Fig 2: miR-221 Down-Regulates transcripts involved in apoptosis, including Pro-Apoptotic protein BIM, and protects DJ-1 knock-down cells from cell death. (A) Compared to cells transfected with negative control pre-miR (pre-miR-NC), SH-SY5Y cells transfected with pre-miR-221 showed a decrease of mRNA transcripts implicated in apoptosis, including BIM, BMF, BNIP3L, and FOXO3A. (B) All three isoforms of BIM protein (BIMEL, BIML, BIMS) were decreased by over-expression of miR-221 and (C) increased by the inhibition of miR-221 using anti-miR-221. (D) Pre-miR-221 transfected cells were treated with 250 uM H2O2 for 24 h. Compared to control, over-expression of miR-221 prevented the oxidative stress mediated induction of pro-apoptotic BIM transcript. (E) Cells were made to stably express non-targeting control (Ctrl) short hairpin RNA (shRNA) or pooled DJ-1 shRNA. Cells were then transduced with either lentiviral control miR (Ctrl miR) or miR-221 for 24 h. Stable DJ-1 KD decreases the levels of mature miR-221, while transduction with lentiviral miR-221 robustly increases its levels. (F) Cells were then treated with the indicated concentrations of MPP+ for 24 h, and cell death was assessed using LDH assay. Compared to control KD cells, stable DJ-1 KD leads to increased cell death, which is rescued when miR-221 is over-expressed. Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) relative to control. ns = not significant. (A, D, F) analyzed using two-way ANOVA with Bonferroni post-hoc multiple comparisons test. (B, C) analyzed using two-tailed student's t-test. (E) Analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test.

Fig 3: miR-221 expression is modulated by wild-type DJ-1 but not its pathogenic mutant. SH-SY5Y cells were transfected with either pooled DJ-1 siRNA (si-DJ-1) or non-targeting control siRNA (si-NT). (A) Immunoblotting shows that DJ-1 is knocked down effectively by pooled DJ-1 siRNA. (B) DJ-1 knock-down (KD) decreases mature miR-221 and (C) precursor pre-miR-221. (D) DJ-1 knockout mouse brains have lower levels of mature miR-221 compared to wild-type mice. (E) DJ-1 knockout mice lack DJ-1 expression. (F) SH-SY5Y cells were transfected with either si-NT or si-DJ-1 for 24 h, followed by transfection with either empty control vector, FLAG-tagged wild-type DJ-1, or FLAG-tagged pathogenic M26I mutant DJ-1. Immunoblotting shows expression of FLAG-tagged wild-type and mutant DJ-1 protein, and the downregulation of endogenous DJ-1 protein in DJ-1 KD cells. (G) Transfection of wild-type DJ-1 but not its pathogenic M26I mutant in DJ-1 KD cells rescued mature miR-221 expression and (H) precursor pre-mir-221 levels. Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) relative to control. (B–D) analyzed using two-tailed student's t-test. (G, H) analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test.

Fig 4: DJ-1 modulates miR-221 expression through the MAPK/ERK1/2 pathway. To investigate whether DJ-1 could modulate miR-221 levels through its effect on the MAPK/ERK pathway, (A) SH-SY5Y cells were treated with MEK1/2 inhibitor, U0126 (50 µM), or vehicle (DMSO) for 24 h. This led to a decrease in levels of active, phosphorylated ERK1/2 without impacting the levels of total ERK1/2 or DJ-1 protein. (B) The inhibition of ERK1/2 phosphorylation led to down-regulation of miR-221 levels. (C) DJ-1 KD cells were transfected with FLAG-tagged hERK1 or vector-only control for 48 h. hERK1 overexpression increased phosphorylation of ERK1 in DJ-1 KD cells (D) and partially rescued the decrease in miR-221 levels. Data are presented as means ± S.E.M. Asterisks denote statistically significant differences (**p ≤ 0.01, ****p ≤ 0.0001) relative to control. (B) analyzed using two-tailed student's t-test. (D) analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test. Veh = vehicle. Vec = vector-only control.