The miR-282-5p regulates larval moulting process by targeting chitinase 5 in Bombyx mori.

Moulting is critical for growth, development and survival in insects. As the main components of cuticle, dynamic change of chitin is consistent with the moulting process. Chitinase is the main enzyme to mediate chitin metabolism in the old cuticle. To avoid over-degrading chitin from the new cuticle, the expression of chitinase must be precisely regulated. In this study, we performed microRNA-sequencing to investigate expression change of microRNAs in silkworm epidermis during the moulting process. A comparative microRNA transcriptomic analysis from different moulting stages and 20-hydroxyecdysone (20E) treatment identified bmo-miR-282-5p as a candidate. By the bioinformatic analysis, chitinase 5 (BmCht5) was predicted to be a target of bmo-miR-282-5p. Meanwhile, a temporal expression analysis revealed that BmCht5 only expressed at moulting D3 stage, whereas bmo-miR-282-5p showed a converse pattern, in which its transcript signal disappeared at this time point. Furthermore, a luciferase assay and agomir treatment demonstrated that bmo-miR-282-5p suppressed transcript of BmCht5 in vivo. As a result, injection of 282-5p agomir triggered 40% death due to moulting failure. In addition, RNA interference (RNAi)-mediated silencing of BmCht5 caused 30% developmental defect. Taken together, our data demonstrate the coordinated regulation of chitinase 5 by conserved miR-282-5p, and the 20E signalling pathway is essential for the normal moulting process in the domesticated silkworm.