Research Paper Volume 13, Issue 1 pp 1369—1382

MicroRNA-6862 inhibition elevates sphingosine kinase 1 and protects neuronal cells from MPP+-induced apoptosis

miR-6862 inhibition attenuates MPP+-induced apoptosis in neuronal cells. Parental control SH-SY5Y cells (“Pare”) as well as stable SH-SY5Y cells, expressing the lentiviral construct encoding the anti-sense of premiR-6862 (lv-antagomiR-6862) or the anti-sense control sequence (lv-antagomiRC), were treated with or without MPP+ (3 mM); Cells were then cultured for applied time periods, the caspase-3/-9 activity (A, B), single strand DNA (ssDNA) contents (C) and mitochondrial depolarization (recording JC-1 intensity at 488 nm, D) were tested. Cell apoptosis was tested by nuclear TUNEL staining assay (E). HCN-2 neuronal cells were transfected with 500 nM of miR-6862 inhibitor (miR-6862i) or the miR inhibitor control (miRiC) for 48h. Cells were then treated with or without MPP+ (3 mM) and cultured for indicated time periods, caspase-3 activity (F) and cell apoptosis (G, H) were tested similarly. Bars stand for mean ± standard deviation (SD, n=5). * P #P + treatment in “Pare” cells or “miRiC” cells. Experiments in this figure were repeated five times, with the similar results obtained. Scale bar= 100 μm (E, G).

Figure 3. miR-6862 inhibition attenuates MPP+-induced apoptosis in neuronal cells. Parental control SH-SY5Y cells (“Pare”) as well as stable SH-SY5Y cells, expressing the lentiviral construct encoding the anti-sense of premiR-6862 (lv-antagomiR-6862) or the anti-sense control sequence (lv-antagomiRC), were treated with or without MPP+ (3 mM); Cells were then cultured for applied time periods, the caspase-3/-9 activity (A, B), single strand DNA (ssDNA) contents (C) and mitochondrial depolarization (recording JC-1 intensity at 488 nm, D) were tested. Cell apoptosis was tested by nuclear TUNEL staining assay (E). HCN-2 neuronal cells were transfected with 500 nM of miR-6862 inhibitor (miR-6862i) or the miR inhibitor control (miRiC) for 48h. Cells were then treated with or without MPP+ (3 mM) and cultured for indicated time periods, caspase-3 activity (F) and cell apoptosis (G, H) were tested similarly. Bars stand for mean ± standard deviation (SD, n=5). * P < 0.05 vs. “Ctrl” treatment in “Pare” cells or “miRiC” cells. #P < 0.05 vs. MPP+ treatment in “Pare” cells or “miRiC” cells. Experiments in this figure were repeated five times, with the similar results obtained. Scale bar= 100 μm (E, G).