Up-stream mechanisms for up-regulation of miR-125b from triclosan exposure to zebrafish (Danio rerio)

Triclosan (TCS) exposure has widely adverse biological effects such as influencing biological reproduction and endocrine disorders. While some studies have addressed TCS-induced expression changes of miRNAs and their related down-stream target genes, no data are available concerning how TCS impairs miRNA expression leading us to study up-stream regulating mechanisms. Four miRNAs (miR-125b, miR-205, miR-142a and miR-203a) showed differential expression between TCS-exposure treatments and the control group; their functions mainly involved fatty acid synthesis and metabolism. TCS exposure led to the up-regulation of mature miR-125b that was concomitant with consistent changes in pri-mir-125b-1 and pri-mir-125b-3 among its 3 pri-mir-125bs. Up regulation of miR-125b originated from direct shear processes involving the two up-regulated precursors, but not pri-mir-125b2. Increased expression of pri-mir-125b-1 and pri-mir-125b-3 resulted from nfe212- and c/ebp alpha-integration with positive control elements of promoters for the two precursors. The overexpression of transcriptional factors, nfe2l2 and c/ebp alpha, initiated the promoter activity for the miR-125b precursor. CpG islands and Nfe2l2 were involved in constitutive expression of mir-1256-1 and mir-125b-3. The activities of two promoter regions, -487 to -1 bp for pri-mir-125b1 and -1327 to +14 bp for pri-mir-125b-3 having binding sites for NFE2 and Nfe212/MAF:NFE2, were higher than other regions, further demonstrating that the transcriptional factor Nfe2l2 was involved in the regulation of pri-mir-125b1 and pri-mir-125b-3. TCS's estrogen activity resulted from its effects on GPER, a novel membrane receptor, rather than the classical ER alpha and ER beta. These results explain, to some extent, the up-stream mechanism for miR-125b up-regulation, and also provide a guidance to future mechanistic study on TCS-exposure.