIGURE 5

Gene Ontology (GO) and KEGG pathway function enrichment analysis for DEmRNAs. The significant molecular functions (A), biological processes (B) and cellular components (C) of DEmRNAs in the ceRNA network were presented using GO analyses. Pathway analyses results of DEmRNAs in the ceRNA network with fold changes >1.5 (D).

FIGURE 6

Regression analyses of the ceRNA network. The correlations of Lnc18qE1-miR-671-5p-Thbs4 (A) and Lnc15qD3-miR-139-3p-Lrp2 (B) were determined by qRT-PCR.