Study On The Relationship Between MiR-279-3p And MiR-4448 In Culex Pipiens Pallens And Its Drug Resistance

Mosquito-borne diseases, including malaria, dengue fever, West Nile fever, have been a major public health problem in the developing, even the developed countries. Currently, there are few vaccines against most such diseases. Chemical control has been the primary control strategy for managing mosquito-borne diseases. Unfortunately, along with the long-term use of chemical insecticides, mosquitoes have developed resistance to multiple insecticides.Studies have shown that the resistance is determined by genes, and up-regulation of several detoxify gene families, as:esterases, P450monooxygenases and glutathione S-transferases, are closely involved in resistance development. However, the regulatory mechanisms are rarely reported. Our preliminary study generated the microRNA profiles for deltamethrin-susceptible (DS) and deltamethrin-resistant (DR) strains of Culex pipiens pallens, and the compared analysis determined several differential-expressed microRNAs. In this study, miR-279-3p and miR-4448-3p, which were significantly up-regulated in deltamethrin-susceptible strains, were selected for further investigation.First, precursor sequences of miR-279-3p and miR-4448were cloned by TA cloning and their expression levels in DS and DR strains were detected by RT-PCR. CYP325BB1and CYP4H31were predicted to be the target genes of these two microRNAs, and their expression level were also detected by RT-PCR. Then, luciferase report assay was used to identify whether miR-279-3p and miR-4448directly regulate CYP325BB1and CYP4H31expression. In vitro, CCK8was used to analyze the influence of the overexpression of CYP325BB1and CYP4H31on sensitivity to deltamethrin in mosquito cell lines. In vivo, microRNA mimics were supplied to mosquitoes by oral feeding and microinjection, then the expression level of their target genes was detected by RT-PCR, and biochemical assays was used to detect the sensitivity to deltamethrin of mosquitoes after the oral feeding and microinjection experiment.The results showed the expression level of miR-279-3p and miR-4448in DS strain was3.3-fold (P<0.01) and5.5-fold (P<0.001) higher than that in DR strain. And the expression level of CYP325BB1and CYP4H31in DS strain was2.3-fold (P<0.01) and4.97-fold (P<0.001) higher than that in DR strain. The results of luciferase report assay confirmed miR-279-3p and miR-4448can directly regulate CYP325BB1and CYP4H31expression levels via binding to its3’UTR. Overexpression of CYP325BB1and CYP4H31made the mosquito cell become less sensitize to deltamethrin. In vivo, overexpression of miR-279-3p and miR-4448reduced the expression level of their target genes and increased the sensitivity to deltamethrin in mosquitoes. Taken together, these results indicated that miR-279-3p and miR-4448may have effect on mosquito deltamethrin-resistance through regulating CYP325BB1and CYP4H31expression.This is the first study that reports microRNA are involved in mosquito insecticides resistance through directly regulating its target genes transcriptional expression, which has important theoretical significance for investigating the mechanisms of mosquito insecticides resistance. In addition, this study provides new theory basis for establishing more target-based control and prevention techniques for mosquito resistance.