Di-n-butyl phthalate promotes monocyte recruitment via miR-137-3p-SP1-MCP-1 pathway.

Since non-covalent bound character and widespread application in numerous products, people are exposed to di-n-butyl phthalate (DBP) at low levels through various ways. Epidemiological studies suggested an association between DBP exposure and atherosclerosis (AS). Still, molecular mechanisms remain unclear. This study aimed to explore the effects of DBP on monocyte recruitment, a key and initial step of AS. EA.hy926 cells were treated with DBP (10-9-10-5 M) or DMSO as control. Chemotaxis assay was applied to investigate THP-1 recruitment. Expression of mRNA /miRNAs and proteins were measured by qRT-PCR and Western blotting, respectively. Levels of monocyte chemotactic protein 1 (MCP-1) in supernatant were detected by ELISA assay. Receptor internalization assay was performed to confirm C-C chemokine receptor type 2 (CCR2) subcellular localization in THP-1 cells and the binding between CCR2 and MCP-1. Dual-luciferase reporter assay was used to analyze the combination between miR-137-3p and specificity protein 1 (SP1), as well as SP1 and MCP-1. Results showed that number of recruited THP-1 cells after EA.hy926 cells treated by DBP was significantly higher than that in the control group due to promoted MCP-1 expression. In addition, expression of MCP-1 was regulated through miR-137-3p-SP1 cascade. Besides, overexpression of miR-137-3p reversed the increased number of recruited THP-1 cells. Our results implied that DBP might promote THP-1 recruitment by targeting miR-137-3p-SP1-MCP-1 in EA.hy926 cells.

Di-n-butyl phthalate regulates vascular smooth muscle cells phenotypic switching by MiR-139-5p-MYOCD pathways.

Di-n-butyl phthalate (DBP) is ubiquitous in environment and has been detected in almost all human bodies. Few data could be found about the effects of DBP on cardiovascular system, though its reproductive toxicities have been studied extensively. This study aimed to explore effects of DBP on phenotypic switching of vascular smooth muscle cells (VSMCs), an essential step during the formation of atherosclerosis (AS). A7r5 cells were employed and exposed to various levels of DBP (10-9, 10-8, 10-7, 10-6, and 10-5 M) or DMSO as control. CCK-8 assay was used to detect the effects of DBP on cell viability. Expressions of mRNA/miRNAs and proteins were measured by qRT-PCR and western blotting, respectively. Bioinformatic analysis and dual-luciferase reporter assay were used to analyze the combination between miR-139-5p and Myocardin (MYOCD). Results revealed that DBP at 10-7 M prompted phenotypic switching from contractile to synthetic of VSMCs by inhibiting contractile VSMCs marker genes via suppressing the expression of MYOCD. Moreover, miR-139c-5p directly targeted MYOCD 3'UTR and modulated MYOCD expression. Besides, DBP inhibited the expression of MYOCD and VSMCs marker genes by upregulating miR-139-5p. Collectively, these data suggested that DBP could promote the phenotypic switching from contractile to synthetic of VSMCs in A7r5 cells through miR-139-5p-MYOCD.