MiR-4448 is involved in deltamethrin resistance by targeting CYP4H31 in Culex pipiens pallens

Background Culex pipiens ( Cx. pipiens ) complex, which acts as a vector of viruses and is widespread and abundant worldwide, including West Nile virus, Japanese encephalitis virus, and Sindbis virus, can cause serious vector-borne 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 to be important regulators of gene expression across a wide variety of organisms, which might function in mosquito deltamethrin resistance. In the present study, we aimed to investigate the regulatory functions of miR-4448 and CYP4H31 in the formation of insecticidal resistance in mosquito Culex pipiens pallens. Methods 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 . 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. 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 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. Graphical Abstract