METTL3-mediated lncRNA HNF1A-AS1/HNF4A-AS1 m6A modification regulates CYP expression .

Interindividual variations in the expression and activity of cytochrome P450 enzymes (CYPs) led to lower therapeutic efficacy or adverse drug events. We previously demonstrated that CYPs are regulated by the long non-coding RNAs (lncRNAs) HNF1A-AS1 and HNF4A-AS1 via transcription factors (TFs) including hepatocyte nuclear factor 1a (HNF1A), hepatocyte nuclear factor 4a (HNF4A), and pregnane X receptor (PXR). However, the upstream mechanisms regulating HNF1A-AS1 and HNF4A-AS1 are poorly understood. N6-methyladenosine (m6A) is a prevalent epi transcriptomic modification in mammalian RNA. Therefore, the aim of this study was to investigate whether m6A modification regulates the expression of HNF1A-AS1 and HNF4A-AS1 and affects CYP expression in HepG2 and Huh7 cells. The methyltransferase-like 3 (METTL3) inhibitor, STM2457, significantly suppressed the expression of HNF1A-AS1 and induced HNF4A-AS1 expression. Consistent with this, a loss-of-function assay of METTL3 in the cell lines resulted in the down-regulation of HNF1A-AS1 and its downstream HNF1A, PXR, and CYPs at the RNA level, as well as the down-regulation of some CYPs proteins, and up-regulation of HNF4A-AS1. The results of gain-of-function experiments showed the opposite trend. Mechanistically, subsequent RNA stability experiments confirmed that METTL3 affected the stability of both lncRNAs, but in opposite ways; that is, METTL3 reduced HNF1A-AS1 stability and increased HNF4A-AS1 stability. Rescue experiments confirmed that the regulation of METTL3 on TFs and CYPs may require the involvement of these two lncRNAs. Altogether, our study demonstrates that METTL3 is involved in TFs-mediated CYP expression by affecting HNF1A-AS1/HNF4A-AS1 stability. Significance Statement While the impact of lncRNAs (HNF1A-AS1 and HNF4A-AS1) on the downstream TF and CYP expression is well studied, the upstream regulation of these two lncRNAs by METTL3 remains unexplored. This study systematically investigated the regulation by METTL3 on the expression of lncRNAs (HNF1A-AS1 and HNF4A-AS1) and their downstream TFs (HNF1A, HNF4A, and PXR) and CYPs in HepG2 and Huh7 cells, revealing that METTL3 is involved in the regulation of lncRNA-TF-CYP expression by affecting the stability of HNF1A-AS1 and HNF4A-AS1.