A Schematic showing the sequences of human miR-106b and miR-93, which were predicted to target the 3’UTRs of OPTN, NDP52, and MFN2. B Schematics representing the reconstructed dual-luciferase reporter plasmids. The WT 3’UTRs of OPTN, NDP52, and MFN2 containing the miRNA-binding sequences were cloned downstream of Renilla luciferase (hRluc). Artificially mutated miRNA-binding sequences (OPTN-Mut, MFN2-Mut, and NDP52-Mut) were also cloned downstream of Renilla luciferase. Mutations are marked in red. C Cells were transfected with reporter plasmids containing the WT 3’UTR of OPTN or a mutant OPTN 3’UTR (OPTN-Mut) along with a miR-106b mimic or miR-93 mimic for 48 h. Firefly luciferase (hluc+) and Renilla luciferase (hRluc) activity levels were measured successively, and the values are presented as the relative fluorescence intensity (hRluc/hluc+). N = 3; **P < 0.01. The quantified results are presented as the mean ± SD. D Cells were transfected with reporter plasmids containing the WT 3’UTR of MFN2 or a mutant MFN2 3’UTR (MFN2-Mut) along with a miR-106b mimic or miR-93 mimic for 48 h. The Renilla luciferase activity was normalized to the firefly luciferase activity. N = 3; ***P < 0.005; ****P < 0.001. The quantified results are presented as the mean ± SD. E Cells were transfected with reporter plasmids containing the WT 3’UTR of NDP52 or a mutant NDP52 3’UTR (NDP52-Mut) along with a miR-106b mimic or miR-93 mimic for 48 h. The normalized luciferase activity (ratio of hRluc to hluc+) was measured in cell lysates. N = 3; ***P < 0.005. The quantified results are presented as the mean ± SD. F, G, I The levels of OPTN, NDP52, and MFN2 in WT cells, three miR-106b-KO cell lines, three miR-93-KO cell lines, and three miR-25-KO cell lines were evaluated by WB analysis. N = 3; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. The data are from three independent tests and are presented as the mean ± SD. H WB analysis of OPTN, NDP52, and MFN2 levels 48 h after transfection with a miRNA mimic NC, a miR-106b mimic, or a miR-93 mimic. Tubulin was used as an endogenous control. N = 3; **P < 0.01; ***P < 0.001; ****P < 0.0001. The data are from three independent tests and are presented as the mean ± SD. J Three cell lines (ShCtrl, shRNF2-1, and shRNF2-2) were transfected with EGFP-C1 or EGFP-Parkin and stimulated with 100 μΜ H2O2 for 0 h or 12 h. The cell lysates were subjected to WB analysis using the indicated antibodies. N = 3; ns, not significant; *P < 0.05; **P < 0.01. The data are from three independent tests and are presented as the mean ± SD.