Figure 5. Experiments in vitro to validate tsRNAs targeting mRNAs by complementarily pairing of the nucleotides. (A) The relative Fzd1 and Duox2 levels were significantly changed after transfecting rno-tRFi-Ser-25a mimics in PC12 cells compared to the control (all P <0.05). (B) The relative expression levels of Mtr and Il1rn were significantly changed after transfecting rno-tRFi-Gln-16a mimics likewise. (C) Schematic representation of the potential binding sites for rno-tRFi-Ser-25a in the Fzd1 3’UTR. Seed sequences of the wild type (Fzd1-WT 3’UTR) and mutant type (Fzd1-mut 3’UTR) luciferase reporter showing the binding site. (D) The relative luciferase activity of the WT and mut reporter constructs, which were cotransfected with either the rno-tRFi-Ser-25a or negative control mimics. Data are presented as the ratio of luciferase activity from the negative control versus the rno-tRFi-Ser-25a mimic-transfected neurons. rno-tRFi-Ser-25a inhibited the luciferase activity of the WT, but not the mut reporter construct. rno-tRFi-Ser-25a directly targeted Fzd1 by binding to the 3’UTR sites. Data are presented as the mean ± SEM and *P<0.05, **P<0.01.