MiR-4448 Is Involved in Deltamethrin Resistance by Targeting CYP4H31 in Culex Pipiens Pallens
Abstract Background: Mosquitoes are vectors of serious diseases affecting human health. Unfortunately, mosquitoes have developed deltamethrin resistance because of its long-term overuse, representing a major challenge to mosquito control. Understanding the molecular regulatory mechanisms of resistance is vital to control mosquitoes. MicroRNAs (miRNAs) are short non‑coding RNAs that have been demonstrated as important regulators of gene expression across a wide variety of organisms, which might function in mosquito deltamethrin resistance. Methods: In the present study, we used quantitative real-time reverse transcription PCR to measure miR-4448 and CYP4H31 (encoding a cytochrome P450) expression levels. The regulatory functions of miR-4448 and CYP4H31 were assessed using Dual-Luciferase reporter assays. Then, oral feeding, RNA interference, and the American Centers for Disease Control and Prevention bottle bioassay were used to determine miR‑4448’s association with deltamethrin resistance by targeting CYP4H31 in vivo. A Cell Counting Kit-8 (CCK-8) was also used to detect the viability of pIB/V5-His-CYP4H31-transfected C6/36 cells after deltamethrin treatment in vitro. Results: MiR-4448 was downregulated in the deltamethrin-resistant strain (DR-strain), whereas CYP4H31 was downregulated in deltamethrin-susceptible strain. negatively regulated CYP4H31 expression was downregulated by miR-4448 recognizing and binding to its 3' untranslated region. Functional verification experiments showed that miR-4448 overexpression resulted in lower expression of CYP4H31. The mortality of miR-4448 mimic-injected DR-strain mosquitoes was higher than that of the controls. CCK-8 assays showed that CYP4H31 decreased cellular resistance to deltamethrin in vitro and the mortality of the DR-strain increased when CYP4H31 was knocked down CYP4H31 in vivo. Conclusions: In mosquitoes, miR-4448 participates in deltamethrin resistance by targeting CYP4H31. The results of the present study increase our understanding of deltamethrin resistance mechanisms.