Role Of CircSTAG1 In Astrocyte Dysfunction And Its Regulatory Mechanisms In Major Depressive Disorder

Part Ⅰ Role of circSTAG1 in regulation of depressive-like behaviors Aims: Major depressive disorder(MDD)is a complex and non-organic mental disorder with a high prevalence,recurrence,and disability rate,which is currently the second most disabling disorder in the world.Due to the disadvantages of classic antidepressants such as slow onset,large side effects and individual differences,it is urgent to develop new antidepressants.Circular RNAs(circ RNAs)are special RNA molecules that are highly expressed in the central nervous system.Existing studies have shown that circ RNAs are involved in regulating a variety of human diseases,but the role of circ RNAs in MDD is rarely reported.In order to explore the pathogenesis of depression and seek new drug therapeutic targets for depression,we further investigated the antidepressant effect of circular RNA STAG1(circSTAG1).Methods:(1)circ RNA sequencing was performed to analyze the differences of circ RNA expression between control and chronic unpredictable stimulation(CUS)mouse hippocampus;(2)Real-time quantitative PCR(q PCR)was used to detect the expression of circ RNAs in mouse plasma,whole blood and hippocampus;(3)Expression of circSTAG1 in human plasma and whole blood was tested by q PCR;(4)q PCR was used to detect the expression of STAG1 message RNA(m RNA)in the mouse hippocampus and human peripheral blood samples;(5)Mouse circSTAG1 was overexpressed by lentivirus vector;(6)Mouse circSTAG1 lentivirus was transfected into 293 T cells and the expression of mouse circSTAG1 in 293 T cells was detected by agar gel electrophoresis;(7)Double-ended sequencing of c DNA confirmed the cyclization of exogenous circSTAG1;(8)circSTAG1 lentivirus was microinjected into the hippocampus to observe depressive-like behaviors of mice.Result:(1)The expression of circSTAG1,circ KALRN,circ DOCK-1 and circ DOCK-2 was decreased in the hippocampus of CUS mice,which was consistent with circ RNA sequencing;(2)circSTAG1 was downregulated in hippocampus,plasma and whole blood of CUS mice;(3)circSTAG1 was downregulated in plasma and whole blood of MDD patients;(4)There was no change in the expression of STAG1 m RNA in the hippocampus of CUS mice and in the whole blood of MDD patients;(5)circSTAG1 was effectively overexpressed by circSTAG1 lentivirus in vitro;(6)Overexpression of circSTAG1 in the hippocampus alleviated depressive-like behaviors of CUS mice.Conclusion: The expression of circSTAG1 was decreased in MDD and CUS mouse samples.Overexpression of circSTAG1 in the hippocampus could inhibit depressivelike behaviors of CUS mice.Part Ⅱ circSTAG1 regulated depressive-like behaviors via ALKBH5Aims: The mechanism of circRNAs involving in disease regulation is complex.In order to explore the specific mechanism of the antidepressant effect of circSTAG1,we further searched for downstream molecules of circSTAG1.N6-methyladenosine(m~6A)modification is the most abundant chemical modification inside m RNA and involved in multiple human diseases.However,the relationship between MDD and RNA m~6 A modification is still not clear.The work in this part is beneficial to understand the potential role of circSTAG1 and RNA m~6 A modification in MDD.Investigation of the regulatory relationship between circSTAG1 and RNA m~6 A modification provides new ideas for MDD treatment strategies.Methods:(1)Flow cytometry was used to classify astrocytes,microglia and neurons from mouse brain,and the expression of circSTAG1 was detected by qPCR;(2)Western blot(WB)was used to detect the expression of GFAP,METTL3,METTL14,WTAP,ALKBH5 and FTO in mouse hippocampus;(3)Diaminobenzidine(DAB)staining and sholl analysis were used to test the number and morphology of astrocytes;(4)The level of RNA m~6 A modification was detected by colorimetric method;(5)Astrocytes were transfected with circSTAG1 lentivirus and small interfering RNA(si RNA),subsequently testing the expression of METTL3,METTL14,WTAP,ALKBH5 and FTO by WB;(6)The cat RAPID algorithm predicted the binding region of ALKBH5 and circSTAG1;(7)The interaction between circSTAG1 and ALKBH5 was confirmed by RNA binding protein immunoprecipitation and RNA pull down assay;(8)Cytoplasmic and nuclear separation and immunofluorescence assay were performed to confirm the subcellular distribution of ALKBH5;(9)ALKBH5 short hairpin RNA(sh RNA)lentivirus was constructed and microinjected into mouse hippocampus to observe depressive-like behaviors.Results:(1)The expression of circSTAG1 was significantly decreased in astrocytes of CUS mice;(2)Overexpression of circSTAG1 in the hippocampus alleviated the abnormal state of astrocytes induced by CUS;(3)Overexpression of circSTAG1 in the hippocampus inhibited the decrease of RNA m~6 A level induced by CUS;(4)In vitro,circSTAG1 did not affect the expression of RNA m~6 A methylases and demethylases;(5)circSTAG1 could combine with ALKBH5,but not other m~6 A methylases and demethylases;(6)Deletion of ALKBH5 domain or mutation of circSTAG1 sequence inhibited the combination of ALKBH5 and circSTAG1;(7)circSTAG1 regulated the distribution of ALKBH5 in astrocyte;(8)Microinjection of circSTAG1 sh RNA lentivirus into mouse hippocampus alleviated depressive-like behaviors of CUS mice.Conclusion: Overexpression of circSTAG1 could inhibit the abnormal function of astrocytes induced by CUS.circSTAG1 reduced the ALKBH5 expression in astrocyte nucleus by binding to cytoplasmic ALKBH5,and knockdown the expression of ALKBH5 alleviated depressive-like behaviors of CUS mice.Part Ⅲ circSTAG1/ALKBH5/FAAH regulated astrocyte loss induced by corticosteroneAims: In order to clarify the role of m~6 A methylation modification in MDD andinvestigate the regulatory mechanism of ALKBH5,we seek to explored the downstreamtarget molecules of ALKBH5.This part provides molecular biological evidence for theregulation of ALKBH5 in MDD and new strategies for the treatment of MDD.Methods:(1)The m~6 A modification of transcripts in mouse hippocampus brain was detected by m~6 A sequencing;(2)The expression of ALKBH5 and FAAH was detected by WB;(3)KEGG enrichment analysis was used to screen downstream FAAH;(4)SRAMP predicted the m~6 A modification site of FAAH m RNA;(5)The FAAH m RNA and m~6A-modified FAAH m RNA fragments were detected by q PCR;(6)The effect of ALKBH5 on FAAH m RNA was verified by luciferase reporter assay;(7)Corticosterone was used to simulate astrocyte loss in MDD;(8)CCK8 assay was used to detect the effects of corticosterone and circSTAG1/ALKBH5/FAAH gene interference or overexpression on astrocyte activity.Results:(1)Abnormal m~6 A modification was mainly observed in the 3’ untranslated region(UTR)of m RNA in CUS mouse hippocampus;(2)M6A level of FAAH m RNA was decreased in CUS mouse hippocampus,and knockdown the expression of ALKBH5 increased the m~6 A methylation of FAAH m RNA in 3’ UTR;(3)The expression of FAAH and FAAH m RNA was increased in CUS mouse hippocampus,and knockdown the expression of ALKBH5 inhibited the expression of FAAH and FAAH m RNA;(4)circSTAG1 inhibited FAAH expression by ALKBH5;(5)In the corticosterone model,downregulated circSTAG1 promoted the expression of FAAH through ALKBH5,and aggravated astrocyte death.Conclusion: circSTAG1 upregulated the m~6 A level of FAAH mRNA through ALKBH5,subsequently inhibited the expression of FAAH.Corticosterone regulated astrocyte viability via circSTAG1/ALKBH5/FAAH axis.