Early Brain microRNA/mRNA Expression is Region-Specific After Neonatal Hypoxic-Ischemic Injury in a Mouse Model

Abstract

Background: MicroRNAs (miRNAs) may be promising therapeutic targets for neonatal hypoxic-ischemic brain injury (HIBI) but targeting miRNA-based therapy will require more precise understanding of endogenous brain miRNA expression. Methods: Postnatal day 9 mouse pups underwent HIBI by unilateral carotid ligation + hypoxia or sham surgery. Next-generation miRNA sequencing and mRNA Neuroinflammation panels were performed on ipsilateral cortex, striatum/thalamus, and cerebellum of each group at 30 min after injury. Targeted canonical pathways were predicted by KEGG analysis. Results: Sixty-one unique miRNAs showed differential expression (DE) in at least one region; nine in more than one region, including miR-410-5p, -1264-3p, 1298-5p, -5,126, and -34b-3p. Forty-four mRNAs showed DE in at least one region; 16 in more than one region. MiRNAs showing DE primarily targeted metabolic pathways, while mRNAs targeted inflammatory and cell death pathways. Minimal miRNA-mRNA interactions were seen at 30 min after HIBI. Conclusion: This study identified miRNAs that deserve future study to assess their potential as therapeutic targets in neonatal HIBI. Additionally, the differences in miRNA expression between regions suggest that future studies assessing brain miRNA expression to guide therapy development should consider evaluating individual brain regions rather than whole brain to ensure the sensitivity needed for the development of targeted therapies.

Keywords: cerebellum; cortex; encephalopathy; next-generation sequencing; striatum.

FIGURE 1

MicroRNAs with significant differential expression at 30 min after hypoxic-ischemic brain injury in the (A) cerebellum, (B) striatum/thalamus, and (C) cortex (n = 4/group for each region). Significance defined by fold-change > 1.5, p value and/or false discovery rate < .05, and average count per million reads >10. Error bars represent standard error of the mean.

FIGURE 2

MicroRNAs (miRNA) with significant differential expression in more than one region. (A) Venn diagram demonstrating the number of miRNAs with significant differential expression and the overlap between each region; (B) differential expression of the nine miRNAs with significant differential expression in more than one region (*regional expression significant, as defined by fold-change > 1.5, p value < .05, and average count per million reads >10); (C) two-way unsupervised clustering of differentially expressed microRNAs demonstrating separation of the majority of the cerebellum samples from the cortex and striatum/thalamus samples. Rows represent microRNA species and columns represent samples. Each square represents expression in a single sample, color-coded for magnitude of change relative to controls; and (D) KEGG pathway analysis of the miRNAs with significant differential expression. Error bars represent standard error of the mean.

FIGURE 3

Messenger RNAs (mRNA) with significant differential expression in more than one region. (A) Venn diagram demonstrating the number of mRNAs with significant differential expression and the overlap between each region; (B) differential expression of the mRNAs with significant differential expression in at least one region (significance defined by fold-change > 1.5 and p value and/or false discovery rate < .05), n = 4/group for each region; and (C) KEGG analysis demonstrating the canonical pathways affected by all of the mRNAs demonstrating significant differential expression. Error bars represent standard error of the mean.

FIGURE 4

Messenger RNA (mRNA) networks in each region representing the mRNA with significant differential expression after hypoxic-ischemic brain injury versus controls and two degrees of connection between those mRNA and the other mRNA analyzed in this study. Not shown are any mRNAs with no, or only one, connection. Node fill color relates to the log fold change of mRNA in hypoxic-ischemic brain injury compared to controls.

FIGURE 5

MicroRNA to messenger RNA (miRNA-mRNA) networks in the cortex. The only two miRNAs that demonstrated significant differential expression at 30 min and were closely linked to mRNAs also significantly altered at 30 min after injury were miR-1195 and -690. Neither the striatum/thalamus nor cerebellum expression data resulted in significant miRNA-mRNA networks.

FIGURE 6

Correlation between Nanostring RNA sequencing and quantitative polymerase chain reaction (qPCR) validation of seven highly expressed mRNAs. The x-axis values are Nanostring log₂ fold-change (FC, hypoxic-ischemic brain injury versus control) values and y-axis are qPCR ΔΔCt log₂FC values. The solid line represents the best-fit regression of the data points with strong correlation (R² = 0.894) and the dashed line represents the ideal correlation line (x = y).