Expansion of plasma MicroRNAs over the first month following human stroke

Abstract

Few have characterized miRNA expression during the transition from injury to neural repair and secondary neurodegeneration following stroke in humans. We compared expression of 754 miRNAs from plasma samples collected 5, 15, and 30 days post-ischemic stroke from a discovery cohort (n = 55) and 15-days post-ischemic stroke from a validation cohort (n = 48) to healthy control samples (n = 55 and 48 respectively) matched for age, sex, race and cardiovascular comorbidities using qRT-PCR. Eight miRNAs remained significantly altered across all time points in both cohorts including many described in acute stroke. The number of significantly dysregulated miRNAs more than doubled from post-stroke day 5 (19 miRNAs) to days 15 (50 miRNAs) and 30 (57 miRNAs). Twelve brain-enriched miRNAs were significantly altered at one or more time points (decreased expression, stroke versus controls: miR-107; increased expression: miR-99-5p, miR-127-3p, miR-128-3p, miR-181a-3p, miR-181a-5p, miR-382-5p, miR-433-3p, miR-491-5p, miR-495-3p, miR-874-3p, and miR-941). Many brain-enriched miRNAs were associated with apoptosis over the first month post-stroke whereas other miRNAs suggested a transition to synapse regulation and neuronal protection by day 30. These findings suggest that a program of decreased cellular proliferation may last at least 30 days post-stroke, and points to specific miRNAs that could contribute to neural repair in humans.

Keywords: Apoptosis; MicroRNAs; ischemic stroke; neuronal plasticity; neuroprotection.

Figure 1.

(a) MicroRNAs with significant differential expression (false discovery rate corrected p < 0.05 stroke vs. control participants) for the discovery cohort at 5, 15, and 30 days post-stroke, and the validation cohort at 15 days post-stroke. The number of significant miRNAs more than doubled from 5 to 15 days post-stroke. The brain- and CSF-enriched miRNAs are highlighted in blue and yellow respectively, identified from prior published data sets. ^– (b) Bi-clustering dendrogram separating miRNAs into two distinct groups based on stroke vs. control log₂ fold-change. This list is limited to the 8 miRNAs dysregulated at all time points as well as all of the brain- and CSF-enriched miRNAs. For a complete dendrogram that includes all dysregulated miRNAs, see supp fig S3. FC – fold-change; FDR p value – false discovery rate corrected p value; Disc – discovery cohort; Val – validation cohort.

Figure 2.

Venn diagram showing the number of microRNAs with significant differential expression between stroke and control participants shared between time points post-stroke in the discovery and validation cohorts.

Figure 3.

MicroRNA expression (stroke vs. controls) from 5–30 days post-stroke for: (a) the eight miRNAs showing significant differential expression at all time points in the discovery and validation cohorts, and (b) the twelve brain-enriched miRNAs with significant differential expression at 1 or more time points in the discovery or validation cohorts. All expression data plotted are from the discovery cohort since longitudinal data was not available for the validation cohort. In panel A the absolute value of the fold-changes for all 8 miRNAs showed significant change over time, suggesting possible decay toward null expression. In panel B the absolute value of the fold changes for all 12 miRNAs showed a significant change from 5 to 15 days post-stroke, suggesting a possible increase in the expression of brain-enriched miRNAs during this time span.

Figure 4.

Pathway analysis for the 8 dysregulated miRNAs between stroke and control participants at all time points in both cohorts. Mir-107 mapped to miR-103-3p and miR-19a-3p mapped to miR-19b-3p since they share the same seed regions. Mir-382-5p and miR-16-2-3p had no experimentally observed data linking them to specific genes. The genes PTEN, PLAG1, ICOS, EZH2, and PTGS2 (COX2) were predicted to have coactivation due to decreased expression of 2 miRNAs (see discussion for further details).

Figure 5.

Theoretical timeline of events related to injury and neural repair over the first month post-stroke in humans. Brain-enriched miRNAs are listed at the specific time points when significant dysregulation was found for stroke vs. control participants. The suspected role for each miRNA is shown based on literature review. Neural repair includes neural outgrowth and synaptogenesis/synaptic pruning, but the timing of these events remains unknown in humans. *miRNAs listed for both repair and injury due to multiple possible roles depending on the downstream genes affected. The framework for this figure was reproduced with permission.