Identification of key genes, pathways, and miRNA/mRNA regulatory networks of CUMS-induced depression in nucleus accumbens by integrated bioinformatics analysis

Abstract

Introduction: Major depressive disorder (MDD) is a recurrent, devastating mental disorder, which affects >350 million people worldwide, and exerts substantial public health and financial costs to society. Thus, there is a significant need to discover innovative therapeutics to treat depression efficiently. Stress-induced dysfunction in the subtype of neuronal cells and the change of synaptic plasticity and structural plasticity of nucleus accumbens (NAc) are implicated in depression symptomology. However, the molecular and epigenetic mechanisms and stresses to the NAc pathological changes in depression remain elusive.

Materials and methods: In this study, treatment group mice were treated continually with the chronic unpredictable mild stress (CUMS) until expression of depression-like behaviors were found. Depression was confirmed with sucrose preference, novelty-suppressed feeding, forced swimming, and tail suspension tests. We applied high-throughput RNA sequencing to assess microRNA expression and transcriptional profiles in the NAc tissue from depression-like behaviors mice and control mice. The regulatory network of miRNAs/mRNAs was constructed based on the high-throughput RNA sequence and bioinformatics software predictions.

Results: A total of 17 miRNAs and 10 mRNAs were significantly upregulated in the NAc of CUMS-induced mice with depression-like behaviors, and 12 miRNAs and 29 mRNAs were downregulated. A series of bioinformatics analyses showed that these altered miRNAs predicted target mRNA and differentially expressed mRNAs were significantly enriched in the MAPK signaling pathway, GABAergic synapse, dopaminergic synapse, cytokine-cytokine receptor interaction, axon guidance, regulation of autophagy, and so on. Furthermore, dual luciferase report assay and qRT-PCR results validated the miRNA/mRNA regulatory network.

Conclusion: The deteriorations of GABAergic synapses, dopaminergic synapses, neurotransmitter synthesis, as well as autophagy-associated apoptotic pathway are associated with the molecular pathological mechanism of CUMS-induced depression.

Keywords: depression; mRNA; miRNA; nucleus accumbens; stress.

Figure 1

CUMS leads the mice to express depression-like behaviors. Mice were subjected to the adaptation for a week, the CUMS for 4 weeks. (A) The values of immobile time in the FST were 170.68±1.28 seconds in CUMS-treated mice (n=12) and 141.60±1.8 seconds in controls (P<0.01). (B) The values of immobile time in the TST were 158.18±1.79 seconds in CUMS-treated mice and 124.94±0.86 seconds in controls (P<0.001). (C) The SPT values were 57.22%±1.9% in CUMS-treated mice (n=12) and 84.23%±0.92% in control mice (P<0.001). (D) The latency to eat food is 560.55±12.25 seconds in CUMS-treated mice and 344.58±7.22 in controls (P<0.001). The results are expressed as mean ± SEM. n=12 per group, ***P<0.001 compared with control. Abbreviations: CUMS, chronic unpredictable mild stress; FST, forced swimming test; TST, tail suspension test; SPT, sucrose preference test; NST, novelty-suppressed feeding test; SEM, standard error of the mean.

Figure 2

The validation of differentially expressed miRNAs in the NAc from mice with CUMS-induced depression-like behaviors and controls. Three upregulated miRNAs and two downregulated miRNAs were involved in different cellular functions and were selected for qRT-PCR analysis. (A) qRT-PCR was used to analyze the relative values of mmu-miR-10a-5p, mmu-miR-144-3p, mmu-miR-219b-5p, mmu-miR-124-5p, and mmu-miR-127-5p from mice with CUMS-induced depression-like behaviors and controls (n=12 per group), in which the samples were used from those tissues for high-throughput sequencing. (B) The relative level of mmu-miR-10a-5p, mmu-miR-144-3p, mmu-miR-219b-5p, mmu-miR-124-5p, and mmu-miR-127-5p from mice with CUMS-induced depression-like behaviors (n=4) and controls (n=4), which were analyzed by high-throughput miRNA sequencing. U6 was set as the internal control. The relative values for control mice were normalized to be 1. The data are expressed as mean ± SEM. *P<0.1, **P<0.01, ***P<0.001. Abbreviations: CUMS, chronic unpredictable mild stress; NAc, nucleus accumbens; SEM, standard error of the mean.

Figure 3

The validation of differentially expressed mRNAs in the NAc from mice with CUMS-induced depression-like behaviors and controls. Notes: Five significantly changed mRNAs involved in different cellular functions were selected for performing qRT-PCR. (A) qRT-PCR was used to analyze the relative values of Hbb-b2, Bdnf, Slc32a1(VGAT), Mbp, and Gad1 from mice with CUMS-induced depression-like behaviors and controls (n=12 per group). The samples contained those tissues for high-throughput sequencing. (B) The relative level of Hbb-b2, Bdnf, Slc32a1(VGAT), Mbp, and Gad1 gene expression from mice with CUMS-induced depression-like behaviors (n=4) and controls (n=4), which were analyzed by high-throughput mRNA sequencing. GAPDH was set as the internal control. The relative values for control mice were normalized to be one. The data are expressed as mean ± SEM. **P<0.01, ***P<0.001. Abbreviations: CUMS, chronic unpredictable mild stress; NAc, nucleus accumbens; SEM, standard error of the mean.

Figure 4

miRNA-144-3p can bind to the Gad1 mRNA predicted region. Notes: (A) miRNA-144-3p targeted to Gad1 was predicted by three miRNA target prediction databases (Targetscan, RNA22, and miRDB). The sequences of these seeds referring to the nucleotides in miRNA positions 2–8 are shown in red. Watson–Crick matches in the seed sequence are shown in blue. (B) Correlation between miRNA-144-3p and its prediction target Gad1 mRNA expression in NAc tissue from mice with depression-like behaviors and controls (r=-0.946; P<0.001). (C) Luciferase reporter assay was performed by the co-transfection of luciferase reporter containing the wild or mutant 3′-UTR of GAD1 mRNA with miR-144-3p mimic or their NC into HEK293T cells. Luciferase activity was determined 48 hours after co-transfection. The data are expressed as mean ± SEM. **P<0.01, ***P<0.001. Abbreviations: NC, negative control; SEM, standard error of the mean.