Fig. 5
ACTN4, ELAVL4 and USP9X are direct targets of miR-142-5p and contribute to miR-142-5p-induced memory impairment and dendritic spine damage. (A) miR-142-5p was predicted using TargetScan, miRDB and miRGate, and the predicted data were subsequently subjected to Venn analysis. (B) Intersecting genes were subjected to GO pathway enrichment analysis, and 10 genes in the dendritic spine-associated actin pathway were selected for validation. miR-142-5p agomir and NC were injected into the normal rat bilateral hippocampus via stereotaxic localization. After 48 h, RT-qPCR showed that the mRNA levels of ELAVL4, PTPN4, USP9X, ACTN4, and SLAIN1 in the hippocampal tissues of the rats in the miR-142-5p agomir intervention group were significantly lower, while the other genes were not significantly different. n = 3–6 rats. unpaired Student’s t test was used. (C, D) RT‒qPCR revealed that ACTN4, ELAVL4 and USP9X mRNA levels were significantly lower in the hippocampal tissues of CCI rats than in those of sham rats. n = 5–8 rats. unpaired Student’s t test was used. (E) RT‒qPCR revealed that the miR-142-5p antagomir significantly reversed the inhibitory effect of plasma_EVCCI on the mRNA levels of ACTN4, ELAVL4 and USP9X in primary hippocampal neurons. ordinary one-way followed by Tukey’s multiple comparisons test was used. (F) Western blot analysis showed that the protein levels of ACTN4, ELAVL4 and USP9X were significantly lower in the hippocampal tissues of rats stereotaxically injected with the miR-142-5p agomir than in those of the NC group. n = 3 rats. unpaired Student’s t test was used. (G) Western blot analysis revealed that ACTN4, ELAVL4 and USP9X protein levels were significantly lower in the hippocampal tissues of CCI rats than in those of sham rats. n = 3–4 rats. unpaired Student’s t test was used. (H) AAV-miR-142-5p sponge significantly improved the decreased ACTN4, ELAVL4 and USP9X protein levels in hippocampal tissues of CCI rats compared to the CCI + AAV-NC group; however, AAV-miR-142-5p exacerbated the decreased protein expression, via Western blot analysis. n = 3–4 rats. ordinary one-way followed by Tukey’s multiple comparisons test was used. The data are shown as the means ± SDs. *P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001