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MiR-4448 is involved in deltamethrin resistance by targeting CYP4H31 in Culex pipiens pallens
Xixi Li1, Shengli Hu1, Haitao Yin1
1Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, 211166, People's Republic of China.
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.
Insights
MicroRNA-4448 (miR-4448) is downregulated in deltamethrin-resistant Culex pipiens mosquitoes, contributing to insecticide resistance by targeting CYP4H31. Understanding this mechanism is key for effective mosquito control strategies.
Area of Science:
- Molecular Biology
- Entomology
- Genetics
Background:
- Culex pipiens mosquitoes are significant vectors of viral diseases like West Nile virus.
- Deltamethrin resistance in mosquitoes poses a major challenge to public health control efforts.
- MicroRNAs (miRNAs) are critical gene regulators potentially involved in insecticide resistance.
Purpose of the Study:
- To investigate the role of miR-4448 and CYP4H31 in deltamethrin resistance in Culex pipiens pallens.
- To elucidate the molecular regulatory mechanism between miR-4448 and CYP4H31 in insecticide resistance.
Main Methods:
- Quantitative real-time reverse transcription PCR to measure gene expression.
- Dual-luciferase reporter assays to assess regulatory functions.
- In vivo and in vitro experiments including RNA interference and CCK-8 assays to determine functional roles.
Main Results:
- miR-4448 was downregulated in deltamethrin-resistant strains, while CYP4H31 was downregulated in susceptible strains.
- miR-4448 directly targets and downregulates CYP4H31 expression.
- Overexpression of miR-4448 increased mosquito mortality to deltamethrin and reduced cellular resistance.
Conclusions:
- miR-4448 plays a crucial role in deltamethrin resistance in mosquitoes by targeting CYP4H31.
- This study enhances understanding of the molecular mechanisms underlying insecticide resistance in disease vectors.
