Fig. 3.

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 (BIM_EL, BIM_L, BIM_S) 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.