ω-3 Polyunsaturated Fatty Acids Up-regulate the Expression of 15-PGDH by Inhibiting miR-26a and miR-26b in Human Cholangiocarcinoma Cells

Running Title: ω-3 PUFAs up-regulate the expression of 15-PGDH in cholangiocarcinoma Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Yao et al ω-3 PUFAs up-regulate the expression of 15-PGDH by inhibiting miR-26a/b ABSTRACT Prostaglandins E 2 (PGE 2) is a pro-inflammatory and pro-tumorigenic lipid mediator that promotes cholangiocarcinoma growth in vitro and in vivo. The NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S)-hydroxyl group of PGE 2 , leading to PGE 2 inactivation. Therefore, induction of 15-PGDH expression may represent a new strategy for the treatment of human cholangiocarcinoma. Here we report that ω-3 polyunsaturated fatty acids (ω-3 PUFAs) up-regulate the expression of 15-PGDH by inhibiting miR26a and miR26b and these effects contribute to ω-3 PUFA-induced inhibition of cholangiocarcinoma growth. Treatment of human cholangiocarcinoma cells (CCLP1 and TFK-1) with ω-3 PUFA (DHA) or transfection of these cells with the Fat-1 gene (encoding Caenorhabditis elegans desaturase which converts ω-6 PUFA to ω-3 PUFA) significantly increased 15-PGDH protein level in cholangiocarcinoma cell lines. On the other hand, DHA treatment or Fat-1 overexpression had little effect on 15-PGDH promoter activity. Human cholangiocarcinoma cells treated with DHA or transfected with Fat-1 expression vector showed reduction of miRNA26a and miRNA26b (both miRNAs target 15-PGDH mRNA thus inhibiting 15-PGDH translation). Consistent with these findings, we observed that overexpression of miR26a or miR26b decreased 15-PGDH protein, reversed ω-3 PUFA-induced accumulation of 15-PGDH protein, and prevented ω-3 PUFA-induced inhibition of cholangiocarcinoma cell growth. Knockdown of 15-PGDH also attenuated ω-3 PUFA-induced inhibition of tumor cell growth. We observed that ω-3 PUFA suppressed miRNA26a and miRNA26b by inhibiting c-myc, a transcription factor that co-regulates gene clusters comprising of miR-26a/b with their host genes CTDSPs. Accordingly, overexpression of c-myc enhanced the expression of miRNA26a/b and prevented ω-3 PUFA-induced inhibition of tumor cell growth. Taken together, our results provide novel evidence for induction of 15-PGDH via suppression of miR26a/b in human cholangiocarcinoma cells and suggest ω-3 PUFA as potential agent for the treatment of human cholangiocarcinoma. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.