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 protects neuronal cells from MPP+. 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, expression of miR-6862 (A) and SphK1 mRNA (B) was tested by qPCR assays, with SphK1 protein expression tested by Western blotting analyses (B, C); Cell viability and death were tested by the CCK-8 assay (D) and the LDH release assay (E), respectively. 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, expression of miR-6862 (F) and SphK1 mRNA (G) was tested by qPCR, with cell death examined by medium LDH release assay (H). 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.

Figure 2. miR-6862 inhibition protects neuronal cells from MPP+. 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, expression of miR-6862 (A) and SphK1 mRNA (B) was tested by qPCR assays, with SphK1 protein expression tested by Western blotting analyses (B, C); Cell viability and death were tested by the CCK-8 assay (D) and the LDH release assay (E), respectively. 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, expression of miR-6862 (F) and SphK1 mRNA (G) was tested by qPCR, with cell death examined by medium LDH release assay (H). 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.