Study On The Role And Mechanism Of CircDYM/CEBPB/ZC3H4 Axis In LPS-induced Microglial Apoptosis

Objective: Microglia are the resident immune cells of the central nervous system,and neuroinflammation mediated by microglia plays an important role in the occurrence and development of a variety of major neurological diseases such as depression.Due to the high accumulation in the central nervous system,circular RNA(circ RNA)has been proved to be closely related to many neurological and psychotic disorders.Our previous study indicated that the overexpression of circular RNA DYM(circDYM)in the hippocampus significantly ameliorated the LPS-induced depression-like behavior of mice,which was accompanied by the inhibition of the neuroinflammation induced by microglia activation.Circ DYM ameliorated depression-like behavior by inhibiting microglial activation.However,microglia apoptosis induced in the course of neuroinflammatory response will further aggravate the neuroinflammatory response.The effect of circDYM on microglia apoptosis and its mechanisms were still unknown.The purpose of this study was to investigate the effects and mechanisms of circDYM in the pathophysiological process of microglia survival,thus providing new theoretical support for the diagnosis and treatment of neuroinflammation-related disorders.Methods:(1)Sucrose preference test,tail suspension test and forced swimming test were used to evaluate the role of circDYM in depression-like behavior induced by lipopolysaccharide;(2)Western blot was used to detect the effects of circDYM on autophagy,apoptosis;(3)Bioinformatics analysis and RNA binding protein immunoprecipitation(RIP)combined with in situ were used to explore the binding between circDYM and CEBPB;(4)Western blot was used to detect the effects of CEBPB on autophagy and apoptosis;(5)The effects of circDYM on the subcellular distribution of CEBPB were investigated by using immunofluorescence and and nuclear fractionation;(6)By overexpression and knockdown of CEBPB using plasmid or si RNA,the effects of CEBPB on ZC3H4 expression was verified;(7)Western blot was used to detect the level of ZC3H4 in hippocampus and BV2 cell treated with LPS;(8)Circ DYM lentivirus and CEBPB plasmid were co-transfected to investigate the effect of circDYM/CEBPB axis on ZC3H4 expression;(9)The autophagic inhibitor 3-MA and inducer rapamycin were used to validate the pathway of LPS-induced apoptosis,respectively;(10)CRISPR Cas9 technology was used to explore the effects of overexpression or knockdown of ZC3H4 on autophagy and apoptosis.(11)Conditional knockout animal model(microglial specific ZC3H4knockdown)was used to investigate the autophagy and apoptosis levels in the hippocampus of LPS-treated mice.Results:(1)Circ DYM overexpression significantly ameliorated the depression-like behavior induced by LPS with concomitant inhibition of microglial autophagy and microglial apoptosis.(2)Overexpression of circDYM reduced microglial autophagy and apoptosis by inhibiting CEBPB translocation from the cytoplasm to the nucleus.(3)Circ DYM reversed the down-regulation expression of ZC3H4 induced by LPS.(4)CEBPB inhibited the expression of ZC3H4 through transcriptional regulation.(5)LPS treatment induced cell autophagy and apoptosis.They were evidenced by the increased expression of BECN1 and LC3B-II while decreased expression of P62 as for autophagic proteins at 24 h,as well as the increased expression of Bax and Caspase3 while decreased expression of Bcl-xl as for apoptotic proteins at 36 h.Furthermore,microglial autophagy contributed to LPS-induced microglial apoptosis as evidenced by the fact that autophagic inhibitor 3-MA inhibited the cell apoptosis.(6)Overexpression of ZC3H4 improved microglial survival by inhibiting autophagy in vitro and in vivo.Conclusions: Circ DYM binded with CEBPB and thereby released the repression of CEBPB’s downstream target gene ZC3H4.Circ DYM/CEBPB/ZC3H4 axis was involved in LPS-mediated microglial apoptosis.This study elucidates the molecular mechanism of circDYM in regulation of microglial apoptosis,and provides novel theoretical support for neuroinflammation in the central nervous system.