Figure 8.

The Apoptotic Inducer Traf3 Is a Target Gene of miR-188-3p

RGCs were transfected with 50 nM of miR-188-3p mimic, Traf3 siRNA, or scramble, 24 h before I/R of 2/2 h. (A) Putative miR-188-3p complementary binding sites in the 3′ UTR region of mouse Traf3. (B) Detection of luciferase activity, 48 h after transfection of miR-188-3p mimic or scramble and the luciferase constructs of Traf3 3′ UTR WT or Traf3 3′ UTR MUT1 (position 94–100 of Traf3 3′ UTR) or Traf3 3′ UTR MUT2 (position 292–298 of Traf3 3′ UTR). The data were expressed as mean ± SEM of five independent experiments. #p < 0.05 versus Traf3 3′ UTR WT/scramble; *p < 0.05 versus Traf3 3′ UTR WT with miR-188-3p mimic. (C–E) miR-188-3p suppresses the expression of Traf3. Traf3 levels of expression were analyzed by quantitative real-time PCR in (C) and by western blot in (D) and (E). The data were expressed as mean ± SEM of five independent experiments. #p < 0.05 versus the scramble group; *p < 0.05 versus the scramble/I/R group. (F and G) The levels of Traf3 were induced by I/R treatment at indicated time points, which were analyzed by western blotting. The data were expressed as mean ± SEM of five independent experiments. #p < 0.05 versus the 0/0 grouop. (H and I) The knockdown of Traf3 expression by Traf3 siRNA attenuates apoptosis upon I/R. Cell apoptosis analysis by flow cytometry in each group. (J–M) Western blot showing that the knockdown of Traf3 significantly suppresses the expression of Traf3 and cleaved caspase3. The data were expressed as mean ± SEM of five independent experiments. #p < 0.05 versus the scramble group; *p < 0.05 versus the scramble/I/R group.