Identifying miR-1174 Targets in Ae. aegypti

We downloaded the 3′-untranslated regions (3’-UTRs) of Ae. aegypti from VectorBase (https://vectorbase.org/vectorbase/app) and predicted the candidate targets of miR-1174 using RNAhybrid [24 (link)], miRanda [25 (link)], TargetScan [26 (link)] and Target Accessibility (PITA) [27 (link)]. The predicted candidate targets were crossed with the upregulated proteins, yielding the final candidate targets. The 3′-UTRs of these candidate target genes were amplified by rapid amplification of cDNA ends (3′-RACEs), and cloned into the psiCHECK™-2 vector after digestion with the restriction enzymes Not I and Xho I. The miR-1174 mimic used for cell transfection was purchased from Thermo Fisher Dharmacon (Thermo Fisher Scientific). The three candidate target genes were finally verified by dual-luciferase reporter assay on GloMax®-Multi Detection System (Promega, Madison, WI, USA), as described in our previous work [28 (link)]. The cell line HEK-293T, vector psiCHECK™-2, and reagents for the dual-luciferase reporter assay (Dual-Glo^® Luciferase Assay Reagent and Dual-Glo^® Stop & Glo^® Reagent) were purchased from Promega.