FIGURE 3.

Mitophagy receptors FUNDC1 and NIX are direct targets of miR-137. A, diagram of WT and mutant luciferase report constructs. The matched base pairs are connected by a vertical line, and the mutated nucleotides in the target sites are underlined. fundc1 and nix 3′ UTR relevant fragments were inserted downstream of the firefly luciferase gene of the pmirGLO vector. B, luciferase reporter assay by the interaction between miR-137 and the predicted microRNA response elements in HEK293 cells. Each WT luciferase construct was co-transfected with control, in-control, miR-137, or in-137. Meanwhile, the mutant luciferase construct was co-transfected with control and miR-137. About 24 h after transfection, luciferase activity was detected. The firefly luciferase activity was normalized to Renilla. Data are shown as the mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, miR-137 represses endogenous FUNDC1 and NIX expression. NC, miR-137 mimics, and in-137 were transfected into HeLa, HEK293, SKNSH, and SY5Y cells, respectively. At 48 h after transfection, cell lysates were prepared and subjected to Western blot analysis by using anti-FUNDC1 and NIX antibodies.