microRNA-14 as an efficient suppressor to switch off ecdysone production after ecdysis in insects.

The precise increase and decrease of hormone ecdysone are critical for accurate development in insects. Most previous works focus on transcriptional activation of ecdysone production; however, little is known about the mechanism of switching off ecdysone biosynthesis after ecdysis. Here, we showed that the precursor microRNA-14 (pre-miR-14) encodes two mature miRNAs in silkworm; both of these two mature miRNAs regulate various genes in the ecdysone-signalling pathway. Bmo-miR-14-5p targets on nine genes whereas Bmo-miR-14-3p targets on two genes in the same pathway. These two mature miRNAs increased immediately after the ecdysis, efficiently suppressing the 20-hydroxyecdysone (20E) biosynthesis, the upstream regulation, and the downstream response genes. Knocking down either of two mature miRNAs or both of them delays moult development, impairing development synchrony in antagomir-treated groups. In addition, overexpressing Bmo-miR-14-5p but not Bmo-miR-14-3p significantly affected the 20E titer and increased the moulting time variation, suggesting that Bmo-miR-14-5p, though it is less abundant, has more potent effects in development regulation than Bmo-miR-14-3p. In summary, we present evidence that a pre-miRNA encodes two mature miRNAs targeting on the same pathway, which significantly improves miRNA regulation efficiencies to programmatically switch off ecdysone biosynthesis.