Research On The Relationship Between MiR-285 And Drug Resistance In Mosquitoes

Mosquitoes can transmit a lot of human diseases,such as malaria,dengue fever,yellow fever,West Nile fever and Japanese encephalitis.Chemical control has always been an preferred means in the circumstances of mosquito-borne disease epidemic and emergency beacause of its killing efficient and fast characteristics.But,insecticide resistance has been developing rapidly with the wide use of insecticides,even becoming a serious threat of mosquito-borne disease prevention and control.Further knowledge of mechanisms about mosquito resistance to insecticides is an important aspect in vector management and control programs.Metabolic resistance is recogonized to be a main resistance mechanism,especially for cytochrome P450.MicroRNAs can affect regulation of gene expression at the post-transcriptional level in many important biological processes.Although antineoplastic drugs resistance has been proved related with regulation of some microRNAs,whether microRNAs are involved in insecticide resistance in mosquitos is poorly understood.Our previous study analyzed microRNA profiles in pyrethroid-susceptible strain and resistant strain of Culex pipiens pallens using high throughput Solexa sequencing.In those differentially expressed microRNAs,miR-285 is upregulated in resistant strain.Using bioinformatic software,quantitative reverse transcription PCR,luciferase reporter assay and microinjection approaches,we conformed that CYP6N23 was the target of miR-285.Then,we studied the relationship between miR-285 and deltamenthrin resistance through microinjection of miR-285 mimic or inhibitor.Last,the relationship between CYP6N23 and deltamenthrin resistance was identified by microinjecting dsRNA against CYP6N23.Target prediction suggested that the seed sequence of miR-285 paired up with the 3'UTR sequence of CYP6N23,CYP6CD2,CYP4D43,CYP9J37.qPCR analysis showed miR-285 was highly expressed in deltamenthrin-resistant strain,while CYP6N23 was highly expressed in deltamenthrin-susceptible strain,suggesting that miR-285 may regulate the expression of CYP6N23.Luciferase report test showed miR-285 could regulte CYP6N23 expression levels in vitro through binding to its 3'UTR.The expressed tendency was opposite about miR-285 and CYP6N23 after microinjection,which suggested miR-285 could regulte CYP6N23 expression in vivo.CDC bottle bioassay showed that miR-285 mimic-injected group was more resistant to deltamethrin compared with the control groups,suggesting miR-285 was related to deltamenthrin resistance.However,mortality rate of miR-285 inhibitor-injected group had no predicted rising,which might be caused by too little change of CYP6N23 expression level.Meanwhile,knockdown of CYP6N23 by injection of dsRNA decreased mortality rate of mosqutioes,and the mRNA level of CYP6A G11 was down-regulated in the mosqutioes,which indicated that CYP6N23 was responsible for insecticide resistance.In this paper,we found that the expression of CYP6AA9 and CYP6AG11 were all increased when CYP6N23 expression was decreased.This result prompted that CYP6N23 and other P450s may coordinately be responsible for resistance to deltamenthrin.But the further study is necessary.Taken together,our results showed that miR-285 might regulate pyrethroid resistance of mosquitos by decreasing the CYP6N23 expression,which further improves the understanding of molecular mechanism of insecticide resistance and provides a new clue for vector management and control.