WTAP promotes microglial M1 polarization by regulating SIRT1

Abstract

Objective To investigate the role and the possible molecular mechanism of RNA methylase, Wilms' tumor 1-associating protein (WTAP), in microglial activation. Methods HMC3 cells was induced to M1 polarization by treatment of 100 ng/mL IFNγ combined with 1 μg/mL LPS and then served as experimental group, and the untreated cells served as control group. The expression of pro-inflammatory cytokines IL-6, IL-1β, TNF-α and N6-methyladenosine (m6A) at mRNA and protein levels was detected by real-time quantitative PCR and Western blotting, respectively. After HMC3 cells were transfected with RNAi lentivirus to knock down WTAP, the knockdown efficiency was detected by real-time quantitative PCR and Western blotting. Western blot assay was employed to measure the expression levels of pro-inflammatory cytokines IL-6 IL-1β and TNF-α in WTAP knockdown HMC3 cells. Western blotting was also adopted to detect the protein levels of silent mating-type information regulation 2 homolog 1 (SIRT1), COP1, USP18, EP4, pro-inflammatory transcription factors and some inflammatory pathway receptors in the knockdown cells. SIRT1 agonist SRT1720 and SIRT1 inhibitor EX527 were employed to treat HMC3 cells in the blank-virus group and the knockdown WTAP group, respectively, and the expression level of C/EBPβ was detected by Western blotting. Results Treatment of 100 ng/mL IFNγ combined with 1 μg/mL LPS induced significant up-regulation of IL-6, IL-1β and TNF-α expression in HMC3 cells as compared with the untreated cells (P < 0.05). WTAP was highly expressed in M1 polarized HMC3 microglia (P < 0.05). WTAP knockdown resulted in obviously decreased expression levels of pro-inflammatory cytokines IL-6, IL-1β and TNF-α (P < 0.05), increased level of SIRT1 (P < 0.05), and reduced pro-inflammatory transcription factor C/EBPβ (P < 0.05). SIRT1 agonist SRT1720 reduced the relatively high expression of C/EBPβ in the blank-virus group (P < 0.01). While, the inhibitor EX527 increased the relative low expression of C/EBPβ in HMC3 cells with low WTAP knockdown (P < 0.05). Conclusion WTAP promotes M1 polarization in HMC3 cells, possibly through regulating the expression of SIRT1 and thus affecting the C/EBPβ signaling pathway.

Keywords

• microglia
• wilms' tumor 1-associating protein
• silent mating-type information regulation 2 homolog 1
• n6-methyladenosine
• polarization