Deep sequencing and miRNA profiles in alcohol-induced neuroinflammation and the TLR4 response in mice cerebral cortex

Abstract

Alcohol abuse can induce brain injury and neurodegeneration, and recent evidence shows the participation of immune receptors toll-like in the neuroinflammation and brain damage. We evaluated the role of miRNAs as potential modulators of the neuroinflammation associated with alcohol abuse and the influence of the TLR4 response. Using mice cerebral cortex and next-generation sequencing (NGS), we identified miRNAs that were differentially expressed in the chronic alcohol-treated versus untreated WT or TLR4-KO mice. We observed a differentially expression of miR-183 Cluster (C) (miR-96/-182/-183), miR-200a and miR-200b, which were down-regulated, while mirR-125b was up-regulated in alcohol-treated WT versus (vs.) untreated mice. These miRNAs modulate targets genes related to the voltage-gated sodium channel, neuron hyperexcitability (Nav1.3, Trpv1, Smad3 and PP1-γ), as well as genes associated with innate immune TLR4 signaling response (Il1r1, Mapk14, Sirt1, Lrp6 and Bdnf). Functional enrichment of the miR-183C and miR-200a/b family target genes, revealed neuroinflammatory pathways networks involved in TLR4 signaling and alcohol abuse. The changes in the neuroinflammatory targets genes associated with alcohol abuse were mostly abolished in the TLR4-KO mice. Our results show the relationship between alcohol intake and miRNAs expression and open up new therapeutically targets to prevent deleterious effects of alcohol on the brain.

Figure 1

Experimental NGS Workflow and small RNA Quality Control. Small RNA libraries were prepared from the cortices of 44 mice (11 mice/condition). Samples were used for the deep sequencing protocol on the Illumina platform. Bioinformatics primary and secondary pipelines were used to detect the miRNAs profile and the differential expression miRNAs analysis (A). The Quality Control score, the Q value in all the nucleotides sequenced, were higher than 39, and the length with the best Q values was 51 nucleotides (B). Mapping Quality of the reads and distribution MAPQ by nucleotide length (21–25nt represent miRNA reads) (C). Number of reads aligned in multiple loci or unique loci in the reference genome sequence (D). Percentage of reads mapping RNA species (E).

Figure 2

miRNA profile and differential expression analysis. The graph shows the most abundant miRNAs in mice cortex samples, where 50% of all the reads are identified as seven miRNAs (miR-181a, miR-26a, miR-30, miR-125a/b, miR-486a/b) (A). We showed the condition-specific patterns of the expression of the miRNAs in the mice cortex samples (B). Validation of high throughput data by sensitivity levels of the NGS and RT-qPCR techniques indicate that miR-181a is the most abundant miRNA species and miR-96 is the lowest in both protocols (C). Analysis of miRNAs’ stability under our specific conditions (D). The profile of the miRNAs with a differential expression in the three study comparisons (TLR4-KO + EtOH vs. KO; WE vs. WT; KO vs. WT) was assessed.

Figure 3

Heatmaps of cortical miRNA expression across alcohol-treated and untreated WT and TLR4-KO mice. The heatmaps show the miRNAs up-regulated (red) or down-regulated (blue) in each study comparison: WT + EtOH vs. WT (A), TLR4-KO vs. WT (B), TLR4-KO + EtOH vs. TLR4-KO (C) and TLR4-KO + EtOH vs. WT-EtOH (D).

Figure 4

RT-qPCR and NGS differential expression analysis. The graphics represent the NGS and RT-qPCR comparisons made in the cortices of the WT and TLR4-KO mice, with or without ethanol treatment (A–L). The NGS black bars denote the mean of the miRNAs raw reads of three independent biological samples (right scale bar). White bars represent the mean ratio of miRNAs obtained by the RT-qPCR analysis in nine individual tissues. Data were normalized with miR-181a and miR-181b. Bars are represented as [mean ± SEM]. *p < 0.05, **p < 0.01, ***p < 0.001 for the NGS data, and ^#p < 0.05, ^##p < 0.01 (Student’s t-test).

Figure 5

Genomic organization, structure and expression of miR-183 and the miR-200 gene clusters involved in alcohol abuse and the TLR4 immune response. (A) Homology sequence with a conserved seed of the miR components of the mmu-miR-183 cluster (miRs-183, -96, -182) on assembly mouse (Chromosome (Chr.) 6, Chr.7 human homolog). (B) The miR-200 family forms two clusters located in different genomic regions: cluster I miR-200s (miR-200b, -200a, -429) and cluster II miR-200s (−200c, −141) located in assembly mouse (Chr. 4 and Chr. 6, respectively) and divided into two functional groups based on their seed sequences. (C) NGS data show that ethanol treatment causes a statistical significant expression (fold change, statistic, p-value (pval), p-value adjusted (padj)) of the selected miRs (miR-96, -182, -183, -200a and -200b). (D) The RT-qPCR shows a differential expression at the levels of miR-183, miR-182, miR-96 (miR-183C), miR-200a and miR-200b in the cortices of the ethanol-treated vs. untreated WT and TLR4-KO mice. n = 9–11 independent experiments. *p < 0.05, **p < 0.01 (Student’s t-test).

Figure 6

The interactome PPI (protein-protein interaction) network of miR-183C and the miR-200 family in alcohol-induced neuroinflammation in the WT and TLR4-KO mice cortex. (A) The diagram shows the different proteins regulated by miR183C and the miR-200 family, and the main proteins involved in alcohol effects (in red), including TLR4 (in blue). (B) The RT-qPCR results show the mRNA expression levels of Nav1.3, Trpv1, Bdnf, Cdca2, Sirt1, Smad3, Mapk14, Lrp6 and Il1r1. n = 10–11 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test) (B).

Figure 7

KEGG pathway and GO enrichment analysis of the differential expressed miR-183C and the miR-200s family in alcohol abuse. (A) The top 15 most enriched KEGG pathways are shown. All the coding functions are annotated against the KEGG database of miR-183C and miR-200s family, which show regulatory pathways, including the functional networks of these clusters. (B) The top 20 enriched GO framework were calculated and plotted and showed the molecular gene functions associated with biological processes and their relationships with miR-183C and the miR-200 family.