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

ABSTRACT

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.