Mono-(2-Ethylhexyl)phthalate Regulates Cholesterol Efflux via MicroRNAs Regulated m⁶A RNA Methylation

Mono-(2-ethylhexyl)phthalate (MEHP) is a major bioactive metabolite which occurs from the widely used industrial plasticizer di(2-ethylhexyl)phthalate, which has been found to be toxic to several human physiological systems, including the cardiometabolic system. Recently, RNA methylation has been shown to be involved in cardio-metabolic regulation. Despite the importance of m⁶A mRNA methylation in physiological processes, studies of RNA methylation associated with endocrine disrupting chemicals are lacking. Here, we investigated the effects of MEHP in a cholesterol efflux pathway and the roles of m⁶A methylation using murine macrophage Raw 264.7 cells. MEHP exposure significantly reduced (P < 0.01 for 50 μM) m⁶A mRNA methylation with decreases in both mRNA (P < 0.01 for 5 and 50 μM) and protein (P < 0.05 for 0.5 μM; P < 0.01 for 5 μM; and P < 0.001 for 50 μM) expression levels of METTL14, a component of the methyltransferase complex. However, m⁵C RNA methylation remained unchanged. MEHP significantly reduced the expression of Scavenger Receptor B type 1 (SR-B1) (P < 0.01 for 5 μM and P < 0.05 for 50 μM). Additionally, we demonstrated that silencing METTL14 with MEHP decreased SR-B1 gene expression compared to the MEHP treatment (P < 0.01) or silencing METTL14 alone (P < 0.05). Furthermore, MEHP significantly promoted the m⁶A modification in SR-B1 (P < 0.001) and activated miRNAs which are predicted to regulate METTL14, such as miR16-1-3p (P < 0.05 for 50 μM MEHP), miR101a-3p (P < 0.05 for 5 and 50 μM MEHP), miR362-3-5p (P < 0.05 for 50 μM MEHP), miR501-5p (P < 0.01 for 50 μM MEHP), miR532-3p (P < 0.05 for 50 μM MEHP), and miR542-3p (P < 0.05 for 50 μM MEHP). Together, these results reveal for the first time that MEHP can modulate RNA methylation to regulate SR-B1 in a cholesterol efflux pathway.