MiR-221 is involved in depression by regulating Wnt2/CREB/BDNF axis in hippocampal neurons

Abstract

Objective: The aim of this study was to investigate the mechanism of miR-221 in depression.

Methods: The molecules expressions were measured by qRT-PCR and western blot. The sucrose preference test (SPT), forced swimming test (FST) and tail suspension test (TST) were used to detect depressive-like symptoms. MTT assay and flow cytometric was used to measure the proliferation and apoptosis of hippocampal neuronal.

Results: MiR-221 expression in the cerebrospinal fluid and serum of major depressive disorder patients and the hippocampus of chronic unpredictable mild stress (CUMS) mice were increased, while the expression of Wnt2, p-CREB and BDNF were decreased. Additionally, silence of miR-221 increased sucrose preference of CUMS mice and shortened the immobility time of CUMS mice in SPT and FST. MiR-221 could targeted regulate Wnt2, and knockdown of Wnt2 reversed the effect of miR-221 inhibitor on the proliferation and apoptosis of hippocampal neurons and countered the promoting effect of miR-221 inhibitor on the expression of Wnt2, p-CREB and BDNF.

Conclusion: MiR-221 could promote the development of depression by regulating Wnt2/CREB/BDNF axis.

Keywords: Major depressive disorder; Wnt2/CREB/BDNF; miR-221.

Figures

Figure 1.

The expression of miR-221 in MDD patients and hippocampus of CUMS mice. (a) The expression of 6 miRNAs in CSF in MDD patients. (b) The expression of 6 miRNAs in serum in MDD patients. (c) SPT. (d) FST. (e) TST. (f) The content of CORT in the serum of CUMS mice. (g) miR-221 expression in hippocampus of CUMS mice. (h) The protein expression of Wnt2, p-CREB and BDNF in hippocampus of CUMS mice. **P < 0.01 vs control.

Figure 2.

Effects of miR-221 on the proliferation and apoptosis of hippocampal neurons in mice. Hippocampal neurons of neonatal mice were transfected with miR-221 mimic. (a) miR-221 expression. (b) The proliferation of hippocampal neuronal. (c) The apoptosis of hippocampal neuronal. Hippocampal neurons of neonatal mice were transfected with miR-221 mimic. (d) miR-221 expression. (e) The proliferation of hippocampal neuronal. (f) The apoptosis of hippocampal neuronal. **P < 0.01 vs pre-NC or NC.

Figure 3.

MiR-221 negatively regulated Wnt2. (a) The binding site between miR-221 and Wnt2. (b) Relative luciferase activity of Wnt2 3’-UTR in HEK293 cells transfected with miR-221 mimic. (c) Relative luciferase activity of Wnt2 3’-UTR in HEK293 cells transfected with miR-221 inhibitor. Hippocampal neurons were transfected with miR-221 mimic or miR-221 inhibitor. (d) Wnt2 protein expression. (e) p-CREB protein expression. (f) BDNF protein expression. **P < 0.01vs pre-NC or NC.

Figure 4.

Knockdown of Wnt2 reversed the effect of miR-221 inhibitor on the proliferation and apoptosis of hippocampal neurons. Hippocampal neurons were divided into NC group, miR-221 inhibitor group, miR-221 inhibitor+si-NC group, and miR-221 inhibitor+si-Wnt2 group. (a) The proliferation of hippocampal neuronal. (b) The apoptosis of hippocampal neuronal. (c) The protein expression of Wnt2, p-CREB and BDNF. **P < 0.01 vs NC, ##P < 0.01 vs miR-221 inhibitor+ si-NC. Hippocampal neurons were divided into NC group, miR-221 mimic group, miR-221 mimic+Lenti-NC group, and miR-221 mimic+Lenti-Wnt2 group. (d) The proliferation of hippocampal neuronal. (e) The apoptosis of hippocampal neuronal. (f) The protein expression of Wnt2, p-CREB and BDNF. **P < 0.01 vs NC, ##P < 0.01 vs miR-221 mimic+Lenti-NC.

Figure 5.

Silence of miR-221 alleviated depressive-like symptoms in CUMS mice. Mice were divided into 4 groups (control group, CUMS group, CUMS+antagomir-control group, and CUMS+miR-221 antagomir group). MiR-221 antagomir was used to inhibit miR-221 expression. (a) SPT. (b) FST. (c) TST. (d) The content of CORT. (e) miR-221 expression. (f) The protein expression of Wnt2, p-CREB and BDNF. **P < 0.01 vs control, ##P < 0.01 vs CUMS+antagomir-ctr.

Figure 6.

Overexpression miR-221 reversed the treatment of fluoxetine on CUMS mice. Mice were divided into 3 groups, including CUMS group, CUMS+fluoxetine group, and CUMS+fluoxetine+miR-221 agomir group. MiR-221 agomir was used to up-regulate miR-221 expression. (a) SPT. (b) FST. (c) TST. (d) The content of CORT. (e) miR-221 expression. (f) The protein expression of Wnt2, p-CREB and BDNF. **P < 0.01 vs CUMS, ##P < 0.01 vs CUMS+ fluoxetine.