miR-275/305 cluster is essential for maintaining energy metabolic homeostasis by the insulin signaling pathway in Bactrocera dorsalis

Abstract

Increasing evidence indicates that miRNAs play crucial regulatory roles in various physiological processes of insects, including systemic metabolism. However, the molecular mechanisms of how specific miRNAs regulate energy metabolic homeostasis remain largely unknown. In the present study, we found that an evolutionarily conserved miR-275/305 cluster was essential for maintaining energy metabolic homeostasis in response to dietary yeast stimulation in Bactrocera dorsalis. Depletion of miR-275 and miR-305 by the CRISPR/Cas9 system significantly reduced triglyceride and glycogen contents, elevated total sugar levels, and impaired flight capacity. Combined in vivo and in vitro experiments, we demonstrated that miR-275 and miR-305 can bind to the 3’UTR regions of SLC2A1 and GLIS2 to repress their expression, respectively. RNAi-mediated knockdown of these two genes partially rescued metabolic phenotypes caused by inhibiting miR-275 and miR-305. Furthermore, we further illustrated that the miR-275/305 cluster acting as a regulator of the metabolic axis was controlled by the insulin signaling pathway. In conclusion, our work combined genetic and physiological approaches to clarify the molecular mechanism of metabolic homeostasis in response to different dietary stimulations and provided a reference for deciphering the potential targets of physiologically important miRNAs in a non-model organism.

MiR-2b-2-5p regulates lipid metabolism and reproduction by targeting CREB in Bactrocera dorsalis

“…Deletion of miR-184-3p leads to termination of oocyte development [ 54 ]. Our previous study also found that the miR-275/305 cluster negatively regulated the transcription of SLC2A1/GLIS2 , eventually ensuring adult metabolic homoeostasis in B. dorsalis [ 55 ]. In this study, depletion of miR-2b-2-5p led to failure of lipid storage and ovarian development, whereas overexpression of miR-2b-2-5p resulted in lipid overaccumulation but displayed a similar reproductive phenotype as miRNA inhibition in B. dorsalis .…”

“…Meanwhile, the expression level of miR-2b-2-5p was relatively low under starvation conditions compared with under feeding conditions. In a previous study, we found that the miR-275/305 cluster is regulated by the insulin signalling pathway in a nutrient-dependent manner [ 55 ]. Whether nutrient signalling, such as insulin or TOR signalling, is involved in the regulation of the miR-2b-2-5p/ CREB axis needs further study.…”

“…Metabolic assays were performed 3 days after antagomir/mimics injection. which have been optimized according to our previous study [ 55 ]. Briefly, five adult flies were homogenized in 1 ml PBST buffer (0.05% Tween 20) and immediately incubated at 70°C for 5 min to inactivate endogenous enzymes.…”

“…Nile Red staining was performed as described in our previous study [ 55 ]. Briefly, fat bodies were fixed in 4% paraformaldehyde (Coolaber, China) and washed with 1×PBS two or three times.…”