Cigarette smoking modulates m6A modification, affecting the induction of CYP1A1 mRNA by regulating human ARNT and AHRR in A549 cells

Abstract

N⁶-Methyladenosine (m⁶A) modification is a common epitranscriptomic mark of eukaryotic RNAs. This modification is installed by a methyltransferase like 3 (METTL3)-METTL14 complex and is eliminated by fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5). Aberrant m⁶A modification is associated with the development and progression of cancer. Cigarette smoking is a major lifestyle habit and risk factor for lung cancer. This study aimed to clarify the effects of cigarette smoking on the expression of m⁶A modification-regulating enzymes and the significance of m⁶A modification in the biological responses to cigarette smoking. Treatment of cigarette smoke extract (CSE) significantly decreased METTL3 and METTL14 protein levels in human lung adenocarcinoma-derived A549 cells. The induction of CYP1A1 mRNA by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a typical ligand of the aryl hydrocarbon receptor (AHR), was attenuated by the knockdown (KD) of METTL3 or ALKBH5, whereas it was enhanced by the KD of FTO. As the underlying mechanisms, significantly decreased expression of AHR nuclear translocator (ARNT) by the KD of METTL3 or ALKBH5, and significantly decreased expression of AHR repressor (AHRR) by the KD of FTO were demonstrated. Formaldehyde-assisted isolation of regulatory elements assay revealed that the KD of METTL3 or ALKBH5 resulted in the compaction of the chromatin structure of ARNT promoter, suggesting that METTL3 and ALKBH5 promote the transcription of ARNT through the rearrangement of chromatin structure. Collectively, we found that CSE treatment decreased METTL3 and METTL14 protein levels, and m⁶A modification have impact on the induction of CYP1A1 by modulating ARNT and AHRR expression.