Circulating MicroRNAs in Extracellular Vesicles as Potential Biomarkers of Alcohol-Induced Neuroinflammation in Adolescence: Gender Differences

Figures

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  Figure 1. Inflammatory-related miRNA profile in the human plasma extracellular vesicles (EVs) deriving from the ethanol-intoxicated adolescents. (A) Human plasma EVs characterization by electron microscopy. Scale bar, 100 nm. (B) Representative immunoblots of the expressions of CD9, CD63, CD81, and calnexin of the human plasma EVs. (C) Graphs represent the expressions of the plasma EV miRNAs (mir-146a-5p, mir-21-5p, mir-182-5p, and mir-183-5p) of the human adolescent females and males after acute ethanol intoxication and the data of the corresponding healthy control individuals. (D) Graphs represent the mir-182-5p expression in the plasma and plasma EVs of the human adolescent females and males after acute ethanol intoxication and the data of the corresponding healthy control individuals. Data represent mean ± SEM, n = 6 independent experiments. * p < 0.05 compared to their respective healthy control individuals, according to the two-way ANOVA followed by the Bonferroni post hoc test.

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  Figure 2. Inflammatory-related miRNA profile in the plasma EVs deriving from the ethanol-treated adolescent wild type (WT) mice. (A) Murine plasma EVs characterization by electron microscopy. Scale bar, 100 nm. (B) Representative immunoblots of the expressions of CD9, CD63, CD81, and calnexin of the murine plasma EVs. (C) Graphs represent the expressions of plasma EV miRNAs (mir-146a-5p, mir-21-5p, mir-182-5p, and mir-183-5p) after ethanol or saline treatment in the adolescent female and male WT mice (postnatal day (PND) 44). (D) Graphs represent the mir-182-5p expression in plasma and plasma EVs after the ethanol or saline treatment in the adolescent female and male WT mice (PND 44). Data represent mean ± SEM, n = 6 independent experiments. * p < 0.05, *** p < 0.001 compared to their respective control counterparts, according to the two-way ANOVA followed by the Bonferroni post hoc test.

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  Figure 3. Inflammatory-related miRNA profile in the plasma EVs deriving from the ethanol-treated adolescent toll-like receptor 4 (TLR4)-knockout (KO) mice. Graphs represent the expression of plasma EV miRNAs (mir-146a-5p, mir-21-5p, mir-182-5p, and mir-183-5p) after the ethanol or saline treatment in the adolescent female and male TLR4-KO mice (PND 44). Data represent mean ± SEM, n = 6 independent experiments. No significant differences were shown, according to the two-way ANOVA followed by the Bonferroni post hoc test.

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  Figure 4. Effects of ethanol in the inflammatory-related miRNA profile in the cerebral cortices and livers of adolescent WT mice. Graphs represent the expression of (A) mir-146a-5p and (B) mir-21-5p after a 24-h and 2-week withdrawal period of ethanol or saline treatment in the female and male adolescent WT mice (PND 44 and PND 58). Data represent mean ± SEM, n = 6 independent experiments. * p < 0.05 compared to their respective control counterparts, according to the two-way ANOVA followed by the Bonferroni post hoc test.

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  Figure 5. Functional analysis of mir-146a-5p and mir-21-5p. (A) Protein–protein interaction for the predicted genes of each miRNA. (B) The principal Reactome pathways were modulated by the STRING protein–protein network interaction analysis, performed with the STRING webserver, and p-values are represented as (−log(p-value)).

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  Figure 6. Effects of ethanol on the target gene profile of mir-146a-5p and mir-21-5p in the cerebral cortices of adolescent WT mice. Graphs represent the expression of Traf6, Stat3, and Camk2a after a 24-h and 2-week withdrawal period of ethanol or saline treatment in the female and male adolescent WT mice (PND 44 and PND 58). Data represent mean ± SEM, n = 6 independent experiments. * p < 0.05 compared to their respective control counterparts, according to the two-way ANOVA followed by the Bonferroni post hoc test.