Role And Regulation Mechanism Of Apobec1-induced RNA Editing In Polarization Of M1 Bone Marrow-derived Macrophages

Objective: M1 macrophages are mainly involved in initiating and maintaining inflammatory responses,and the polarization of M1 macrophages is closely associated with inflammatory bowel disease(IBD)and other related diseases.The editing enzyme Apolipoprotein B m RNA Editing Enzyme Catalytic Subunit 1(Apobec1)-mediated cytidine to uridine(C>U)RNA editing is common in macrophages and plays a potentially important role in macrophage polarization,but the specific regulatory mechanism remains to be elucidated.The current study aimed to analyze the role and mechanism of Apobec1-mediated C>U RNA editing in M1 macrophage polarization.Methods: Bone marrow-derived macrophages(BMDM)were harvested and cultured from mouse femur and tibia.After 7 consecutive days of stimulation by using Macrophage Colony Stimulating Factor(M-CSF)naive BMDM maturized into M0 macrophages.LPS(100 ng/ml)was used to induce BMDM.Total RNA from BMDM was extracted before(0 hr)and 6 hours(6 hr)after LPS stimulation,and subjected to RNA-Seq.The RNA-sequencing reads were mapped to the mouse reference genome(mm10)using RNA STAR.Differential gene expression was analyzed using edge R.Differential RNA editing sites were identified using Varscan2,with selected candidates validated by Sanger sequencing.The level of m RNA or protein was quantitated by q PCR or Western-blot,and function enrichment analysis was performed on the differential expressed or edited genes using the Enrichr software.The effect of RNA editing on gene expression was analyzed in vitro using the firefly luciferase and Renilla reporter,site-directed mutagenesis of the editing site,and transfection of recombinant plasmids for 36 hours in human 293 T cells.An RNA-Seq dataset of IBD mouse model(PRJAN508301)was downloaded and analyzed to analyze the effect of Il12 b knockout on IBD.The RNA pull-down coupled with mass spectrometry method was used to analyze the effect of RNA editing on RNA binding proteins(RBPs).Results:(1)With the knockout of Apobec1 gene,M1 BMDM cells showed dramatic morphological changes,and Apobec1 knockout up-regulated pro-apoptotic factors such as(apoptotic protease activating factor-1)Apaf1 and(Bcl2-associated X)Bax genes and down-regulated apoptosis inhibitor such as(B-cell lymphoma-2 like 1)Bcl2l1.(2)Apobec1knockout significantly reduced the expressions of inflammatory factors Il12 b,Il1b,or markers of M1 macrophages such as Cd80 and Cd86,and up-regulated the M2-like macrophage marker mannose receptor c1-like protein 1(Mrc1).(3)The expression of Apobec1 was down-regulated during the early stage of M1 macrophage polarization.And RNA-Seq results showed that WT without LPS stimulation(WT 0hr)had the largest number of differential C>U RNA editing sites,followed by WT with LPS stimulation(WT 6hr),and the Apobec1 knockout groups(A1 KO 0hr and A1 KO 6hr)had the lowest number of C>U differential RNA editing sites.RNA editing of genes such as Il12b、Oxct1、and Mmd increased during LPS stimulation of BMDM,and was almost diminished by Apobec1 knockout,suggesting these genes were potentially important targets of Apobec1-mediated RNA editing.GO analysis showed that the genes with down-regulated RNA editing were significantly enriched in Il12-related signaling pathways upon Apobec1 knockout in LPS stimulated BMDM(A1KO 6hr vs.WT 6hr).(4)The reporter gene assay showed that the expression of edited Il12b3’UTR was about 2.1 times higher than the unedited one;BMDM q PCR results showed that Il12 b expression in WT was about 5.5 times compared with A1 KO upon LPS-stimulation(A1 KO 6hr vs.WT 6hr).Spearman correlation analysis showed a significant positive correlation between Il12 b 3’UTR editing and Il12 b m RNA expression.(5)RNA-Seq results showed that the expressions of Il12 b and Apobec1 were up-regulated in IBD mice,while Il12 b knockout reduced the immune response in IBD mice compared to controls.(6)RNA pull-down coupled Mass spectrometry revealed 81 unique RBPs potentially binding to unedited Il12 b 3’UTR,420 unique RBPs potentially binding to edited Il12 b 3’UTR,and common 323 RBPs shared by the two.RBPs binding to the unedited Il12 b 3’UTR were mainly related to the immune system process and viral response,while RBPs binding to the edited Il12 b 3’UTR were mainly related to cellular localization,NF-KB signaling pathway,and splicesome.And Protein-Protein interaction(PPI)analysis found possible protein-protein interaction networks in BMDM M1 polarization,including a group consisting of three proteins,namely EEF1 G,EIF4G1 and EIF3 A,which were eukaryotic transcription factors to facilitate protein translation.Conclusion: Our results demonstrated that Apobec1-mediated C>U RNA editing plays an important role in M1 BMDM polarization,in which Il12 b was a potential key target.And our results also suggested that Apobec1-mediated C>U RNA editing may alter the binding of RBPs to Il12 b 3’UTR,and increase the expression of Il12 b to promote M1 polarization.