Abstract
BackgroundMicroglia are key regulators of the inflammatory response in the brain. Adenosine in RNAs can be converted to m6A (N6-methyladenosine), which regulates RNA metabolism and functions as a key epitranscriptomic modification. The m6A modification pattern and m6A-related signatures under pro-inflammatory and anti-inflammatory conditions of microglia remain unclear.MethodsPrimary rat microglia were differentiated into pro-inflammatory M1-like (M1-L), anti-inflammatory M2-like (M2-L), and resting, unstimulated (M0-L) phenotypes. m6A mRNA and lncRNA epitranscriptomic microarray analyses were performed, and pathway analysis was conducted to understand the functional implications of m6A methylation in mRNAs and lncRNAs. The m6A methylation level and gene expression of mRNAs and lncRNAs were subsequently verified by m6A Me-RIP and qRT-PCR.ResultsA total of 1588 mRNAs and 340 lncRNAs, 315 mRNAs and 38 lncRNAs, and 521 mRNAs and 244 lncRNAs were differentially m6A methylated between M1-L and M0-L (M1-L/M0-L), M2-L and M0-L (M2-L/M0-L), M2-L and M1-L (M2-L/M1-L), respectively. Furthermore, 4902 mRNAs, 4676 mRNAs, and 5095 mRNAs were identified distinctively expressed in M1-L/M0-L, M2-L/M0-L, and M2-L/M1-L, respectively. Pathway analysis of differentially m6A methylated mRNAs and lncRNAs in M1-L/M0-L identified immune system, signal transduction, and protein degradation processes. In contrast, the distinct m6A methylated mRNAs in M2-L/M0-L were involved in genetic information processing, metabolism, cellular processes, and neurodegenerative disease-related pathways. We validated m6A methylation and the expression levels of five mRNAs and five lncRNAs, which were involved in upregulated pathways in M1-L/M0-L, and five mRNAs involved in upregulated pathways in M2-L/M0-L.ConclusionsThese findings identify a distinct m6A epitranscriptome in microglia, and which may serve as novel and useful regulator during pro-inflammatory and anti-inflammatory response of microglia.
Highlights
Microglia are non-neuronal cells, which belong to the glial population of central nervous system (CNS) cells
We found that only 5.0% of hypo-methylated/upregulated genes and 6.9% of hypermethylated/downregulated genes in M1-L/Rest phenotype unstimulated (M0-L) are hyper-methylated/downregulated and hypo-methylated/ upregulated in M2-L/M1-L, respectively
Only a single gene (Diaph3) was hypo-methylated/downregulated in M1-L/M0-L and hyper-methylated/upregulated in M2-L/M0-L. These results revealed that 83.3% (106 genes) of differentially m6A-associated genes in M1-L/M0-L were negatively correlated with m6A-associated genes in M2-L/M1-L, suggesting that these genes might play an important role in the inflammatory response of microglia
Summary
Microglia are non-neuronal cells, which belong to the glial population of central nervous system (CNS) cells. As the resident immune cells of the brain parenchyma, microglia act as central communicators between the nervous and immune systems to coordinate homeostatic and immune surveillance functions of the CNS [1, 2]. The comparison of microglial gene expression profiles with the transcriptomes of other peripheral immune cells demonstrated that microglial are unique within the innate immune cell repertoire [5]. The comparison of microglial gene expression profiles with the transcriptome of other brain cells revealed that microglia are distinct from other CNS cell populations [10]. Microglia are key regulators of the inflammatory response in the brain. The m6A modification pattern and m6A-related signatures under pro-inflammatory and antiinflammatory conditions of microglia remain unclear
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