Research Paper Volume 12, Issue 22 pp 22794—22813

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.