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. 2018 May;22(5):2558-2568.
doi: 10.1111/jcmm.13491. Epub 2018 Mar 4.

METTL3 regulates alternative splicing of MyD88 upon the lipopolysaccharide-induced inflammatory response in human dental pulp cells

Zhihui Feng  1 , Qimeng Li  1 , Runsha Meng  1 , Baicheng Yi  1 , Qiong Xu  1
Affiliations

METTL3 regulates alternative splicing of MyD88 upon the lipopolysaccharide-induced inflammatory response in human dental pulp cells

Zhihui Feng et al. J Cell Mol Med. 2018 May.

Abstract

Dental pulp inflammation is a widespread public health problem caused by oral bacterial infections and can progress to pulp necrosis and periapical diseases. N6-methyladenosine (m6A) is a prevalent epitranscriptomic modification in mRNA. Previous studies have demonstrated that m6A methylation plays important roles in cell differentiation, embryonic development and stress responses. However, whether m6A modification affects dental pulp inflammation remains unknown. To address this issue, we investigated the expression of m6A and N6-adenosine methyltransferase (METTL3, METTL14) as well as demethylases (FTO, ALKBH5) and found that the levels of m6A and METTL3 were up-regulated in human dental pulp cells (HDPCs) stimulated by lipopolysaccharide (LPS). Furthermore, we knocked down METTL3 and demonstrated that METTL3 depletion decreased the expression of inflammatory cytokines and the phosphorylation of IKKα/β, p65 and IκBα in the NF-κB signalling pathway as well as p38, ERK and JNK in the MAPK signalling pathway in LPS-induced HDPCs. The RNA sequencing analysis revealed that the vast number of genes affected by METTL3 depletion was associated with the inflammatory response. Previous research has shown that METTL3-dependent N6-adenosine methylation plays an important role in mRNA splicing. In this study, we found that METTL3 knockdown facilitated the expression of MyD88S, a splice variant of MyD88 that inhibits inflammatory cytokine production, suggesting that METTL3 might inhibit the LPS-induced inflammatory response of HDPCs by regulating alternative splicing of MyD88 VSports手机版. These data shed light on new findings in epitranscriptomic regulation of the inflammatory response and open new avenues for research into the molecular mechanisms of dental pulp inflammation. .

Keywords: METTL3; MyD88; N6-methyladenosine; alternative splicing; dental pulp inflammation; lipopolysaccharide V体育安卓版. .

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Figures

Figure 1
Figure 1
The total m6A content and METTL3 expression levels were increased in LPS‐treated HDPCs. HDPCs were treated with 1 μg/ml LPS for 3, 6, 12 and 24 hrs, and cells without LPS stimulation were used as controls. (A) The total m6A content was detected by quantifying m6A RNA methylation. (B) qPCR was performed to determine the mRNA expression levels of METTL3, METTL14, FTO and ALKBH5 in LPS‐treated HDPCs. GAPDH was used as an internal control. (C, D) METTL3 protein expression was assessed by Western blotting. Vinculin was used as an internal control. The data are expressed as the mean ± S.E.M. (n = 3). Statistically significant difference relative to the control (time 0), *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2
Figure 2
METTL3 depletion down‐regulated cytokine expression in LPS‐treated HDPCs. METTL3 knockdown in transfected HDPCs was confirmed by qPCR (A) and immunoblotting (B). Mock, cells transfected with only transfection reagent; Control, cells transfected with negative control siRNA; siMETTL3, cells transfected with METTL3 siRNA. (C, D) HDPCs were transfected with siRNAs (METTL3 or the negative control) and then treated with or without 1 μg/ml LPS for 24 hrs. The supernatant was collected and examined using a cytokine array. CC, cells transfected with negative control siRNA; CL, cells transfected with negative control siRNA and treated with LPS; MC, cells transfected with METTL3 siRNA; ML, cells transfected with METTL3 siRNA and treated with LPS. (E–I) The supernatants were collected, and the expression of IL‐6, IL‐8, GRO, Gro‐α and RANTES proteins was assessed by ELISA. siCTRL, cells transfected with negative control siRNA; siMETTL3, cells transfected with METTL3 siRNA. The data are presented as the mean ± S.E.M. (n = 3). Statistically significant difference relative to the siCTRL group (*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 3
Figure 3
METTL3 inhibition down‐regulated LPS‐induced NF‐κB and MAPK signalling pathway activation in HDPCs. HDPCs were transfected with siRNAs (METTL3 or the negative control) and then treated with 1 μg/ml LPS for 0, 15, 30, 60 and 120 min. (A) The phosphorylation of IKKα, IKKβ, IκBα and p65 in the NF‐κB signalling pathway was examined by Western blotting. The relative quantitative analysis of the phosphorylation of IKKα, IKKβ, IκBα and p65 compared to that of the siCTRL group. (B) The phosphorylation of JNK, ERK and p38 in the MAPK signalling pathway was examined by Western blotting. The relative quantitative analysis of the phosphorylation of JNK, ERK and p38 compared to that of the siCTRL group. siCTRL, cells transfected with negative control siRNA; siMETTL3, cells transfected with METTL3 siRNA. The data are presented as the mean ± S.E.M. (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4
Figure 4
METTL3 inhibition down‐regulated the LPS‐induced inflammatory response of HDPCs. Total RNA obtained from the siCTRL and siMETTL3 cells with or without LPS treatment for 24 hrs was subjected to RNA sequencing. (A) The genes that were differentially expressed following LPS treatment or following METTL3 knockdown (MC vs. CC, or ML vs. CL) with a Log2 ratio >1.5 or <−1.5 are shown in the heatmap. CC, cells transfected with negative control siRNA; CL, cells transfected with negative control siRNA and treated with LPS; MC, cells transfected with METTL3 siRNA; ML, cells transfected with METTL3 siRNA and treated with LPS. Gene Ontology (B) and KEGG pathway (D) analyses of genes that were differentially expressed with a Log2 ratio <−1.5 ordered according to gene enrichment. Gene Ontology (C) and KEGG pathway (E) analyses of genes that were differentially expressed with a Log2 ratio >1.5 ordered according to gene enrichment.
Figure 5
Figure 5
METTL3 inhibition facilitated MyD88S mRNA expression. HDPCs were transfected with siRNAs (METTL3 or the negative control) and then treated with 1 μg/ml LPS for 0, 15, 30, 60 and 120 min. (A) RT‐PCR was performed to amplify an mRNA product specific to MyD88S or mRNA product specific to GAPDH. Quantitation of the bands is depicted in (B) GAPDH and (C) MyD88S. (D) RT‐PCR was performed to amplify products from both MyD88L and MyD88S using primers that bracketed MyD88 exon 2. Quantitation of the bands is depicted in (E) MyD88L and (F) MyD88S. siCTRL, cells transfected with negative control siRNA; siMETTL3, cells transfected with METTL3 siRNA. The data are presented as the mean ± SEM (n = 3). Statistically significant difference relative to the siCTRL group (*P < 0.05; **P < 0.01; ***P < 0.001).
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