Fig 2: Functional implications for miRNA target enrichment in specific hierarchical clusters.(A) Distribution of the number of predicted miR-133ab murine target genes in the six hierarchical clusters. Compared to other hierarchical clusters, cluster E was significantly enriched for predicted murine targets of miR-133ab (Fisher's exact test, p<0.05). (B) Expression profiles of miR-133a and miR-133b, along with (C) those of their respective protein targets in cluster E. qRT-PCR was performed to detect and measure the relative abundance of miR-133a and miR-133b in the SCN. Information regarding predicted miRNA targets was extracted from the broadly conserved microRNA families from the TargetScanMouse database. (D–F) Western blot analysis of three predicted miR-133ab targets in cluster E, including (D) SH3GL2, (E) SYT1, and (F) SV2A, in Neuro2A cells transfected with either microRNA inhibitors against miR-133a (A-) or miR-133b (B-), microRNA mimics for miR-133a (A) or miR-133b (B), or microRNA inhibitor negative controls (C). Actin was used as the loading control in Western blot analysis. Values in each graph represent the median relative abundance of the protein examined normalized to actin expression (n = 3 per CT). The asterisk (*) denotes the presence of a faster-migrating, non-specific band.

Fig 3: The effect of prenatal exposure to VPA on the expression of v-SNARE proteins: VAMP1/2, Syp, Syn1, p-Syn1(Ser62/Ser67), and Syt1. The level of mRNA of Vamp1, Vamp2, Syp, Syn1, and Syt1 in the hippocampus (A) and cerebral cortex (C) of the control and VPA-exposed rats is presented. The level of mRNA was measured by real-time PCR and calculated by the ??Ct method with Actb (ß-actin) as a reference gene. The data represent the mean values ± SEM from n = (4–5) independent experiments in the hippocampus and n = (4–6) in the cerebral cortex. The immunoreactivity of the v-SNARE proteins in the control and the VPA-exposed rats was monitored using a Western blot analysis. Densitometric analysis and representative pictures of VAMP1/2, Syp, Syn1, p-Syn1, and Syt1 in the hippocampus (B) and cerebral cortex (D) are shown. Results were normalized to GAPDH levels. Data represent the mean values ± SEM from n = (3–9) independent experiments in both the hippocampus and cerebral cortex. * p < 0.5, ** p < 0.01, *** p < 0.001 vs. control.