Low Expression of YTH Domain-Containing 1 Promotes Microglial M1 Polarization by Reducing the Stability of Sirtuin 1 mRNA

Abstract

The N6-methyladenosine (m6A) modification is the most abundant posttranscriptional mRNA modification in mammalian cells and is dynamically modulated by a series of "writers," "erasers," and "readers." Studies have shown that m6A affects RNA metabolism in terms of RNA processing, nuclear export, translation, and decay. However, the role of the m6A modification in retinal microglial activation remains unclear. Here, we analyzed the single-cell RNA sequencing data of retinal cells from mice with uveitis and found that the m6A-binding protein YTH domain-containing 1 (YTHDC1) was significantly downregulated in retinal microglia in the context of uveitis. Further studies showed that YTHDC1 deficiency resulted in M1 microglial polarization, an increased inflammatory response and the promotion of microglial migration. Mechanistically, YTHDC1 maintained sirtuin 1 (SIRT1) mRNA stability, which reduced signal transducer and activator of transcription 3 (STAT3) phosphorylation, thus inhibiting microglial M1 polarization. Collectively, our data show that YTHDC1 is critical for microglial inflammatory response regulation and can serve as a target for the development of therapeutics for autogenic immune diseases.

Keywords: RNA stability; SIRT1; YTHDC1; m6A; microglia cells.

FIGURE 1

scRNA-seq analysis of Aire^{– /–} retinal microglia. (A) Bar charts showing the expression levels of m6A writers, erasers and readers in WT and Aire^–/– retina microglia (n = 4). (B) Violin plots showing the expression levels of m6A writers, erasers and readers in WT and Aire^–/– retinal microglia. *P < 0.05 by Student’s t-test; n.s., no significance. The data are presented as the mean ± SD.

FIGURE 2

scRNA-seq analysis of EAU mouse retinal microglia and ALKBH5 and YTHDC1 protein expression levels in M1 microglia. (A) Violin plots showing the expression level of m6A genes in retinal microglia at 0, 14, 21, and 28 days. (B) Line plots showing the expression levels of m6A genes in retinal microglia at 0, 14, 21, and 28 days. (C,D) Western blot analysis of ALKBH5 and YTHDC1 in LPS (1 μg/ml)-induced BV2 cells after 24 h compared with those of the control (n = 3). (E) Quantitative m6A RNA methylation analysis (colorimetric) of LPS (1 μg/ml)-induced BV2 cells at 24 h compared with that of the control (n = 3). *P < 0.05 and ^**P < 0.01 by Student’s t-test; n.s., no significance. The data are presented as the mean ± SD.

FIGURE 3

YTHDC1 depletion facilitates BV2 microglia M1 activation and migration in vitro. (A–C) The transfection efficiency of YTHDC1 knockdown in BV2 cells was measured by qRT-PCR (A) and Western blotting (B,C). (D,E) Western blot analysis of iNOS, COX2 and TNF-α in the different groups. (F) Immunofluorescence analysis of iNOS in the different groups. (G,H) Wound healing assays in the different groups. (I,J) Transwell assays in the different groups. NC: cells transfected with negative control shRNA; ShYTHDC1 (1, 2, and 3) or ShYTHDC1: cells transfected with YTHDC1 shRNA. *P < 0.05, ^**P < 0.01, and ^***P < 0.001 by Student’s t-test. The data are presented as the mean ± SD. (n = 3). Scale bar, 200 μm.

FIGURE 4

SIRT1 is the target of YTHDC1 in BV2 cells. (A) RT-qPCR analysis of COP1, IκB, USP18, EP4, SIRT1, and SOCS1 in ShYTHDC1-transfected BV2 cells compared to unloaded lentivirus-transfected BV2 cells after treatment with LPS. (B,C) Western blot analysis of COP1, USP18, EP4, and SIRT1 in ShYTHDC1-transfected BV2 cells compared to unloaded lentivirus-transfected BV2 cells after treatment with LPS. (D,E) Western blot analysis of SIRT1 protein expression in BV2 cells treated with different concentrations of SRT1720 compared with the control group. (F,G) Western blot analysis of SIRT1, iNOS and COX2 in the different groups. *P < 0.05, ^**P < 0.01, and ^***P < 0.001 by Student’s t-test. The data are presented as the mean ± SD (n = 3).

FIGURE 5

STAT3 phosphorylation is regulated by SIRT1 in BV2 after YTHDC1 silencing. (A) RT-qPCR analysis of some proinflammatory transcription factors in YTHDC1-knockdown BV2 cells after treatment with LPS. (B,C) Western blot analysis of the protein levels of IRF8, STAT3, cEBPβ, and EGR1 in YTHDC1-knockdown BV2 cells after treatment with LPS. (D,E) Western blot analysis of p-STAT3 in NC- or shYTHDC1-transfected BV2 cells after treatment with LPS. (F,G) Western blot analysis of STAT3 and p-STAT3 in the different groups. (H,I) Western blot analysis of ac-STAT3 in the different groups. *P < 0.05, ^**P < 0.01, and ^***P < 0.001 by Student’s t-test. The data presented as the mean ± SD (n = 3).

FIGURE 6

YTHDC1 promoted the stability of SIRT1 mRNA in BV2 cells. (A) RT-qPCR analysis of SIRT1 in NC- or shYTHDC1-transfected BV2 cells after treatment with LPS. β-actin served as a loading control. (B) RT-qPCR analysis of SIRT1 mRNA levels in the cytoplasm and nucleus of NC- or shYTHDC1-transfected BV2 cells after treatment with LPS. (C) RT-qPCR analysis of SIRT1 and STAT3 mRNA levels in NC- or shYTHDC1-transfected BV2 cells subjected to ActD (1 μg/ml) treatment for the indicated times after treatment with LPS. (D) MeRIP analysis followed by qRT-PCR was used to assess the m6A modification of SIRT1 in NC- or shYTHDC1-transfected BV2 cells. (E) Quantitative detection of m6A RNA methylation in NC- or shYTHDC1-transfected BV2 cells after treatment with LPS. *P < 0.05, ^**P < 0.01, and ^***P < 0.001 by Student’s t-test. The data are presented as the mean ± SD (n = 3).