| Anxiety disorder is one of the most common mental disorders,and its incidence has been increasing gradually in recent years.Anxiety disorder patients not only have strong inner worries which is inconsistent with the true facts,but also face multiple organ system function damage and even social withdrawal,so anxiety disorder is always an important psychological topic.Currently,benzodiazepines,serotonin reuptake inhibitors and cognitive behavioral therapy are the main treatment methods for anxiety disorders,but the above methods are difficult to meet both the high effectiveness and strong specificity at the same time.The main reason for this phenomenon is that the mechanism of anxiety disorder is not well studied,and the pathophysiological process of anxiety disorder is poorly understood,which hampers the development of new drugs and therapeutic innovation.Research suggests that the pathogenesis of anxiety disorder may involve a very complex dynamic process,Genome-Wide Association Studies(GWAS)also show that most anxiety disorders may be the comprehensive result of small effects of multiple genes.micro RNA(miRNA)in short non-coding RNAs can simultaneously regulate the expression of up to hundreds of genes through their seed sequences,and form a network regulation pattern,which is exactly matched with the multi-gene regulation mechanism of psychosomatic diseases.Studies have shown that the central miRNA mechanism has an important contribution to the functional regulation of stress response,and its disorder may be related to the etiology of anxiety and mood disorders.There have been many studies on miRNA in depression and schizophrenia,but the relationship between anxiety and miRNA remains unclear.How do miRNA play the role in anxiety disorders or what the potential mechanism is remains to be further explored.Clarifying whether miRNA is involved in the pathophysiological process of anxiety disorder and its possible mechanism will provide new ideas for advancing the diagnostic window of anxiety disorder,and updating the drug methods for disease treatment.MethodsMale C57BL/6 mice(6-8 weeks old,22-28g)were randomly divided into two groups:CRS group;Control group.After being adapted to the feeding environment for a week.Chronic restraint stress(CRS)was administered 2 hours a day for 10 consecutive days to construct CRS group model,while the Control group did not perform any operation.Subsequently,behavioral tests were conducted on the two groups of mice through elevated cross maze test,light and dark box test and open field test to verify the effects of the model.After confirming the stable expression of anxiety-like behavior in the mice constructed from this model,subsequent relevant experiments were conducted.Fresh brain tissues of hippocampus,prefrontal lobe and amygdala were collected from two groups of mice for miRNA sequencing,and the absolute value of FC was greater than 2,P< 0.05 was used as the screening criterion,and Edge R analysis was performed on miRNA expression in each brain region.GO functional annotation and KEGG pathway were used to analyze the differentially expressed miRNAs in different brain regions in order to screen key brain regions.After selecting key brain regions,quantitative Real-time PCR(q RT-PCR)detection was performed on the top 5 most significant miRNAs in this selected brain region,so as to confirm the reliability of sequencing results again,the most typical miRNA was then selected for the follow-up intervention experiments based on relevant literature,and experimental data.The miR-128-2-5p was selected as the key target molecule in the amygdala,and the miR-128-2-5p simulator agmoir was injected into the mouse amygdala(±3.00,-1.22,-4.60)with stereoscopic injection apparatus,to up-regulate the expression of miR-128-2-5p,and then the CRS model was constructed.The model construction was grouped into three groups:agmoir group(modeling under over expression of miR-128-2-5p),NC group(modeling only),and Control group(no intervention and no modeling).Anxiety-like behavior was detected by elevated cross maze test,open field test and light and dark box test.The expression changes of downstream target genes of miR-128-2-5 were detected by Western blot(WB)and q RT-PCR.Results1.Changes of miRNA expression in different brain regions of anxiety disorder mice(1)The results of the elevated cross maze,open field and light and dark box tests showed that mice in the CRS group showed more obvious anxiety-like behavior than those in the control group after the chronic restraint stress method was constructed.(2)The expression profiles of miRNA in hippocampus,prefrontal cortex and amygdala were different between the CRS group and control group.GO functional annotation and KEGG pathway analysis showed that the function of miRNA differentially expressed in hippocampus was mainly concentrated in metabolism and cancer-related functions.The prefrontal tissues mainly focused on the glycan biosynthetic pathway,AMPK signaling pathway and cancer-related functions.The miRNAs in amygdala were mainly focused on axon guidance,transcription factor activity,and cell-gated ion channels.2.The regulation of amygdala miR-128-2-5p on anxiety-like behavior in mice(1)q RT-PCR results of key miRNA molecules in amygdala: miR-128-2-5p,miR-10b-5p,miR-200a-3p,miR-200b-3p,and miR-429-3p showed that the difference of miR-128-2-5p was the most significant,which was highly consistent with sequencing results.(2)The expression level of miR-128-2-5p in mouse amygdala was significantly up-regulated by injecting the miR-128-2-5p simulator agmoir into mouse amygdala.(3)The expression level of miR-128-2-5p was up-regulated by injecting the miR-128-2-5p simulator agmoir into the mouse amygdala,and the anxiety-like behavior was significantly reduced.3.Possible mechanism of amygdala miR-128-2-5p in regulating anxiety disorders(1)The downstream target genes of miR-128-2-5p were predicted using miRanda,PITA and RNAhybrid software,and 23 candidate genes were obtained.bmpr2 and irf6 were identified as key target genes.(2)The 3’UTR region of miR-128-2-5p contains binding sites of bmpr2 and irf6,and dual luciferase reporting method has verified the targeting binding effect of bmpr2,irf6 and miR-128-2-5p.(3)Upregulated expression of miR-128-2-5p in the amygdala by injection of miR-128-2-5p simulator agmoir in mice significantly reduced the expression levels of bmpr2 and irf6 genes and proteins in amygdala.Conclusion1.Chronic restraint stress model can be used to create anxiety model mice.Differential miRNA function and pathway enrichment in the amygdala of mice in the anxiety model group are highly correlated with that in anxiety disorders,suggesting that the amygdala may be an important brain region regulating anxiety-like behaviors.2.Up regulate the expression of amygdala miR-128-2-5p can reduce anxiety-like behavior in mice,suggesting that amygdala miR-128-2-5p may be a key molecule in the regulation of anxiety disorders.3.After up-regulation of miR-128-2-5p expression in the amygdala,the expression of target genes bmpr2 and irf6 and its protein in the amygdala was significantly decreased,suggesting that miR-128-2-5p may affect anxiety-like behavior by regulating the transcription of target genes bmpr2 and irf6. |
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