ADAR1 Regulates DARPP-32 Through MiR-874-3p To Participate In Chronic Stress-induced Cognitive Decline

Objective Chronic unpredictable stress(CUS)BALB/c mouse model and mouse adrenal medulla pheochromocytoma(PC12)cell line were used to investigate whether adenosine deaminase acting on RNA 1(ADAR1)regulates DARPP-32 through mi R-874-3p to participate in the molecular mechanism of chronic stress-induced cognitive impairment.Methods 1.Male SPF BALB/c mice weighing 15-20 g with postnatal 21 day were selected.Mice were divided into CUS model group and blank control group by random number method.Blank control group mice lived in normal feeding environment,CUS model group mice were fed stably in standard environment of animal laboratory for 1w and experienced chronic stress stimulation in different ways for 4 w.Open field test and rotarod test were used to assess the success of modelling.Then BALB/c mice were intraperitoneally injected with ADAR1 inducer/inhibitor,interferon-γ(IFN-γ)as ADAR1 inducer,and Pentostatin(PEN)as ADAR1 inhibitor.The corresponding control group was given the same amount of normal saline.The mice were divided into6 groups(n=13-15/group): blank control group(C),drug intervention control group[ADAR1 inducer intervention control group(C+ADAR1 inducer)and ADAR1 inhibitor intervention control group(C+ADAR1 inhibitor)],stress model group(CUS),stress model drug intervention group [ADAR1 inducer intervention model group(CUS+ADAR1 inducer),ADAR1 inhibitor intervention model group(CUS+ADAR1inhibitor)].Continuous drug intervention was performed for 7 days.After the intervention on the fifth day,the new object recognition experiment and the new object localization experiment were performed until the end of administration.Western blot(WB)was used to detect the protein expression levels of ADAR1 and dopamine-and c AMP-regulated phosphoprotein of Mr 32 k Da(DARPP-32)in the prefrontal cortex and hippocampus of experimental mice.The expression levels of Adar1,Darpp-32 m RNA and mi R-874-3p in the prefrontal cortex and hippocampus were measured using quantitative real time polymerase chain reaction(q RT-PCR).2.PC12 cells were treated with ADAR1 inducer(IFN-γ)and ADAR1 inhibitor(EHNA)for drug intervention,PC12 cells were divided into blank control group(C),ADAR1 inducer intervention group(ADAR1 inducer)and ADAR1 inhibitor intervention group(ADAR1 inhibitor).ADAR1 high/low expression PC12 cell model and mi R-874-3p high/low expression PC12 cell model were further constructed and grouped as follows:blank control group(C),negative control group(NC),ADAR1 high expression group(ADAR1 pc DNA),ADAR1 low expression group(ADAR1 si),mi R-874-3p high expression group(mi R-874 mimics)and mi R-874-3p low expression group(mi R-874inhibitor).The protein and m RNA expressions of ADAR1,DARPP-32 and mi R-874-3p were detected by Western blot and q RT-PCR.3.Bio-informatics is used to analyze the stem-loop structure of mmu-pre-mi R-874,to predict the potential site of mmu-pre-mi R-874 pre-m RNA with A-I editing and RNA interference binding site of mmu-mi R-874-3p in Darpp-32 m RNA and Adar1 m RNA,to predict the potential molecular mechanism of Adar1 affecting Darpp-32 expression through mi R-874-3p.Results 1.Before the drug intervention,compared to control BALB/c mice,model BALB/c mice had a significantly lower total distance travelled and a significantly lower mean speed of movement in the open field experiment;significantly lower discrimination index in new object recognition and new object localization experiments.There was no significant difference in the time spent on the rotarod between the model group and the blank control group in the rotarod experiment.After the administration of ADAR1 inducer,compared with stress model group mice,the discrimination index of BALB/c mice in the ADAR1 inducer intervention model group in the new object positioning and new object recognition experiments was significantly increased.2.The results of WB and q RT-PCR in vivo experiments showed that compared with the control BALB/c mice,the protein and m RNA expression levels of ADAR1 and DARPP-32 in the prefrontal cortex and hippocampus of stress model group mice were significantly decreased,and the expression of mi R-874-3p was significantly increased.Compared with stress model group mice,the expression levels of ADAR1 and DARPP-32 protein and m RNA in the prefrontal cortex and hippocampus of the ADAR1 inducer intervention model group were significantly increased,and the expression of mi R-874-3p was significantly decreased.3.The results of q RT-PCR in vitro showed that the expression levels of Adar1 and Darpp-32 m RNA in PC12 cells of ADAR1 inducer group were significantly higher than those in blank control group;The expression levels of Adar1 and Darpp-32 m RNA in the ADAR1 inhibitor group were significantly decreased.4.The effects of ADAR1 high/low expression on the expression of mi R-874-3p and DARPP-32: Western blot and q RT-PCR results showed that both ADAR1 and DARPP-32 protein and m RNA expression were significantly increased/decreased in ADAR1 high/low expressing PC12 cells compared to control PC12 cells;ADAR1high/low expression of mi R-874-3p was significantly reduced/increased in ADAR1high/low expressing cells.5.High/low expression of mi R-874-3p affected the expression of Adar1 and Darpp-32:q RT-PCR results showed that Adar1 and Darpp-32 m RNA expression was significantly lower/elevated in mi R-874-3p high/low expressing cells compared to control PC12 cells.6.The pre-m RNA of mmu-pre-mi R-874 had potential sites for A-I editing,mmu-874-3p has 7 binding sites between site 316 and site 322 in the 3’UTR of Darpp-32 m RNA,and 7 binding sites between site 1092 and site 1098 in the 3’UTR of Adar1 m RNA.Conclusions 1.ADAR1 inducer can alleviate the decreased cognitive function of BALB/c mice exposed to chronic stressors,and reverse the abnormal expression of DARPP-32 and mi R-874-3p in the prefrontal cortex and hippocampus of the mice exposed to chronic stress;2.The expression of ADAR1 can affect the expression of DARPP-32 and mi R-874-3p in PC12 cells,and the expression of mi R-874-3p can affect the expression of ADAR1 and DARPP-32 in PC12 cells;3.Adar1 can potentially act on pri-m RNA or pre-m RNA of mi R-874-3p through A-to-I editing,affect the biosynthesis of mature mi R-874-3p,and then affect the expression of Darpp-32 through RNA interference.