| Background:Major depressive disorder(MDD)is a severe mental illness characterized by low mood,cognitive impairment,and impaired social function.Depression can be triggered by a variety of factors with overlapping causal pathways,and its pathogenesis has not been completely elucidated so far.Due to lack of reliable biomarkers,depression is difficult to be diagnose and has a high rate of misdiagnosis.Furthermore,antidepressant drugs also have problems such as slow action,obvious side effects,and poor therapeutic effects.Therefore,it is important to deeply study the pathogenesis of depression and find reliable biomarkers and therapeutic targets for clinical practice.Due to their closed-loop structure,circular RNAs(circRNAs)are more stable and have a longer half-life than its linear transcripts in cells.Their most common and currently widely used function is as micro RNA(miRNA)sponges,and several studies have reported that circRNAs are involved in the development and progression of disease by targeting to miRNAs.Transcriptome analysis has revealed aberrant expression of circRNAs in peripheral blood in humans and animal models of depression.The association of miRNAs with depression has been widely reported.Previous studies found that serum miR-182-5p expression was upregulated in depressed patients and negatively correlated with brain-derived neurotrophic factor(BDNF).Animal studies also revealed that the up-regulation of hippocampal miR-182-5p was associated with a decrease in BDNF in the chronic unpredictable mild stress(CUMS)model of rats.Moreover,dual luciferase assays confirmed that BDNF could bind to miR-182-5p.However,these studies did not clarify the specific phenotypes through which depression is affected by miR-182-5p via targeting BDNF and whether circRNA could be involved in the pathogenesis of depression as a miR-182-5p sponge.BDNF is a peptide growth factor whose signaling pathway is involved in the pathogenesis of depression.Decreased BDNF expression and signaling can impair or hinder neuroplasticity,promote neuronal atrophy and a decrease in the number and function of synapse,which induces depression-like behavior.Synaptophysin(SYP),growth-associated protein 43(GAP43)and postsynaptic density protein 95(PSD95)are proteins that are closely associated with synaptic transmission.Their expressions were reduced in depressed patients and animal models of depression,and increased after treatment.The hippocampus is engaged in mood regulation and is a critical brain region for the development of depression.Previous studies have found that neuroplasticity impairments of depression are particularly common in the hippocampus.Given the structural stability,miRNA sponges and association with depression of circRNAs,we desired to investigate their expression and role in depression,and whether circRNA plays a role in depression via the miR-182-5p/BDNF axis regulating synaptic plasticity.Our study was conducted in the following three parts.Part 1 Peripheral blood circPTK2 expression in patients with depression Objective:In order to identify the clinical characteristics and the expression level of circRNAs in peripheral blood of MDD patients,and select the significantly expressed circRNA.to evaluate the ability of circRNA to discriminate depression,and to analyze the relationship between circRNA and severity of disease,suicidal tendency.Methods:Adult patients with MDD admitted to the psychological clinic of Changchun Sixth Hospital from June 24,2020 to April 21,2021 were collected as the case group,while healthy people who underwent physical examination in the First Hospital of Jilin University from September 16,2020 to March 30,2021 were recruited as the control group.MDD was diagnosed by two psychiatrists according to DSM-5,and the case and control groups both met the inclusion criteria.Sociodemographic and medical history data of the subjects were collected,and their morning fasting venous blood in 2ml was also collected to isolate peripheral blood mononuclear cells(PBMCs).The circRNAs expression in PBMCs of depressed patients and controls was detected by fluorescence quantitative PCR,and significantly expressed circRNA were selected.The discriminatory ability of circRNA for depression was evaluated by the receiver operating characteristic(ROC)curve,and the relationship between circRNA and the severity of depression and suicidal tendency was analyzed.Results:A total of 50 patients with MDD and 40 age-and sex-matched healthy controls were included.The mean age of onset of MDD was 31.86 years,with 41 patients(82.0%)with early onset of adulthood(18-44 years).Depression was more prevalent in females(62.0%),and students with depression accounted for a proportion(18.0%)over18 years old.Patients tend to have a high level of education and a high percentage of non-only children.The mean 24-item Hamilton Depression Scale(HAMD-24)score was 40.60 points,and 60.0% of the patients were severely depressed and 80.0% had suicidal tendencies.The mean Hamilton Anxiety Scale(HAMA)score was 25.86 points,and 47 patients(94.0%)had definite anxiety.Bioinformatic analysis revealed that human circ USP42(hsa＿circ＿0007823),circPTK2(hsa＿circ＿0002483),circ KIAA0907(hsa＿circ＿0000140)could bind with human miR-182-5p,and they all had mouse homologous sequences(note: homologous mouse circRNAs were also able to bind to mouse miR-182-5p).Real-time fluorescence quantitative PCR revealed that the expression of circPTK2 and circ KIAA0907 was significantly reduced in the peripheral blood of MDD patients(n = 31)compared to controls(n = 24-25),while the expression of circ USP42 was not significantly reduced.ROC curve analysis revealed that circPTK2 was better than circ KIAA0907 in recognizing MDD.Among the three circRNAs,circPTK2 with the greatest expression difference,the highest abundance and the better identification of MDD was selected for our follow-up study.After expanding the sample size,the expression of circPTK2 in MDD patients(n = 50)was still significantly lower than that in healthy controls(n = 40).ROC curve showed that circPTK2 had a good discriminatory ability for depression.Correlation analysis revealed that circPTK2 expression was not associated with HAMD-24 and HAMA scale scores,but negatively correlated with the suicide scores of cognitive impairment factor and the day-night variability factor in the HAMD-24 assessment.Conclusions:Circ PTK2 and circ KIAA0907 are significantly reduced in MDD patients.Circ PTK2 is involved in the pathogenesis of depression and may be a peripheral biomarker of depression.Part 2 The establishment and behavioral analysis of CUMS mice modelObjective:To determine whether the CUMS depression model of mice was successfully established,the duration of depression-like behavior,to evaluate the applicability of behavioral tests in CUMS,and to select appropriate behavioral assessment methods.Methods:C57BL/6 mice were randomly assigned into the CUMS group and the control group.Mice in the CUMS group were randomly exposed to various low-intensity social and environmental stressors 2-3 times per day for 4 weeks.The mice in the control group were housed normally without any treatment.After CUMS modeling,both groups were subjected to behavioral tests for 1-3 weeks to observe the results of CUMS model and the duration of depressive symptoms.Behavioral tests included: sucrose consumption test(SCT),which calculated the sucrose consumption as a percentage of total fluid intake;novelty suppressed feeding test(NSFT),which recorded the latency to feed and the food consumption in five minutes;tail suspension test(TST),which counted the immobility time of mice during the last 4 minutes;forced swimming test(FST),which counted the immobility time of mice during the last 4 minutes;open field test(OFT),the total distance or average speed and the time spent in center of mice were counted.Results:After CUMS modeling,a total of 10 CUMS mice and 7 control mice were subjected to behavioral tests for 3 weeks.At the first week after CUMS modeling,there were no statistical differences between the two groups in TST,the food consumption in five minutes of NSFT,the total distance and time spent in center of OFT,and SCT.However,CUMS mice showed longer latency to feed of NSFT and longer immobility time of FST than control mice.At second weeks after CUMS modeling,CUMS mice showed the latency to feed of NSFT and immobility time of FST were still longer than the control group.In addition,the mice with CUMS had less food consumption in five minutes of NSFT compared to the control group.However,there was still no statistical difference between the two groups in TST.At the third week after CUMS modeling,no statistical differences were observed between two groups in TST,the latency to feed of NSFT,and the total distance of OFT,and an approximate statistical difference in the time spent in center of OFT.The CUMS mice had less food consumption in five minutes of NSFT,longer immobility time of FST,and less sucrose consumption of SCT than the control group.Conclusions:We successfully established a depression model of CUMS in mice,which could sustain depression-like behavior for more than 3 weeks.FST,OFT,and NSFT were applicable to the behavioral assessment of CUMS mice,and SCT and TST were not applicable.Part 3 The role and mechanism of circPTK2 in a mouse depression model Objective:To investigate the expression levels of circPTK2,miR-182-5p,BDNF,and neuroplasticity-related proteins in the hippocampus of CUMS mice.To identify whether circPTK2 is involved in depression pathogenesis through the circPTK2/miR-182-5p/BDNF axis that regulates hippocampal synaptic plasticity.Methods:C57BL/6 mice were randomly divided into the CUMS and control groups,and behavioral tests were performed to assess the behavior of both groups.The expression levels of circPTK2(mmu＿circ＿0005779),miR-182-5p in hippocampus of CUMS mice and control mice were measured by fluorescence quantitative PCR.The discriminative power of circPTK2 for depression-like behavior in mice was assessed by the ROC curve,and the correlation between circPTK2 and behavioral results was analyzed.RNase R and Actinomycin D experiments were performed to determine the stability of circPTK2.The circ Control-enhanced green fluorescent protein(EGFP)adeno-associated virus(AAV)and circPTK2-EGFP AAV were constructed and injected into the hippocampus of C57BL/6 mice by stereotaxic injection technique.After 3-4 weeks,when the transcriptional expression of AAV gene reached its peak,immunofluorescence staining was performed to observe whether the hippocampal injection position was correct and whether AVV co-localized with neuronal cells.Fluorescence quantitative PCR was used to measure whether circPTK2-EGFP AAV overexpressed circPTK2.The behavioral tests were conducted to observe the behavior of each group of mice before and after hippocampal stereotaxic injection of circ Control-EGFP AAV and circPTK2-EGFP AAV into CUMS mice and control mice.After AAV injection,fluorescence quantitative PCR was performed to measure miR-182-5p expression,and dual luciferase assays were used to validate circPTK2 could bind to miR-182-5p.Western Blot was carried out to determine the expressions of BDNF,SYP,GAP43,and PSD95.Results:Before the beginning of CUMS,there was no difference in body weight between the CUMS group(10 mice)and the control group(8 mice).After the first,second,third and fourth weeks of CUMS modeling,the body weight of CUMS mice was lighter than that of control mice.After CUMS modeling,behavioral tests revealed that CUMS mice had significantly longer latency to feed of NSFT and immobility time of FST than control mice.The relative expression of circPTK2 in the hippocampus of CUMS mice was significantly lower than that of control mice,while the relative expression of miR-182-5p was significantly higher.ROC curve analysis showed that circPTK2 had good discriminatory ability for depression-like behavior in mice.However,the relative expression of circPTK2 did not correlate with the immobility time of FST and latency to feed of NSFT.Stability experiments revealed that circPTK2 was more resistant to RNase R than PTK2 m RNA;PTK2 m RNA expression gradually decreased with the extension of actinomycin D treatment,while circPTK2 was slightly enriched.Immunofluorescent staining of AAV hippocampal stereotaxic injections showed that both circ Control-EGFP and circPTK2-EGFP AAV mice showed extensive expression of EGFP(green)in the hippocampus,and both were co-located with neuronal cells(red).Fluorescence PCR of hippocampal tissue showed that circPTK2-EGFP mice expressed significantly higher levels of circPTK2 than control mice.Before hippocampal stereotaxic injection of AAV,CUMS mice showed significantly longer immobility time of FST than control mice.After 3-4 weeks of circ Control-EGFP AAV and circPTK2-EGFP AAV injection,the immobility time of FST was still significantly increased in CUMS mice compared to control mice,and significantly decreased after administration of circPTK2 AAV compared to the CUMS mice after administration of circ Control AAV.The average speed of OFT was slowed in CUMS mice compared to control mice,and was increased after overexpression of circPTK2.The dual luciferase assay confirmed that circPTK2 could bind to miR-182-5p.Fluorescence quantitative PCR revealed that miR-182-5p expression in hippocampal tissue of CUMS mice was significantly increased,and was decreased after microinjection of circPTK2 AAV in hippocampus.Western Blot showed that the expression level of BDNF in hippocampus of CUMS mice was significantly decreased compared with that of control mice,and was significantly increased after overexpression of circPTK2.In addition,the expression of SYP,GAP43 and PSD95 in the hippocampus of CUMS mice were also significantly decreased,and they were significantly increased after overexpression of circPTK2.Conclusions:Circ PTK2 expression was decreased in the hippocampus of CUMS mice,and overexpression of circPTK2 could improve depressive-like behavior in mice.Circ PTK2 ameliorates depressive-like behavior via the miR-182-5p/BDNF axis regulating hippocampal synaptic plasticity. |
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