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 directly binds to and silences SphK1 in SH-SY5Y neuronal cells. miRNA-6862 putatively targets SphK1 mRNA 3’-UTR (untranslated region, at position of 113-120) (A). miRNA-6862 (fluorescence-tagged) locates in the cytoplasm of SH-SY5Y cells (B). RNA pull down showed a directing binding between biotinylated-miR-6862 and SphK1 mRNA in SH-SY5Y cells (C); Stable SH-SY5Y cells with the lentiviral construct encoding the premiR-6862 sequence (lv-premiR-6862) or the nonsense miRNA sequence (lv-miRC) were established, expression of listed genes (mRNA and protein) was shown (D, F, G). The relative SphK1 3’-UTR luciferase reporter activity was tested as well (E). SH-SY5Y cells were transfected with 500 nM of wile-type (“WT-“) or the mutant (“Mut1-”/“Mut2-”) miRNA-6862 mimics (sequences were listed in H), control cells were transfected with nonsense control miRNA (“miRC”), after 48h the relative SphK1 3’-UTR luciferase reporter activity (I) and SphK1 mRNA expression (J) were tested. Stable SH-SY5Y cells with the lentiviral construct encoding the anti-sense of premiR-6862 (lv-antagomiR-6862) or the anti-sense control sequence (lv-antagomiRC) were established, expression of listed genes was shown (K–N). The relative SphK1 3’-UTR luciferase reporter activity was tested as well (L). “Pare” stands for the parental control cells (same for all Figures). Data were presented as mean ± standard deviation (SD, n=5). * P

Figure 1. miR-6862 directly binds to and silences SphK1 in SH-SY5Y neuronal cells. miRNA-6862 putatively targets SphK1 mRNA 3’-UTR (untranslated region, at position of 113-120) (A). miRNA-6862 (fluorescence-tagged) locates in the cytoplasm of SH-SY5Y cells (B). RNA pull down showed a directing binding between biotinylated-miR-6862 and SphK1 mRNA in SH-SY5Y cells (C); Stable SH-SY5Y cells with the lentiviral construct encoding the premiR-6862 sequence (lv-premiR-6862) or the nonsense miRNA sequence (lv-miRC) were established, expression of listed genes (mRNA and protein) was shown (D, F, G). The relative SphK1 3’-UTR luciferase reporter activity was tested as well (E). SH-SY5Y cells were transfected with 500 nM of wile-type (“WT-“) or the mutant (“Mut1-”/“Mut2-”) miRNA-6862 mimics (sequences were listed in H), control cells were transfected with nonsense control miRNA (“miRC”), after 48h the relative SphK1 3’-UTR luciferase reporter activity (I) and SphK1 mRNA expression (J) were tested. Stable SH-SY5Y cells with the lentiviral construct encoding the anti-sense of premiR-6862 (lv-antagomiR-6862) or the anti-sense control sequence (lv-antagomiRC) were established, expression of listed genes was shown (KN). The relative SphK1 3’-UTR luciferase reporter activity was tested as well (L). “Pare” stands for the parental control cells (same for all Figures). Data were presented as mean ± standard deviation (SD, n=5). * P < 0.05 vs. “lv-miRC”/“miRC”/“lv-antagomiRC” cells. Experiments in this figure were repeated five times with similar results obtained.