Multifactorial approach to broaden target identification using miR-2940 from the mosquito Aedes aegypti

miRNAs are part of an ever-expanding field of systems controlling eukaryotic gene regulation. In the dengue mosquito, Aedes aegypti, the miRNA aae-miR-2940-5p has been shown to be important in the maintenance of the endosymbiont Wolbachia pipientis and necessary for contribution to the dengue restriction phenotype. Currently only a few mRNA targets for aae-miR-2940-5p have been identified. Here we conduct a multifactorial approach to identify targets for the miRNA. Using biotinylated synthetic aae-miR-2940-5p in a cell line model and also injection of mimics in adult female mosquitoes we conducted high throughput sequencing and differential gene expression analyses to implicate direct mRNA targets of aae-miR-2940-5p within both datasets. Favorable miRNA/mRNA interactions were then screened in silico and manually validated using RT-qPCR. These genes represent a broad responsibility in many host processes. While these techniques indicate reasonable success in the prediction of direct miRNA-mRNA interactions, several significantly upregulated genes in aae-miR-2940-5p injections were also identified. These included Imd pathway effectors DefA (200%), DefD (170%) and CTLGA5 (100%). We coupled together these results provide a reasonable insight into the broad regulatory network of aae-miR-2940-5p.