Study On The Relationship Between Mosquito MiR - 278-3p And Drug Resistance

Mosquito-borne disease, including malaria, dengue fever, West Nile virus, filariasis and Japanese encephalitis, csuses serious hazard to human health. Chemical control has always been an important means of mosquito control strategy, for its killing speed and convenient characteristics. But with large-scale and unreasonable use of insecticide, insecticide resistance has been produced. WHO predicted that insecticide resistance will become a maor obstacle in the prevention of mosquito-borne disease. Therefore, the study of resistance mechanism and discovery of new resistance genes is still the focus in prevention and control of mosquito-borne diseases. Metabolic resistance is one of the main mechanisms of insecticide resistance, most researches focused on the changes in the expression levels of insecticide metabolism related enzymes, and less involved in the post transcriptional gene regulation mechanism. miRNA is an important factor on post-transcriptional regulation, which can regulate genes through degradating mRNA expression.In our previous study, mi R-278-3p was found low expression in the deltamethrin-resistant strains of Culex pipiens pallens. Based on the result, we predicted its target gene CYP6AG11, detected the both expression level through qPCR, and validated the relationship between miR-278-3p and CYP6AG11 through luciferase report; then, we identified the relationship between CYP6AG11 and deltamenthrin resistance using strategies of genetic over expression and RNAi; last, we studied the relationship between miR-278-3p and deltamenthrin resistance through gene injection of miR-278-3p, while detected the expression level of CYP6AG11 through qPCR.Target prediction suggested that the seed sequence of miR-278-3p paired up with the 3’UTR sequence of CYP6AG11. QPCR analysis showed miR-278-3p was highly expressed in deltamenthrin-sensitive strain, while CYP6AG11 was highly expressed in deltamenthrin-resistant strain, which suggested that miR-278-3p may regulate the expression of CYP6AG11. Luciferase report showed miR-278-3p could regulte CYP6AG11 expression levels through binding to its 3’UTR.Cytotoxicity assay showed that over-expression of CYP6AG11 via transfection comparably increased cell viability in C6/36 cells under deltamethrin treatment, and the mRNA level of CYP6AG11 was up-regulated in C6/36 cells. Meanwhile, knockdown of CYP6AG11 by injection of siRNA increased mortality of mosqutioes, and the mRNA level of CYP6AG11 was down-regulated in the mosqutioes.The expression level of CYP6AG11 was significantly lower in the group injected with mi R-278-3p mimic than in the other groups, suggesting that mi R-278-3p could regulate the expression level of CYP6AG11 in mosquitoes. CDC bottle bioassay showed that miR-278-3p-injected group were more susceptible to deltamethrin compared with the other groups.The expression level of CYP6AG11 and miR-278-3p in the field mosquito populations were detected, qPCR analysis demonstrated that the expression level of miR-278-3p was 2.1-fold and 1.7-fold higher in the deltamethrin-susceptible strain in Shanghe and Pingyin population; while the expression level of CYP6AG11 was respectively 21.3-fold and 2.0-fold higher in the deltamethrin-resistant strain.In this study, our results showed that miR-278-3p might regulate pyrethroid resistance of mosquitos by decreasing the expression level of CYP6AG11. Our study provided a new scientific evidence to clarify the molecular mechanism of insecticide resistance.