Function And Mechanism Of Ultra-Conserved LncRNA Uc.173-in Regulating Microglial Activation During Depression

Background:Depression is a neuropsychiatric disease characterized by persistent and spontaneous feelings of sadness with an increasing prevalence worldwide,which is the important cause of disability and suicide.At present,about one-third of clinical patients do not respond well to standard antidepressant treatment,indicating that much remains unknown about the pathogenesis of depression.Recent studies have found the important role of neuroimmune mechanisms in the pathology of depression.As resident immune cell in the central nervous system,microglia perform various functions such as immune surveillance,phagocytosis,synaptic pruning,and secretion of cytokines and chemokines.Previous studies have observed microglial activation and neuroinflammation in the brains of depressed patients and animal models of depression,while elimination of microglia or inhibiting the activation of microglia could alleviate depressive symptoms,suggesting that the regulation of neuroimmunological abnormalities mediated by microglia may be an effective therapeutic target for depression.Long non-coding RNAs have been shown to play a role in regulating the function of microglia in a variety of neurological diseases,therefore,it is also likely to play an important role in the neuroimmune mechanism of depression.Currently,the researches on lncRNA in depression are still limited.This study intends to provide a new experimental basis for the pathogenesis and therapeutic targets of depression by studying the regulatory role of key lncRNA molecules in neuroimmune response of depression.Objectives:Part Ⅰ:To explore the expression of lncRNA uc.173-in patients with depression and animal models of Depression;Part Ⅱ:To explore the effect of lncRNA uc.173-on the microglial activation and depression-like behavior in mice;Part Ⅲ:To explore the regulatory effect of lncRNA uc.173-on UBE2b and the role of UBE2b in the inflammatory response of microglia.Methods:Part Ⅰ:（1）Patients with clinically diagnosed depression and healthy controls were included,and their baseline clinical data and peripheral blood samples were collected;（2）Enzyme-linked immunosorbent assay（ELISA）was performed to detect the levels of serum inflammatory cytokines;（3）The correlations between the severity of depression and the levels of inflammatory cytokines were analyzed;（4）Peripheral monocytes were isolated by magnetic bead sorting,and differential expression genes between two groups were screened by transcriptome sequencing;（5）Primary microglia cells were extracted and activated to the anti-inflammatory type,then the differential genes were screened by microarray;（6）After constructing models of lipopolysaccharide（LPS）injection and chronic restraint stress（CRS）,flow cytometry was used to isolate microglia of depressed mice,and quantitative polymerase chain reaction（QPCR）was performed to detect the expression of differential gene.Part Ⅱ:（1）LncRNA uc.173-was overexpressed and knocked down by lentivirus and primary microglia were transfected,then the m RNA expression of inflammatory markers of microglia after uc.173-overexpression and knockdown was detected by QPCR;（2）CX3CR1^{cre ER}:uc.173-^shmice were constructed and the knockdown efficiency of uc.173-in microglia was verified by RNAscope after tamoxifen induction;（3）Tail suspension test,forced swimming test,sucrose preference test,light/dark box test,open field test and Y maze were used to detect the effect of lncRNA uc.173-conditional knockdown on behavior changes of mice after the construction of LPS and CRS model;（4）Immunofluorescence（IF）and ELISA were performed to detect the microglial activation and the detection of inflammatory factors in medial prefrontal cortex（m PFC）and hippocampus of depressed mice after conditional knockdown of lncRNA uc.173-;（5）IF and western blotting（WB）were used to detect the synaptic damage,expression of neurotrophic factors and hippocampal neurogenesis in depressed mice after conditional knockdown of lncRNA uc.173-.Part Ⅲ:（1）Dual-luciferase reporter gene assay was performed to detect the position relationship between uc.173-and UBE2b;（2）The regulatory effect of uc.173-on UBE2b was detected by QPCR;（3）QPCR and WB were used to detect the expression of UBE2b after microglia activation;（4）After transfected with UBE2b overexpressing and interfering lentivirus,the expression level of inflammatory markers in microglia were detected using QPCR.Results:Part Ⅰ:（1）A total of 107 subjects were enrolled,including 51 healthy controls and 57patients with depression,the scores of hamilton depression scale were significantly different between two groups,while no significant differences in baseline clinical data such as age,sex,body mass index and education level were observed;（2）Compared with healthy controls,the levels of IL-1β,TNF-αand IL-6 were significantly increased and the level of IL-4 was significantly decreased in the serum of depression cases;（3）The spearman correlation analysis showed that the levels of IL-1β,TNF-αand IL-6 were positively correlated with the severity of depression,while the level of IL-4 was negatively correlated with the severity of depression;（4）Peripheral monocytes of subjects from two groups were isolated and transcriptome sequencing indicated that the expression of lncRNA uc.173-was decreased in the monocytes of depressed patients;（5）Microarray screening results showed that the expression of lncRNA uc.173-in microglia was significantly up-regulated after the activation to anti-inflammatory type,then QPCR confirmed the results of transcriptome sequencing and microarray screening.Pearson correlation analysis indicated a significant positive correlation between the level of uc.173-in monocytes and IL-4 level in serum;（6）Flow cytometry was used to isolate microglia from the brain of LPS and CRS modeling mice,and QPCR observed that the expression level of lncRNA uc.173-in microglia was significantly decreased in depressed mice.Part Ⅱ:（1）lncRNA uc.173-was overexpressed and knocked down by lentivirus in primary microglia,QPCR showed that the expressions of Arg1,Ym-1,Fizz1 and CD206were significantly up-regulated and the expressions of TNF-α,IL-1βand inducible nitric oxide synthase（i NOS）were significantly downregulated after the overexpression of lncRNA uc.173-,while the expressions of arginase 1（Arg1）,chitinase-3-like protein 3（Ym-1）,resistin-like molecule alpha（Fizz1）and CD206 were significantly downregulated and the expressions of TNF-α,IL-1βand i NOS were significantly upregulated after the knockdown of lncRNA uc.173-;（2）CX3CR1^{cre ER}:uc.173-^sh mice were constructed,and RNAscope staining showed that the expression of uc.173-in microglia cells was decreased after the induction of tamoxifen;（3）Tail suspension teat and forced swimming test showed that the immobility time in CX3CR1^{cre ER}:uc.173-^sh group after LPS injection was significantly longer than that in uc.173-^sh group;IF showed that compared with the uc.173-^sh group,the amount of microglia was significantly increased and the process of microglia was shortened in the medial prefrontal cortex（m PFC）and hippocampus of CX3CR1^{cre ER}:uc.173-^sh mice after LPS injection;ELISA showed that the expressions of pro-inflammatory cytokines TNF-α,IL-1βand IL-6 were significantly increased and the expressions of anti-inflammatory cytokine IL-4 were significantly decreased in m PFC and hippocampus of CX3CR1^{cre ER}:uc.173-^sh LPS group,compared with those in uc.173-^sh LPS group;（4）Tail suspension teat and forced swimming test showed that the immobility time in CX3CR1^{cre ER}:uc.173-^sh group was significantly longer than that in uc.173-^sh group after CRS,sucrose preference test showed that the sucrose preference index of CX3CR1^{cre ER}:uc.173-^shmice was significantly lower than that of uc.173-^sh mice after CRS;IF showed that compared with the uc.173-^sh CRS group,the amount of microglia was significantly increased and the process of microglia was significantly shortened in m PFC and hippocampus of CX3CR1^{cre ER}:uc.173-^sh mice after CRS;ELISA showed that compared with those in uc.173-^sh CRS group,the expressions of pro-inflammatory cytokines TNF-α,IL-1βand IL-6 were significantly increased and the expressions of anti-inflammatory cytokine IL-4 were significantly decreased in m PFC and hippocampus of CX3CR1^{cre ER}:uc.173-^sh CRS group;（5）Compared with uc.173-^sh CRS group,CX3CR1^{cre ER}:uc.173-^sh CRS group significantly decreased synapsin-1 expression in m PFC,BDNF expression in hippocampus,and Brdu⁺cells and Brdu⁺-Neun⁺cells in DG region of hippocampus.Part Ⅲ:（1）Dual luciferase reporter gene assay showed that lncRNA uc.173-was bound to the 3’UTR region of UBE2b;（2）QPCR showed that overexpression of uc.173-in microglia significantly decreased the expression of UBE2b,and knockdown of uc.173-in microglia significantly increased the expression of UBE2b;（3）WB and QPCR showed UBE2b expression was upregulated in pro-inflammatory microglia and down-regulated in anti-inflammatory microglia;（4）After the overexpression of UBE2b,QPCR showed that the m RNA expressions of Arg1,Ym-1,Fizz1 and CD206 in microglia were significantly down-regulated while the m RNA expressions of TNF-α,IL-1βand i NOS were significantly up-regulated,and WB showed that the expression of TNF-αwas significantly increased;after the knockdown of UBE2b,QPCR showed that m RNA expressions of Arg1,Fizz1 and insulin like growth factor-1（IGF1）in microglia were significantly upregulated while m RNA expressions of TNF-α,IL-1βand i NOS were significantly downregulated,and WB showed that the expression of TNF-αwas significantly decreased.Conclusion:The expressions of peripheral proinflammatory factors IL-1β,TNF-αand IL-6 were significantly increased,while the expression of anti-inflammatory factor IL-4 was significantly decreased in patients with depression,which were significantly correlated with the severity of depression.The expression of ultra-conserved lncRNA uc.173-was significantly decreased in peripheral monocyte of patients with depression and in microglia of depressed mice,and significantly increased in anti-inflammatory microglia triggered by IL-4.Knockdown of lncRNA uc.173-expression in microglia in vitro can promote the activation of microglia to pro-inflammatory type,and knockdown of lncRNA uc.173-in vivo can significantly promote the activation of microglia and neuroinflammatory response in m PFC and hippocampus,increase synaptic damage and decrease neurogenesis,which aggravate depression-like behavior in mice after LPS and CRS modeling.In addition,lncRNA uc.173-negatively regulates the expression of UBE2b,and the inhibition of UBE2b expression can promote the anti-inflammatory activation of microglia.This study suggests that lncRNA uc.173-affects the pathological process of depression by regulating microglia activation.