Metamorphosis is one of the most important physiological processes in insects. It is regulated by a serial of ecdysone cascade genes. Recently, lots of microRNAs (miRNAs) were investigated in insects; however, their function in metamorphosis is largely unknown. In the present study, the dynamics of a small RNA population was investigated by RNA sequencing from the midgut of a lepidopteran pest Spodoptera litura during larval–pupal metamorphosis. A total of 101 miRNAs were identified, and 75 miRNAs were differentially expressed during the metamorphic process. The relationship between these differentially expressed miRNAs and 12 ecdysone cascade genes was analyzed by four classical software programs, and a multiple‐to‐multiple regulatory network was found to exist between these miRNAs and their targets. Among them, miR‐14‐3p and its two targets (EcR and E75) were chosen for further validation. MiR‐14‐3p had higher expression level in the 6th instar larvae as compared with either the prepupae or pupae, which was opposite to that of both EcR and E75, two ecdysone cascade genes. Luciferase reporter assay confirmed that both EcR and E75 were regulated by miR‐14‐3p. Interestingly, the 3′ untranslated regions are nearly identical to each other among different transcript variants of the ecdysone cascade genes, including EcR, USP, E75, E74, E78, E93, Hr3, Hr4, Hr39, Krh1 and Ftzf1. Thus, different transcript variants of one ecdysone cascade gene could be regulated by the same miRNA. The above data suggest that the ecdysone signaling pathway is under the tight control of miRNA. These findings expand our understanding of the mechanism of insect metamorphosis and may also provide a novel possibility for the control of pest insects in the future.

中文翻译：

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多个 microRNA 控制斜纹夜蛾中肠中的蜕皮激素信号

变态是昆虫最重要的生理过程之一。它受一系列蜕皮激素级联基因的调控。最近，在昆虫中研究了大量的微小RNA（miRNA）。然而，它们在变态中的功能在很大程度上是未知的。在本研究中，通过对鳞翅目害虫斜纹夜蛾 (Spodoptera litura) 幼虫-蛹变态过程中肠的 RNA 测序，研究了小 RNA 种群的动态。共鉴定出101个miRNA，75个miRNA在变质过程中差异表达。通过四个经典软件程序分析了这些差异表达的 miRNA 与 12 个蜕皮激素级联基因之间的关系，发现这些 miRNA 与其靶标之间存在多对多调控网络。他们之中，选择 miR-14-3p 及其两个靶标（EcR 和 E75）进行进一步验证。与预蛹或蛹相比，MiR-14-3p 在 6 龄幼虫中的表达水平更高，这与两个蜕皮激素级联基因 EcR 和 E75 的表达水平相反。荧光素酶报告基因检测证实 EcR 和 E75 均受 miR-14-3p 调控。有趣的是，在蜕皮激素级联基因的不同转录变体中，3' 非翻译区几乎彼此相同，包括 EcR、USP、E75、E74、E78、E93、Hr3、Hr4、Hr39、Krh1 和 Ftzf1。因此，一种蜕皮激素级联基因的不同转录变体可以由相同的 miRNA 调节。以上数据表明蜕皮激素信号通路受到miRNA的严格控制。