Clinical and preclinical evaluation of miR-144-5p as a key target for major depressive disorder

Abstract

Background: Neuronal abnormalities are closely associated with major depressive disorder (MDD). Available evidence suggests a role for microRNAs (miRNAs) in regulating the expression of genes involved in MDD. Hence, miRNAs that can be potential therapeutic targets need to be identified.

Methods: A mouse model of chronic unpredictable stress (CUS) was used to evaluate the function of miRNAs in MDD. miR-144-5p was screened from the hippocampi of CUS mice based on sequencing results. Adenovirus-associated vectors were used to overexpress or knockdown miR-144-5p in mice. BpV(pic) and LY294002 were used to determine the relationship between miR-144-5p target genes PTEN and TLR4 in neuronal impairment caused by miR-144-5p deficiency. Western blotting, immunofluorescence, ELISA immunosorbent assay, and Golgi staining were used to detect neuronal abnormalities. Serum samples from healthy individuals and patients with MDD were used to detect miR-144-5p levels in the serum and serum exosomes using qRT-PCR.

Results: miR-144-5p expression was significantly decreased within the hippocampal dentate gyrus (DG) of CUS mice. Upregulation of miR-144-5p in the DG ameliorated depression-like behavior in CUS mice and attenuated neuronal abnormalities by directly targeting PTEN and TLR4 expression. Furthermore, miR-144-5p knockdown in normal mice led to depression-like behavior via inducing neuronal abnormalities, including abnormal neurogenesis, neuronal apoptosis, altered synaptic plasticity, and neuroinflammation. miR-144-5p deficiency-mediated neuronal impairment was mediated by PI3K/Akt/FoxO1 signaling. Furthermore, miR-144-5p levels were downregulated in the sera of patients with MDD and associated with depressive symptoms. Consistently, serum exosome-derived miR-144-5p levels were decreased in patients with MDD.

Conclusion: miR-144-5p plays a vital role in regulating neuronal abnormalities in depression. Our findings provide translational evidence that miR-144-5p is a new potential therapeutic target for MDD.

Keywords: depression; miR-144-5p; neuroinflammation; neuronal destruction.

FIGURE 1

miR‐144‐5p is downregulated in CUS mice and validation of miR‐144‐5p target genes. (A) Heatmap of differentially expressed miRNAs in the hippocampus of CUS mice compared with the control group. n = 3 in each group. Fold change ≥1.5, p < 0.05. (B) The levels of miR‐144‐5p, miR‐211‐5p, miR‐33‐3p, and miR‐466b‐3p in the hippocampus. n = 8 in each group. The levels of miR‐144‐5p in CA1 (C) and DG (D). n = 8 in each group. *p < 0.05, **p < 0.01, ***p < 0.001 versus Ctrl. (E) Enrichment analysis of miR‐144‐5p target genes with KEGG. (F) Bioinformatical prediction of miR‐144‐5p target genes. (G,H) Results of putative seed‐matching sites and luciferase reporter assays verified on 293 T cells. n = 3 in each group. *p < 0.05, ***p < 0.001 versus WT+NC, ^## p < 0.01, ^### p < 0.001 versus WT+ mmu‐miR‐144‐5p.

FIGURE 2

Upregulation of miR‐144‐5p rescues depression‐like phenotypes in CUS mice. (A) Experimental paradigm for AAV‐miR‐144‐5p intervention. (B) Schematics of AAV‐miR‐144‐5p. (C) The infection efficiency of AAV‐miR‐144‐5p. n = 6 in each group. **p < 0.01 versus WT/Uninjected group, ^## p < 0.01 versus S/AAV‐Ctrl group. (D–H) Behavioral effects of expressed AAV‐miR‐144‐5p in the DG. Upregulation of miR‐144‐5p relieved depressive‐like phenotypes in CUS mice as measured by the SPT (D), TST (E), FST (F), and OFT (G,H). n = 24 in each group. **p < 0.01, ***p < 0.001 versus WT/Uninjected group, ^# p < 0.05, ^## p < 0.01 versus S/AAV‐Ctrl group.

FIGURE 3

Upregulation of miR‐144‐5p promotes neuronal neurogenesis in the DG. (A) Representative bands acquired from an experiment of TLR4, PTEN, PI3K, p‐Akt, and p‐p65. (B) Overexpression of miR‐144‐5p reduced levels of TLR4, PTEN, and p‐p65 while reversing decrease in PI3K and p‐Akt induced by CUS. n = 6 in each group. ***p < 0.001 verus WT/Uninjected group, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus S/AAV‐Ctrl group. (C–F) Representative photomicrographs showing and DCX⁺ and Nestin⁺ cells in the DG. Scale bar: 50 μm. n = 6 in each group. **p < 0.01, ***p < 0.001 verus WT/Uninjected group, ^# p < 0.05 versus S/AAV‐Ctrl group.

FIGURE 4

Upregulation of miR‐144‐5p rescues neuronal apoptosis and synaptic plasticity impairments and abrogates neuroinflammatory responses in CUS mice. (A) Representative bands acquired from an experiment of Bcl‐2 and Bax. (B) Overexpression of miR‐144‐5p increased levels of Bcl‐2 while preventing increase in Bax induced by CUS. n = 6 in each group. ***p < 0.001 versus WT/Uninjected group, ^# p < 0.05, ^## p < 0.01 versus S/AAV‐Ctrl group. (C) Representative bands acquired from an experiment of SYP and PSD95. (D) Overexpression of miR‐144‐5p reversed the reduction in the levels of SYP and PSD95 induced by CUS. n = 6 in each group. ***p < 0.001 versus WT/Uninjected group, ^## p < 0.01, ^### p < 0.001 versus S/AAV‐Ctrl group. (E) Representative images of the dendritic spine in Golgi staining. Scale bar: 2 μm. (F) Overexpression of miR‐144‐5p alleviated synaptic plasticity impairment induced by CUS. n = 6 in each group. ***p < 0.001 versus WT/Uninjected group, ^### p < 0.001 versus S/AAV‐Ctrl group. (G) Representative bands acquired from an experiment of iNOS. (H) Overexpression of miR‐144‐5p suppressed levels of iNOS. n = 6 mice per group. ***p < 0.001 versus WT/Uninjected group, ^### p < 0.001 versus S/AAV‐Ctrl group. Levels of cytokines such as TNF‐α (I), IL‐6 (J), and IL‐1β (K) were detected by ELISA. n = 6 mice per group. ***p < 0.001 versus WT/Uninjected group, ^## p < 0.01, ^### p < 0.001 versus S/AAV‐Ctrl group. (L) Representative photomicrographs of CD86/Iba1 staining. Scale bar: 50 μm.

FIGURE 5

PI3K/Akt/FoxO1 signaling is involved in behavioral dysfunctions caused by miR‐144‐5p deficiency. (A) Representative bands acquired from an experiment of PTEN, PI3K, p‐Akt, p‐FoxO1, TLR4, and p‐p65. (B) Administration of bpV(pic) increased PI3K, p‐Akt, and p‐FoxO1 and decreased p‐p65 expression in mice with knockdown miR‐144‐5p. LY294002, as an inhibitor of PI3K, reversed the increase in p‐FoxO1 expression and restored the reduction in TLR4 and p‐p65 expression induced by bpV(pic) effect. n = 6 in each group. **p < 0.01 versus Vehicle group, ^# p < 0.05, ^## p < 0.01 versus BPV group. (C–G) Behavioral evaluation in the SPT (C), TST (D), FST (E) and OFT (F,G). n = 18 in each group. **p < 0.01 versus Vehicle group, ^# p < 0.05, ^## p < 0.01 versus BPV group.

FIGURE 6

miR‐144‐5p is downregulated in MDD patients. (A) Levels of miR‐144‐5p were decreased in the serum of patients with MDD (n = 24) compared with HC subjects (n = 24). **p < 0.01 versus HC. (B) ROC analysis of miR‐144‐5p to separate MDD patients from HC subjects. (C) Correlation between miR‐144‐5p levels and HAMD‐24 score utilizing Pearson's correlation analysis. (D) Correlation between miR‐144‐5p levels and HAMA score utilizing Pearson's correlation analysis.