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.

中文翻译：

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Mono-(2-Ethylhexyl)phthalate 通过 MicroRNAs 调控的 m6A RNA 甲基化调节胆固醇流出。

邻苯二甲酸单（2-乙基己基）酯（MEHP）是一种主要的生物活性代谢物，它来自广泛使用的工业增塑剂邻苯二甲酸二（2-乙基己基）酯，已发现它对包括心脏代谢系统在内的几种人体生理系统有毒。最近，RNA 甲基化已被证明与心脏代谢调节有关。尽管 m6A mRNA 甲基化在生理过程中很重要，但缺乏对与内分泌干扰化学物质相关的 RNA 甲基化的研究。在这里，我们使用鼠巨噬细胞 Raw 264.7 细胞研究了 MEHP 在胆固醇流出途径中的作用以及 m6A 甲基化的作用。MEHP 暴露显着降低（50 μM 的 P < 0.01）m6A mRNA 甲基化，mRNA（5 和 50 μM 的 P < 0.01）和蛋白质（0.5 μM 的 P < 0.05；5 μM 的 P < 0.01；对于 50 μM，P < 0.001) METTL14（甲基转移酶复合物的组成部分）的表达水平。然而，m5C RNA 甲基化保持不变。MEHP 显着降低了 Scavenger Receptor B type 1 (SR-B1) 的表达（5 μM P < 0.01，50 μM P < 0.05）。此外，我们证明与 MEHP 处理（P < 0.01）或单独沉默 METTL14（P < 0.05）相比，用 MEHP 沉默 METTL14 降低了 SR-B1 基因表达。此外，MEHP 显着促进 SR-B1 中的 m6A 修饰（P < 0.001）并激活被预测调节 METTL14 的 miRNA，例如 miR16-1-3p（对于 50 μM MEHP，P < 0.05）、miR101a-3p（P < 0.001） 5 和 50 μM MEHP 为 0.05）、miR362-3-5p（50 μM MEHP P < 0.05）、miR501-5p（50 μM MEHP P < 0.01）、miR532-3p（50 μM MEHP P < 0.05）和 miR542-3p (P < 0. 05 为 50 μM MEHP）。总之，这些结果首次揭示了 MEHP 可以调节 RNA 甲基化以调节胆固醇流出途径中的 SR-B1。