Role And Mechanism Of MiR-144-5p In The Pathogenesis Of Depression

Background: Depression is a common psychosomatic disorder that manifests as a lack of pleasure,behavioral despair and depressed mood.The onset of depression is associated with genetic and environmental factors,with stress being an important risk factor for the development of depression.The clinical diagnosis of depression relies heavily on subjective affective scales and lacks reliable clinical biomarkers.The main treatment is currently pharmacological,with antidepressants having a slow onset and limited effect in clinical treatment.The highly heterogeneous presentation of depression illustrates the complexity of the development of the disorder.Current research suggests that neurogenesis,synaptic plasticity,and neural damage are important pathological mechanisms of depression.As a class of small non-coding RNAs,micro RNAs(miRNAs)can regulate the expression of genes.The changes in miRNA diversity in brain tissue,serum and cerebrospinal fluid of depressed patients further suggest that miRNAs may be involved in the pathological mechanisms of depression.Based on this,the molecular mechanism of the pathogenesis of depression related to miRNA remains to be studied.In this study,we constructed a chronic unpredicted stress(CUS)-induced depression model in mice to investigate the mechanism of miR-144-5p in the hippocampal dentate gyrus(DG)that regulates depression-like behavior in mice,providing a potential target for the treatment of depression.Methods:(1)RNA sequencing was performed to analyze the differentially expressed miRNAs in the hippocampus of the CUS group mice compared to the normal control group.RNA sequencing was performed to analyze the differentially expressed miRNAs in the hippocampus of CUS mice compared to normal controls.q PCR validation,bioinformatic analysis,target gene prediction and dual luciferase were used sequentially to investigate whether miR-144-5p is involved in regulating the pathogenesis of depression and to predict its downstream targets,PTEN and TLR4.(2)Adeno-associated viruses overexpressing miR-144-5p were constructed and the corresponding viruses were injected into the hippocampal DG region of each group of mice by brain stereotaxic localization,and behavioral assessments were performed two weeks later.Using q PCR to detect miR-144-5p levels,immunofluorescence staining to detect neurogenesis indicators,western bolt to detect target gene signaling pathways,Golgi staining to detect synaptic plasticity and ELISA to detect inflammatory factor indicators.(3)Adeno-associated viruses with knockdown miR-144-5p were constructed and the corresponding viruses were injected into the hippocampal DG region of each group of mice by brain stereotaxic localization,and behavioral assessments were performed two weeks later.Using q PCR to detect miR-144-5p levels,immunofluorescence staining to detect neurogenesis indicators,western bolt to detect target gene signaling pathways,Golgi staining to detect synaptic plasticity and ELISA to detect inflammatory factor indicators.(4)The mice with miR-144-5p knockdown were treated with PTEN inhibitor bp V(pic)or combined with PI3K/Akt signaling inhibitor LY294002,and the behavioral evaluation and target gene pathway indexes were detected by western blot.(5)q PCR was used to detect the levels of serum and exosomal miR-144-5p in normal healthy individuals and MDD patients.Results:(1)RNA sequencing analysis revealed 17 differentially expressed miRNAs in the hippocampal brain region of CUS mice compared to normal mice.The expression of miR-144-5p was significantly downregulated in the hippocampal brain region of CUS mice.Bioinformatic analysis revealed that miR-144-5p plays an important role in synaptic plasticity,which is an important pathophysiological mechanism of depression.The downstream target genes of miR-144-5p were also predicted by the target gene prediction software Target Scan and miRWalk.Compared to normal mice,CUS mice showed reduced levels of miR-144-5p in both hippocampal CA1 and DG regions,an important brain region for studying the pathogenesis of depression and a significant decrease in miR-144-5p levels in this region,which was used as a follow-up region.Dual luciferase assay identified that miR-144-5p can target the 3’UTR region that binds PTEN and TLR4.(2)Overexpression of miR-144-5p levels in the hippocampal DG region of depressed mice improved depression-like behaviors compared to controls:increased the percentage of sucrose consumption,reduced resting time of tail suspension and forced swimming,and increased the total distance of activity;promoted neurogenesis: increased the number of DCX-positive cells in the DG region(promoting differentiation of neural stem cells to neurons)and Nestin-positive cells(promoting neural stem cell proliferation);inhibited apoptosis: increased Bcl-2 and suppressed Bax levels;improved synaptic plasticity: increased SYP,PSD95 levels and dendritic spine density;reduced neuroinflammation: decreased i NOS,TNF-α,IL-6 and IL-1β levels and inhibited M1 polarization.(3)Knockdown of miR-144-5p levels in the DG region of the hippocampus of normal mice induced depression-like behaviors compared to controls: decreased the percentage of sucrose consumption,increased the resting time of tail suspension and forced swimming,and decreased the total distance of activity;inhibited neurogenesis: decreased the number of DCX-positive cells in the DG region(inhibited differentiation of neural stem cells to neurons)and the number of Nestin-positive cells(inhibited neural stem cell proliferation);promoted apoptosis:decreases Bcl-2 and increases Bax levels;impaired synaptic plasticity: decreased SYP,PSD95 levels and dendritic spine density;promoted neuroinflammation: increased levels of i NOS,TNF-α,IL-6 and IL-1β and promoted M1 polarization.(4)Behavioral assessment showed that bp V(pic)treatment significantly improved depression-like behaviors caused by miR-144-5p knockdown: increased percentage of sucrose consumption,decreased resting time in tail suspension and forced swimming,and increased total distance of activity compared to controls.Combined LY294002 treatment reversed this treatment effect: decreased the percentage of sucrose consumption,increased resting time of tail suspension and forced swimming,and decreased the total distance of activity.Western blot results showed that bp V(pic)treatment increased PI3 K,p-Akt,p-Fox O1 levels and decreased TLR4,p-p65 levels.Combined LY294002 treatment reversed the changes in the levels of p-Akt,p-Fox O1,TLR4 and p-p65 caused by bp V(pic).In miR-144-5p knockdown mice,triggering of TLR4 signaling was associated with elevated PI3K/Akt/Fox O1 activation.(5)The results of peripheral blood q PCR showed that miR-144-5p levels were decreased in the serum of CUS mice compared to normal mice,and in depressed patients compared to normal healthy individuals.(5)The q PCR results of peripheral blood showed that miR-144-5p levels were decreased in the serum of CUS mice compared to normal mice,and in MDD patients compared to normal healthy individuals.ROC curve analysis showed an area under the curve of 0.741(95% CI,0.602-0.881),specificity 0.667 and sensitivity 0.792.Results of the symptom scale assessment showed that decreased levels of miR-144-5p were associated with depressive symptom severity: HAMD-24(r =-0.4426,p = 0.03),HAMA(r =-0.428,p = 0.04).Levels of the serum-derived exosome miR-144-5p were also significantly decreased in MDD patients compared to normal healthy individuals.Conclusion:(1)CUS induced depressive-like behaviors in mice,accompanied by neurogenesis,neuronal damage and synaptic plasticity impairment in the DG region of the hippocampus.(2)Overexpression of miR-144-5p levels in the hippocampal DG region of depressed mice alleviated depression-like behavior in mice,accompanied by increased neurogenesis,alleviated neuronal damage,and reduced impairment of synaptic plasticity.(3)Knockdown of miR-144-5p in the hippocampal DG region of normal mice induced depressive-like behavior and led to decreased neurogenesis,neuronal damage and synaptic plasticity impairment.(4)The damage caused by miR-144-5p reduction in the hippocampal DG region of mice is mediated by PI3K/Akt/Fox O1 signaling.(5)The expression of miR-144-5p in peripheral serum of MDD patients is decreased,and this level is negatively correlated with the severity of depression.