MicroRNA-125a-3p is involved in early behavioral disorders in stroke-afflicted rats through the regulation of Cadm2

Abstract

Ischemic strokes carry a significant risk of mortality and recurrent vascular events. Recent studies suggest that changes in microRNAs (miRNAs or miRs) may affect the development of the stroke. However, few studies have investigated the role of miRNAs in behavioral disorder in early stroke. In the present study, animal models of middle cerebral artery occlusion (MCAO) are used, as well as a cell model of neurite outgrowth to further investigate the role of miRNAs in targeting synapse-associated proteins expression in early stroke. The authors used miRNA expression microarrays on RNA extracted from the cortex tissue samples from the rats of MCAO and control rats. Reverse transcription‑quantitative polymerase chain reaction was conducted to verify the candidate miRNAs discovered by microarray analysis. Data indicated that miR‑125a was significantly increased in the cortex of the model of MCAO, which were concomitant with that rats of MCAO at the same age displayed significant behavioral deficits. Bioinformatics analysis predicted the cell adhesion molecule 2 (Cadm2, mRNA) neurite outgrowth-associated protein is targeted by miR‑125a. Overexpression of miR‑125a reduced the level of Cadm2 expression in PC12 cell injury induced by free-serum. In contrast, inhibition of miR‑125a using miR‑125a inhibitors significantly resulted in higher levels of Cadm2 expression. In conclusion, miR‑125a is involved in the behavioral disorder of animal models of MCAO by regulation of Cadm2.

Figure 1

miR-125a was increased in the cortex of ischemic rats. (A) The expression of miRNAs in cortex was measured using microarray analysis in control group (left) and model group (right). (B) Longa score in control group and model group. (C) The expression of miR-125a in cortex were measured using reverse transcription-quantitative polymerase chain reaction in the cortex, data were presented as the mean ± standard deviation, n=3 in each group. ^*P<0.05 vs. normal group. miR, microRNA; MCAO, middle cerebral artery occlusion; Cadm2, cell adhesion molecule 2.

Figure 2

Cadm2 was decreased in the cortex of ischemic rats. (A) The expression of Cadm2 in cortex was measured by laser confocal microscopy (original magnification, ×200). (B) Longa score in control group and model group. The expression of Cadm2 was measured using a western blot assay in the cortex, and the immunoreactive bands were quantified with ImageJ software. By using western blot analysis, the expression of the Cadm2 protein was efficiently decreased in the model group, compared with the control group. ^*P<0.05 and ^**P<0.01 vs. control. MCAO, middle cerebral artery occlusion; Cadm2, cell adhesion molecule 2.

Figure 3

miR-125a inhibited Cadm2 expression (original magnification, ×200). (A) Control group, (B) model group (serum-free), (C) miR-125a mimic group, (D) miR-125a inhibitor group. (E) Percentage of Cadm2-positive cells. One-way analysis of variance was used to analyze the differences. ^**P<0.01 and ***P<0.001; ^#P<0.05 and ^###P<0.001 as indicated. miR, microRNA. Cadm2, cell adhesion molecule 2.

Figure 4

microRNA-125a inhibited neurite outgrowth in PC12 cells. (A) Spindle cell rate, (B) cell differentiation rate D1, (C) cell differentiation rate D2, (D) average protrusion length of cell. One-way analysis of variance test was used to analyze the variances between the groups ^*P<0.05 and ^***P<0.001 as indicated. NC, negative control.

Figure 5

miR-125a regulates the expression of Cadm2. (A) (a) The target sites of miR-125a in the Cadm2 3′-UTR were predicted by bioinformatics software, TargetScan and miRNA.org. (b) The target sites of miR-125a in Cadm2-WT 3′-UTR and Cadm2-MUT 3′-UTR. (c) The construction profile of the psiCHECK2-Cadm2 vector is presented in the diagram that contained the miR-125a-3p target sites in Cadm2 3′-UTR. (B) Expression of target protein Cadm2 in the PC12 cells were analyzed with the western blot assay, and the bands were quantified with ImageJ software. One-way analysis of variance was used to analyze the variances. By western blotting, the expression of the Cadm2 protein was efficiently decreased by miR-125a mimics, compared with the control group, and may be increased obviously when treated with miR-125a inhibitor. (C) The miR-125a mimic, inhibitor, mimic NC or inhibitor NC were co-transfected with Cadm2-WT 3′-UTR or Cadm2-MUT 3′-UTR into PC12 cells. miR-125a targeted the expression of Cadm2. One-way analysis of variance test was used to analyze the variances between groups. ^*P<0.05, ^**P<0.01 and ^***P<0.001 as indicated. miR, microRNA; UTR, untranslated region; WT, wild-type; NC, negative control; Cadm2, cell adhesion molecule 2.