Post-transcriptional regulatory mechanisms play important roles in the regulation of long-chain (≥ C₂₀) polyunsaturated fatty acid (LC-PUFA) biosynthesis. Here, we address a potentially important role of the miR-15/16 cluster in the regulation of LC-PUFA biosynthesis in rabbitfish Siganus canaliculatus. In rabbitfish, miR-15 and miR-16 were both highly responsive to fatty acids affecting LC-PUFA biosynthesis and displayed a similar expression pattern in a range of rabbitfish tissues. A common potential binding site for miR-15 and miR-16 was predicted in the 3′UTR of peroxisome proliferator-activated receptor gamma (pparγ), an inhibitor of LC-PUFA biosynthesis in rabbitfish, and luciferase reporter assays revealed that pparγ was a potential target of miR-15/16 cluster. In vitro individual or co-overexpression of miR-15 and miR-16 in rabbitfish hepatocyte line (SCHL) inhibited both mRNA and protein levels of Pparγ, and increased the mRNA levels of Δ6Δ5 fads2, Δ4 fads2, and elovl5, key enzymes of LC-PUFA biosynthesis. Inhibition of pparγ was more pronounced with co-overexpression of miR-15 and miR-16 than with individual overexpression in SCHL. Knockdown of miR-15/16 cluster gave opposite results, and increased mRNA levels of LC-PUFA biosynthesis enzymes were observed after knockdown of pparγ. Furthermore, miR-15/16 cluster overexpression significantly increased the contents of 22:6n-3, 20:4n-6 and total LC-PUFA in SCHL with higher 18:4n-3/18:3n-3 and 22:6n-3/22:5n-3 ratio. These suggested that miR-15 and miR-16 as a miRNA cluster together enhanced LC-PUFA biosynthesis by targeting pparγ in rabbitfish. This is the first report of the participation of miR-15/16 cluster in LC-PUFA biosynthesis in vertebrates.

中文翻译：

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miR-15/16 簇通过靶向 pparγ 参与了脊椎动物 LC-PUFA 生物合成的调控，正如在兔鱼 Siganus canaliculatus 中所证明的那样。

转录后调控机制在长链（≥ C ₂₀）多不饱和脂肪酸（LC-PUFA）生物合成的调控中起重要作用。在这里，我们解决了 miR-15/16 簇在调节兔鱼Siganus canaliculatus LC-PUFA 生物合成中的潜在重要作用。在兔鱼中，miR-15 和 miR-16 都对影响 LC-PUFA 生物合成的脂肪酸高度敏感，并在一系列兔鱼组织中显示出相似的表达模式。miR-15 和 miR-16 的共同潜在结合位点在过氧化物酶体增殖物激活受体 γ（pparγ）的 3'UTR 中被预测，该受体是兔鱼中 LC-PUFA 生物合成的抑制剂，荧光素酶报告基因分析显示pparγ是 miR-15/16 簇的潜在目标。兔鱼肝细胞系 (SCHL) 中 miR-15 和 miR-16 的体外单独或共过表达抑制了 Pparγ 的 mRNA 和蛋白质水平，并增加了LC 关键酶Δ6Δ5 fads2、Δ4 fads2和elovl5的 mRNA 水平-PUFA 生物合成。在SCHL 中，与单个过表达相比，miR-15 和 miR-16 的共过表达对pparγ 的抑制更明显。敲除 miR-15/16 簇得到相反的结果，敲除 pparγ后观察到 LC-PUFA 生物合成酶的 mRNA 水平增加. 此外，miR-15/16 簇过表达显着增加 SCHL 中 22:6n-3、20:4n-6 和总 LC-PUFA 的含量，其中 18:4n-3/18:3n-3 和 22:6n- 3/22:5n-3 比率。这些表明 miR-15 和 miR-16 作为 miRNA 簇通过靶向兔鱼中的pparγ共同增强了 LC-PUFA 的生物合成。这是关于 miR-15/16 簇参与脊椎动物 LC-PUFA 生物合成的第一份报告。