METTL3-mediated m6 A modification of circPRKAR1B promotes Crohn's colitis by inducing pyroptosis via autophagy inhibition

Abstract

Background: The roles of circRNA and N6-methyladenosine (m⁶ A) methylation in Crohn's disease (CD) have drawn much attention. Therefore, this investigation aimed to discover how the m⁶ A modification of circRNAs contributes to CD progression.

Methods: The study performed circRNA sequencing on colon samples from four CD patients and four normal controls (NCs) to screen for dysregulated circRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the candidate circRNA expression and determine its correlation to CD-associated inflammatory indicators. In vivo and in vitro investigations were conducted to examine the functions and pathways of circPRKAR1B in CD, besides investigating the m⁶ A modification role in circRNA expression modulation.

Results: The RNA-seq revealed that hsa_circ_0008039 (circPRKAR1B) was the most significant upregulated circRNA and was identified as the candidate circRNA for further examinations. Relative circPRKAR1B expression was significantly upregulated in CD colon tissues and closely related to CD-associated inflammatory indices. The circPRKAR1B expression and function were regulated by methyltransferase-like 3 (METTL3)-mediated m⁶ A methylation. In vitro studies indicated that circPRKAR1B promoted pyroptosis mediated by NLRP3 inflammasome (NLRP3; nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3) and impaired autophagy by interacting with the RNA-binding protein (RBP) SPTBN1, (SPTBN1; spectrin beta, non-erythrocytic 1). The in vivo investigations revealed the treatment effects of si-circPRKAR1B and si-METTL3 in colitis models of IL-10-deficient mice.

Conclusion: Our study reveals that METTL3-mediated m⁶ A modification of circPRKAR1B promotes Crohn's colitis by aggravating NLRP3 inflammasome-mediated pyroptosis via autophagy impairment in colonic epithelial cells.

Keywords: Crohn's disease; SPTBN1; autophagy; circPRKAR1B; m6A modification; pyroptosis.

FIGURE 1

The circPRKAR1B as circRNA candidate. (A) Overlap of circRNAs in RNA‐seq (left) and circBase (right). (B) Composition of circRNAs in terms of genomic origin. (C) Correlations between samples according to the 3D PCA plot distribution. (D) Length distribution of detected circRNAs. (E) CircRNA distribution on chromosomes/scaffolds. Volcano plots (F), heatmap (G) and histogram (H) showing circRNA expression profiles in colon tissues from CD patients and NCs according to RNA‐seq (threshold: p < .05 and |log2FC| > 1). (I) Relative circPRKAR1B expression in CD patients and NCs according to qRT‐PCR analysis. Correlations between circPRKAR1B levels and CD‐associated inflammatory indicators, including CRP (J), CDAI (K), SES‐CD (L) and faecal calprotectin (M), in CD patients. circRNA, circular RNA; PCA, principal component analysis; CD, Crohn's disease; NC, normal control; FC, fold change; qRT‐PCR, quantitative real‐time polymerase chain reaction; CRP, C‐reactive protein; CDAI, Crohn's disease activity index; SES‐CD, simple endoscopic score for Crohn's disease. The data are presented as mean ± SD. ***p < .001 by Student's t‐tests.

FIGURE 2

Characterization of circPRKAR1B. (A) FISH analysis of circPRKAR1B in colon tissues from CD patients and NCs; scale bar: 20 μm. (B) Relative circPRKAR1B expression in isolated epithelial cells from CD patients and NCs according to qRT‐PCR. (C) The circular structure of circPRKAR1B back‐spliced by exons 2 and 4. (D) Sanger sequencing confirmed the circular structure in NCM460 cells. (E) RNase R treatment experiments with qRT‐PCR in NCM460 and HCoEpic cell lines. Actinomycin D assay (F) and agarose gel electrophoresis (G) analyses in NCM460 cell lines. (H) Basic information on circPRKAR1B from the circBank database. (I) The predicted secondary structures of circPRKAR1B based on the RNAfold website. (J) Localization of circPRKAR1B in NCM460 and HCoEpic cell lines according to FISH analysis; scale bar: 10 μm. (K) Expression of circPRKAR1B in the cytoplasm and nucleus according to qRT‐PCR. circRNA, circular RNA; CD, Crohn's disease; NC, normal control; qRT‐PCR, quantitative real‐time polymerase chain reaction; MFE, minimum free energy; FISH, fluorescence In Situ hybridization; ns, non‐significant, ***p < .001, ****p < .0001 by Student's t‐tests.

FIGURE 3

circPRKAR1B is regulated by METTL3‐mediated m⁶A methylation. (A) m⁶A methylation modification sites predicted by SRAMP and RMBase 2.0. (B) m⁶A sites predicted with high confidence by SRAMP. (C) Relative m⁶A levels in isolated epithelial cells from CD patients and NCs. (D) Expression of m⁶A methyltransferases, including METTL3/14 and WTAP, in isolated epithelial cells from CD patients and NCs. (E) Impacts of three si‐circPRKAR1B sequences on the expression of circPRKAR1B and PRKAR1B mRNA according to qRT‐PCR. Efficacy of three designed si‐METTL3 sequences according to qRT‐PCR (F) and western blotting (G). The impacts of si‐METTL3 on m⁶A levels according to qRT‐PCR (H) and circPRKAR1B expression according to MeRIP‐qPCR (I) and qRT‐PCR (J) were determined in NCM460 and HCoEpic cell lines. METTL3, methyltransferase‐like 3; METTL14, methyltransferase‐like 14; m⁶A, N6‐methyladenosine; CD, Crohn's disease; NC, normal control; WTAP, Wilms tumour 1‐associated protein; qRT‐PCR, quantitative real‐time polymerase chain reaction; RIP, RNA immunoprecipitation. The data are presented as mean ± SD. ***p < .001 by Student's t‐tests.

FIGURE 4

circPRKAR1B interacts with SPTBN1. (A) Silver staining of the RNA pull‐down assay results. According to MS analysis of the RNA pull‐down products, deregulated proteins were subjected to GO (B) and KEGG enrichment (C) analyses. (D) Binding potential between circPRKAR1B and SPTBN1 by the catRAPID database. The interaction assessment between circPRKAR1B and SPTBN1 by RNA pull‐down western blotting (E) and RIP assays (F). (G) Correlation between circPRKAR1B and SPTBN1 levels in colon tissues from CD patients. (H) Relative SPTBN1 expression in isolated epithelial cells from CD patients and NCs. SPTBN1, spectrin beta, non‐erythrocytic 1; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; CD, Crohn's disease; NC, normal control; RIP, RNA immunoprecipitation. The data are expressed as mean ± SD; ns, non‐significant, ***p < .001 by Student's t‐tests.

FIGURE 5

Impacts of circPRKAR1B and m⁶A modification on cell autophagy. The assessment of the impacts of si‐circPRKAR1B, si‐METTL3 and si‐circ + si‐SPTBN1 treatment on epithelial cell autophagy by western blotting (A), grayscale analysis of LC3B II/I (B), immunofluorescence (C; scale bar: 20 μm), autophagic flux analysis via mCherry‐GFP‐LC3B confocal microscopy (D; scale bar: 25 μm) and autophagosome analysis via TEM (E; scale bar: 1 μm). METTL3, methyltransferase‐like 3; m⁶A, N6‐methyladenosine; TEM, transmission electron microscope. The data are presented as mean ± SD. ***p < .001 by one‐way ANOVA.

FIGURE 6

Impacts of circPRKAR1B and m⁶A modification on NLRP3 inflammasome‐mediated pyroptosis. The assessment of the impacts of si‐circPRKAR1B and si‐METTL3 treatment on NLRP3 inflammasome‐mediated pyroptosis using western blotting (A), grayscale analysis of blots (B), immunofluorescence (C; scale bar: 20 μm, white arrows: NLRP3 inflammasome specks and D; scale bar: 25 μm), TUNEL staining (E; scale bar: 25 μm), pyroptosis analysis via SEM (F; scale bar: 100 and 20 μm) and proinflammatory cytokine (IL‐1β/18) level analyses via ELISA (G). METTL3, methyltransferase‐like 3; m⁶A, N6‐methyladenosine; SEM, scanning electron microscopy; NLRP3, nucleotide‐binding oligomerization domain, leucine‐rich repeat and pyrin domain‐containing 3; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; ELISA, enzyme‐linked immunosorbent assay; IL, interleukin. The data are presented as mean ± SD. ***p < .001 by one‐way ANOVA.

FIGURE 7

In vivo circPRKAR1B and m⁶A modification functions in IL‐10‐KO mice. The assessment of the impacts of si‐circPRKAR1B and si‐METTL3 treatment on the relative circPRKAR1B expression (A), disease activity index (B), colon length (C), pathological inflammatory score from HE staining (D) and inflammatory cytokines including TNF‐α (E), IFN‐γ (F) and IL‐17 (G). The impacts of si‐circPRKAR1B and si‐METTL3 treatment on autophagy and NLRP3 inflammasome‐mediated pyroptosis were assessed by immunofluorescence (H and I; scale bar: 50 and 20 μm), western blotting (J), grayscale analysis of blots (K) and autophagosome analysis via TEM (L; scale bar: 1 μm). SPTBN1, spectrin beta, non‐erythrocytic 1; KO, knockout; METTL3, methyltransferase‐like 3; m⁶A, N6‐methyladenosine; HE, haematoxylin and eosin; TEM, transmission electron microscopy; TNF‐α, tumour necrosis factor‐α; IFN‐γ, interferon‐γ; IL‐17, interleukin‐17; NLRP3, nucleotide‐binding oligomerization domain, leucine‐rich repeat and pyrin domain‐containing 3. The data are presented as mean ± SD. *p < .05, **p < .01, ***p < .001 by one‐way ANOVA.

FIGURE 8

Graphical illustration of how METTL3‐mediated m⁶A modification of circPRKAR1B promotes Crohn's colitis by inducing pyroptosis via autophagy inhibition. SPTBN1, spectrin beta, non‐erythrocytic 1; METTL3, methyltransferase‐like 3; m⁶A, N6‐methyladenosine; HE, haematoxylin and eosin; IL‐1β, interleukin‐1β; NLRP3, nucleotide‐binding oligomerization domain, leucine‐rich repeat and pyrin domain‐containing 3; RBP, RNA‐binding protein.