Inhibition of MicroRNA-182/183 Cluster Ameliorates Schizophrenia by Activating the Axon Guidance Pathway and Upregulating DCC

Abstract

Schizophrenia (SZ) is a complex disorder caused by a variety of genetic and environmental factors. Mounting evidence suggests the involvement of microRNAs (miRNAs) in the pathology of SZ. Accordingly, the current study set out to investigate the possible implication of the miR-182/183 cluster, as well as its associated mechanism in the progression of SZ. Firstly, rat models of SZ were established by intraperitoneal injection of MK-801. Moreover, rat primary hippocampal neurons were exposed to MK-801 to simulate injury of hippocampal neurons. The expression of miR-182/183 or its putative target gene DCC was manipulated to examine their effects on SZ in vitro and in vivo. It was found that miR-182 and miR-183 were both highly expressed in peripheral blood of SZ patients and hippocampal tissues of SZ rats. In addition, the miR-182/183 cluster could target DDC and downregulate the expression of DDC. On the other hand, inhibition of the miR-182/183 cluster ameliorated SZ, as evidenced by elevated serum levels of NGF and BDNF, along with reductions in spontaneous activity, serum GFAP levels, and hippocampal neuronal apoptosis. Additionally, DCC was found to activate the axon guiding pathway and influence synaptic activity in hippocampal neurons. Collectively, our findings highlighted that inhibition of the miR-182/183 cluster could potentially attenuate SZ through DCC-dependent activation of the axon guidance pathway. Furthermore, inhibition of the miR-182/183 cluster may represent a potential target for the SZ treatment.

Figure 1

A schematic figure of the in vivo (a) and in vitro (b) experiment design.

Figure 2

miR-182 and miR-183 are highly expressed in SZ. (a) RT-qPCR was carried out to measure the expression of miR-182/183 in the peripheral blood sample of SZ patients (n = 59) and healthy individuals (n = 48). (b) RT-qPCR was conducted to determine the expression of miR-182/183 in the serum samples from SZ (n = 8) and normal (n = 8) rats. (c) RT-qPCR was performed to determine the expression of miR-182/183 in the hippocampal tissues from SZ (n = 8) and normal (n = 8) rats. The experimental data were measurement data, expressed as the mean ± standard deviation, and analyzed by independent sample t-test. ^∗p < 0.05vs. the normal group.

Figure 3

Inhibition of miR-182/183 cluster improves SZ at different levels: (a) miR-182/183 cluster transfection efficiency (n = 8/group); (b) open field test (left), PPI test (middle), and Morris water maze test (right) in SZ rats (n = 8/group); (c) representative micrographs showing Nissl body in hippocampal tissues from SZ rats. MK-801-exposed hippocampal neurons were treated with miR-182/183 mimic or AntimiR-182/183; (d) expression of miR-182 and miR-183 in the MK-801-exposed hippocampal neurons determined by RT-qPCR; (e) quantitative analysis of apoptosis of MK-801-exposed hippocampal neurons detected by Hoechst 33342 staining. The data in the figure were measurement data, expressed as the mean ± standard deviation, and analyzed by one-way ANOVA. ^∗p < 0.05vs. the AgomiR control or miR-182/183 mimic-NC control; ^#p < 0.05vs. the scrambled control or AgomiR control group. The cell experiments were repeated three times independently.

Figure 4

miR-182/183 targets DCC and negatively regulates DCC: (a) Venn diagram of the predicted target genes of miR-182 and miR-183 by the RNA22 database. The blue circle represents the prediction result of miR-182, the red circle represents the prediction result of miR-183, and the middle part represents the intersection of the two sets of data. (b–d) GO function analysis of miRNA target gene prediction results; (b–d) enrichment results of three GO functional groups: biological process (BP), cellular component (CC), and molecular function (MF). The abscissa indicates the generation, and the ordinate indicates the name of entry identifiers. The circle size and color in the figure indicate the number of genes in the entry identifier and the p value, respectively. (e) KEGG enrichment analysis of metabolic pathway in relation to miRNA target gene prediction results; (f) Western blot analysis of DCC protein in the hippocampal tissues of SZ (n = 8) and normal (n = 8) rats; (g) the binding site between miR-182/183 and DCC predicted by the bioinformatics website https://cm.jefferson.edu/rna22/; (h) binding of miR-182/183 to DCC determined by dual-luciferase reporter assay; (i) Western blot analysis of DCC protein in the hippocampal neurons treated with miR-182/183 mimic or AntimiR-182/183. The data in the figure were measurement data, expressed as the mean ± standard deviation, and analyzed by an independent sample t-test. ^∗p < 0.05vs. the normal or miR-182/183 mimic-NC group. ^#p < 0.05vs. the AntimiR control group. The cell experiments were repeated three times independently.

Figure 5

DCC overexpression reverses the promoting effect of the miR-182/183 cluster on the progression of SZ: (a) expression of miR-182 and miR-183 in the hippocampal tissues of SZ rats with AgomiR-182/183+oe-DCC determined by RT-qPCR along with Western blot analysis of DCC protein in the hippocampal tissues of SZ rats with AgomiR-182/183+oe-DCC (n = 8/group); (b) open field test (left), PPI test (middle), and Morris water maze test (right) in SZ rats (n = 8/group); (c) apoptosis in hippocampal neurons in response to AgomiR-182/183+oe-DCC. The data in the figure were measurement data, expressed as the mean ± standard deviation, and analyzed by an independent sample t-test. ^∗p < 0.05vs. the AgomiR-182/183+oe-NC or miR-182/183 mimic+oe-NC group. The cell experiments were repeated three times independently.

Figure 6

DCC activates the axon guidance pathway and thus affects synaptic activity in hippocampal neurons. (a) Expression of DCC and axon guidance pathway-related protein PLC in hippocampal neurons. (b) ELISA was used to detect the expression of PIP₂ and IP₃ in hippocampal neurons. The data in the figure were measurement data, expressed as the mean ± standard deviation. Comparison between two groups was analyzed by the independent sample t-test; comparison among multiple groups was analyzed by one-way ANOVA, followed by Tukey's post hoc test. ^∗p < 0.05vs. the oe-NC, miR-182/183 mimic NC, or oe-DCC+DMSO group. ^#p < 0.05vs. the AntimiR control group. The cell experiments were repeated three times independently.

Figure 7

Schematic diagram of the mechanism by which miR-182/183 cluster affects SZ. miR-182 and miR-183 are highly expressed in SZ. Inhibition of miR-182/183 cluster can upregulate DCC, thereby activating the axon guidance pathway and ultimately preventing the occurrence and development of SZ.