20-Hydroxyecdysone (20-HE) plays essential roles in coordinating developmental transitions of insects through responsive protein-coding genes and microRNAs (miRNAs). The involvement of single miRNAs in the ecdysone-signalling pathways has been extensively explored, but the interplay between ecdysone and the majority of miRNAs still remains largely unknown. Here, by small RNA sequencing, we systematically investigated the genome-wide responses of miRNAs to 20-HE in the embryogenic cell lines of Bombyx mori and Drosophila melanogaster. Over 60 and 70 20-HE-responsive miRNAs were identified in the BmE cell line and S2 cell line, respectively. The response of miRNAs to ecdysone exhibited a time-dependent pattern, and the response intensity increased with extending exposure to 20-HE. The relationship between ecdysone and the miRNAs was further explored through knockdown of ecdysone-signalling pathway genes. Specifically, ecdysone regulated the cluster miR-275 and miR-305 through the coordination of BmEcR-B and downstream BmE75B, and the interaction between BmEcR and miR-275 cluster was strengthened by the feedback regulation of BmE75B. Ecdysone induced miR-275-3p and miR-305-5p through the ecdysone response effectors (EcREs) at the upstream of the pre-miR-275 cluster. Overall, the results might help us further understand the relationship between ecdysone signalling pathways and small RNAs in the development and metamorphosis of insects.

20-羟基蜕皮激素（20-HE）在通过响应性蛋白编码基因和microRNA（miRNA）协调昆虫的发育过渡中起重要作用。蜕皮激素信号途径中涉及单个miRNA的问题已得到广泛研究，但是蜕皮激素与大多数miRNA之间的相互作用仍然很大程度上未知。在这里，通过小RNA测序，我们系统地研究了家蚕和果蝇果蝇细胞系中miRNA对20-HE的全基因组反应。在BmE细胞系和S2细胞系中分别鉴定出60多种和70种20-HE反应性miRNA。miRNA对蜕皮激素的反应表现出时间依赖性，并且反应强度随着暴露于20-HE而增加。蜕皮激素和miRNA之间的关系通过蜕皮激素信号通路基因的敲低进一步探索。具体而言，通过蜕皮激素的协调调节的簇的miR-275和miR-305 BmEcR-B和下游BmE75B，和之间的相互作用BmEcR和miR-275簇通过的反馈调节加强BmE75B。蜕皮激素通过pre-miR-275簇上游的蜕皮激素反应效应因子（EcREs）诱导了miR-275-3p和miR-305-5p。总体而言，结果可能有助于我们进一步了解蜕皮激素信号传导途径与昆虫发育和变态中的小RNA之间的关系。