Qiao Huili, Tong Ziqian, Wang Yuanzhuo, Yang Juanjuan, Sun Yanyan, Shi Huixuan, Liu Zhuo, Duan Jianping, Li Dandan, Kan Yunchao
Henan Key Laboratory of Insect Biology in Funiu Mountain, Henan International Joint Laboratory of Insect Biology, College of Life Science, Nanyang Normal University, Nanyang, Henan, 473061, China.
School of Resourses and Enviroment, Henan Institute of Science and Technology, Xinxiang, Henan, 453003, China.
BMC Genomics. 2025 Mar 31;26(1):317. doi: 10.1186/s12864-025-11499-9.
20-Hydroxyecdysone (20E) is an important hormone that regulates insect development and metamorphosis. The fat body of insects plays a crucial role in nutrient storage and energy metabolism and is considered the exchange center for regulating insect development. The fat body undergoes remarkable transformation during insect metamorphosis and is primarily regulated by 20E. microRNAs (miRNAs) have been identified in different insects and have multiple functions in various physiological processes. However, the interaction of 20E and miRNAs in fat body regulation remains unclear.
We constructed six small RNA libraries using Bombyx mori fat body treated with 20E. Expression and functional analyses were conducted to identify 20E-responsive miRNAs. In total, 431 miRNAs were identified, including 389 known and 42 novel miRNAs. Differential expression analysis revealed significant expression changes in the expression of 40, 9, and 18 miRNAs at 2 h, 6 h, and 12 h after 20E treatment, respectively. The expression of 10 miRNAs was validated using quantitative real-time PCR. miR-34-5p is a highly conserved miRNA among the 10 validated miRNAs, and autophagy-related gene 1 (Atg1) was considered a target gene of miR-34-5p. The expression analysis of miR-34-5p and Atg1 exhibited an opposite expression pattern in the fat body after the 20E treatment. Dual-luciferase assay indicated that miR-34-5p could inhibit Atg1 expression by targeting a binding site in CDS region of Atg1. In larval fat body, overexpressing miR-34-5p by injecting miR-34-5p agomir suppressed the expression of Atg1 and autophagy, whereas knocking down miR-34-5p by injecting miR-34-5p antagomir induced the expression of Atg1 and autophagy. Meanwhile, Atg1 silencing by RNAi also inhibited autophagy. These results indicate that miR-34-5p participates in 20E-induced autophagy in B. mori fat body by interacting with Atg1.
We systematically identified and functionally characterized miRNAs associated with 20E regulation in the fat body of B. mori. miR-34-5p is involved in 20E-induced autophagy in B. mori by regulating its target gene Atg1. These results provide insight into the role of sophisticated interactions between miRNAs, 20E regulation, and autophagy in fat body remodeling and insect metamorphosis.
20-羟基蜕皮酮（20E）是一种调节昆虫发育和变态的重要激素。昆虫的脂肪体在营养储存和能量代谢中起关键作用，被认为是调节昆虫发育的交换中心。在昆虫变态过程中，脂肪体会发生显著变化，且主要受20E调控。微小RNA（miRNA）已在不同昆虫中被鉴定出来，并在各种生理过程中具有多种功能。然而，20E与miRNA在脂肪体调控中的相互作用仍不清楚。
我们用20E处理家蚕脂肪体构建了6个小RNA文库。通过表达和功能分析来鉴定对20E有反应的miRNA。总共鉴定出431个miRNA，包括389个已知的和42个新的miRNA。差异表达分析显示，在20E处理后2小时、6小时和12小时，分别有40个、9个和18个miRNA的表达发生了显著变化。用定量实时PCR验证了10个miRNA的表达。miR-34-5p是10个经验证的miRNA中高度保守的miRNA，自噬相关基因1（Atg1）被认为是miR-34-5p的靶基因。20E处理后，miR-34-5p和Atg1在脂肪体中的表达分析呈现出相反的表达模式。双荧光素酶测定表明，miR-34-5p可通过靶向Atg1编码区的一个结合位点来抑制Atg1的表达。在幼虫脂肪体中，通过注射miR-34-5p激动剂过表达miR-34-5p可抑制Atg1的表达和自噬，而通过注射miR-34-5p拮抗剂敲低miR-34-5p则诱导Atg1的表达和自噬。同时，通过RNA干扰使Atg1沉默也抑制了自噬。这些结果表明，miR-34-5p通过与Atg1相互作用参与了20E诱导的家蚕脂肪体自噬。
我们系统地鉴定了家蚕脂肪体中与20E调控相关的miRNA，并对其进行了功能表征。miR-34-5p通过调节其靶基因Atg1参与了20E诱导的家蚕自噬。这些结果为深入了解miRNA、20E调控和自噬之间复杂的相互作用在脂肪体重塑和昆虫变态中的作用提供了思路。
