Analysis Of The Differentially Expressed CircRNAs Of M2 Microglia

Objective: Neuroinflammation contributes a lot to the pathogenesis of neurodegenerative diseases.As a kind of innate immune cell of the central nervous system,microglia could regulate the advancement of neuroinflammation.Microglia could respond to multiple inflammatory injuries and then activate into different phenotypes,such as proinflammatory M1 microglia and anti-inflammatory M2 microglia,affecting the course of the disease.The differential expression of circ RNAs was detected in activated microglia by circ RNA-seq.Analyzing the functions of these circ RNAs could provide a promising target for microglial activation,regulating the process of neuroinflammation and therefore modulating the development of diseases.Methods: BV2 microglia were treated with IL-4 for 8 hours,and the cells were collected for circ RNA-seq.The obtained circ RNAs were analyzed by CIRI2 and CIRCexplorer2,and the differential expression of circ RNA was measured by the DESeq method.The biological functions of these circ RNAs were analyzed by KEGG analysis and GO analysis.The stability of circ FOCAD and circ ADGRE1 was determined by RNase R assay,and the subcellular localization of circ ADGRE1 was measured by FISH assay.The prediction of the secondary structure of circ FOCAD and circ ADGRE1 was detected by an online website,RNA fold.The CRISPRCas13 d system specifically knocked down the expression of circ ADGRE1.BV2 microglia were transfected with circ ADGRE1 lentivirus.Seventy-two hours after transfection,BV2 cells were administrated with LPS and IL-4for 8 hours,respectively,and then the total cells were collected to extract RNA to detect the expression of circ ADGRE1,m ADGRE1,and inflammatory factors.The expression trends of circ FOCAD and circ ADGRE1 and m FOCAD and m ADGRE1 were detected by RT-q PCR at different time points.Results: The results of circ RNA-seq combined with CIRI2 and CIRCexplorer2 analysis showed that more than 4000 circ RNAs were detected in IL-4-treated BV2 microglia.These circ RNAs located on the different sites of chromosomes.The length of circ RNA was largely concentrated between 200 bp and 1000 bp.Most of them are derived from the exon sequence of the protein coding region.DESeq analysis showed that there were 120 circ RNA expressions with significant differences,among which 50 circ RNA expressions were increased and 70 circ RNA expressions were decreased.The potential biological functions of their parental genes were shown by GO analysis that these genes are primarily involved in protein metabolism,and KEGG analysis showed that the parental genes of circ RNA were significantly involved in the HIPPO signaling pathway.Knockdown of circ ADGRE1 contributed little to the expression of pro-inflammatory factors upon LPS treatment in BV2 cells,while the expression of anti-inflammatory factors ARG1 increased in response to IL-4 stimulation.Conclusion:1.The expression of circ RNAs was altered in M2 microglia.2.The differentially expressed circ RNA might be involved in protein catabolic process and participate in the HIPPO signal pathway.3.circ ADGRE1 and circ FOCAD were resistant to RNase R digestion.Over 100 mi RNA binding sites are predicted to combine with circ ADGRE1 or circ FOCAD.The expression of m ADGRE1 and circ ADGRE1 altered over time,but are not synchronized.4.Initial studies found that the knockdown of circ ADGRE1 could promote the expression of ARG1.