Cannabinoids shift the basal ganglia miRNA m6A methylation profile towards an anti-inflammatory phenotype in SIV-infected Rhesus macaques

Abstract

BackgroundEpitranscriptomic modifications modulate diverse biological processes like regulation of gene expression, abundance, location and function. N6-methyladenosine (m6A) methylation has been shown to regulate various diseases, including cancer and inflammation. While there is evidence that m6A modification is functionally relevant in neural development and differentiation, the role of m6A modification in HIV neuropathogenesis is unknown. MethodsHere, we used anti-N6-methyladenosine (m6A) antibody immunoprecipitation and microarray profiling to identify m6A modifications in miRNAs in basal ganglia (BG) of Rhesus macaques (RMs) that were uninfected (VEH) and SIV-infected on combination anti-retroviral therapy (cART) and either VEH-treated (VEH/SIV/cART), or THC:CBD-treated (THC:CBD/SIV/cART). Ingenuity pathway analysis was conducted to understand the biological implications of miRNA m6A methylation in HIV neuropathogenesis. Finally, to understand the functional significance of m6A modifications in miRNAs, we overexpressed FAM-labeled wild-type or m6A-modified miR-194-5p in SCC-25 cells and determined its impact on the expression of its target, STAT1, an interferon-stimulated transcription factor known to drive persistent neuroinflammation in several neurodegenerative diseases. ResultsHIV/SIV infection promoted an overall hypomethylated miRNA m6A profile. While the overall hypomethylated m6A profile was not significantly impacted by THC:CBD, specific miRNAs predicted to target proinflammatory genes showed markedly reduced m6A methylation levels compared to the VEH-treated RMs. Additionally, specific BG tissue miRNAs bearing m6A epi-transcriptomic marks were transferred and detected in BG-derived extracellular vesicles. Mechanistically, the DRACH motif in the seed region of miR-194-5p was significantly m6A hypomethylated in THC:CBD/SIV/cART RMs. In SCC-25 cells, unlike wild-type miR-194-5p, transfected m6A-modified miR-194-5p mimics failed to downregulate STAT1 protein expression. Further, compared to VEH/SIV/cART RMs, THC:CBD administration significantly reduced m6A methylation of 44 miRNAs directly involved in regulating CNS network genes. ConclusionsThese results underscore the need for investigating the qualitative, and posttranscriptional modifications in RNA along with the more traditional, quantitative alterations in pathological conditions or in response to disease modifying treatments. Our findings indicate that m6A epitranscriptomic marks in the seed nucleotide region can impair miRNA function and that cannabinoids may preserve it by reducing m6A methylation levels. Finally, these findings provide a novel mechanistic (miRNA m6A hypomethylation) explanation underlying the anti-neuroinflammatory effects of phytocannabinoids in HIV/SIV infection.