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 overexpression augments MPP+-induced neuronal cell death. Parental control SH-SY5Y cells (“Pare”) as well as stable SH-SY5Y cells, with the lentiviral construct encoding the premiR-6862 sequence (lv-premiR-6862) or the nonsense miRNA sequence (lv-miRC), were treated with or without MPP+ (3 mM), cells were then cultured for applied time periods, SphK1 mRNA and protein expression was tested (A); Cell viability and death were tested by CCK-8 assay (D) and LDH release assay (E), respectively; Caspase-9 activation (D), single strand DNA (ssDNA) contents (E) and mitochondrial depolarization (JC-1 dye assay, F) were tested, with cell apoptosis tested by nuclear TUNEL staining assay (G). HCN-2 neuronal cells were transfected with 500 nM of miR-6862 mimic or the miR control mimic (“miRC”) for 48h. Cells were then treated with or without MPP+ (3 mM) and cultured for indicated time periods, SphK1 mRNA expression (H), medium LDH contents (I), and cell apoptosis (by recording TUNEL-positive nuclei, (J) were tested. Bars stand for mean ± standard deviation (SD, n=5). * P #P + treatment in “Pare” cells or “miRC” cells. Experiments in this figure were repeated five times, with the similar results obtained.

Figure 4. miR-6862 overexpression augments MPP+-induced neuronal cell death. Parental control SH-SY5Y cells (“Pare”) as well as stable SH-SY5Y cells, with the lentiviral construct encoding the premiR-6862 sequence (lv-premiR-6862) or the nonsense miRNA sequence (lv-miRC), were treated with or without MPP+ (3 mM), cells were then cultured for applied time periods, SphK1 mRNA and protein expression was tested (A); Cell viability and death were tested by CCK-8 assay (D) and LDH release assay (E), respectively; Caspase-9 activation (D), single strand DNA (ssDNA) contents (E) and mitochondrial depolarization (JC-1 dye assay, F) were tested, with cell apoptosis tested by nuclear TUNEL staining assay (G). HCN-2 neuronal cells were transfected with 500 nM of miR-6862 mimic or the miR control mimic (“miRC”) for 48h. Cells were then treated with or without MPP+ (3 mM) and cultured for indicated time periods, SphK1 mRNA expression (H), medium LDH contents (I), and cell apoptosis (by recording TUNEL-positive nuclei, (J) were tested. Bars stand for mean ± standard deviation (SD, n=5). * P < 0.05 vs. “Ctrl” treatment in “Pare” cells or “miRC” cells. #P < 0.05 vs. MPP+ treatment in “Pare” cells or “miRC” cells. Experiments in this figure were repeated five times, with the similar results obtained.