Figure 1.

MiR-7 is a major suppressor of α-Syn expression. (A). Predicted binding sites of miRs on the 3′-UTR of α-Syn mRNA (∼2500 bp). The putative binding sites of different miRs are annotated at the approximate positions on the α-Syn mRNA 3′-UTR. These sites were predicted by TargetScan (47) or provided by miRTarBase (48). Sites highlighted by the red arrows were previously reported. (B). MiR-7 inhibited luminescence of a dual luciferase reporter bearing the gene of α-Syn mRNA 3′-UTR downstream the Renilla luciferase gene. Equal amount of each pCG-pri-miR plasmid was co-transfected with the luciferase reporter. PCG-pri-miR-9 was tested as a negative control. Luciferase levels were recorded 48 h after transfection. Mean Renilla/firefly values and SEM from six independent repeats are shown. (C). Single-nucleotide mutation of miR-7 binding site inactivated the inhibitive effects of miR-7. The nucleotide on the third position of miR-7 targeted seed regions on α-Syn mRNA 3′-UTR gene was mutated individually. The mutants were numbered according to the binding sites from 5′ to 3′ of α-Syn mRNA 3′-UTR. Luciferase levels were measured 48 h after the co-transfection of pCG-pri-miR-7–1 plasmids with reporters bearing wild-type or mutated α-Syn mRNA 3′-UTR gene. The luciferase levels were relative to co-transfection of pCG-pri-miR-9 with wild-type reporter. Mean Renilla/Firefly values and SEM from three independent repeats are shown. (D andE). α-Syn expression was inhibited by upregulated miR-7. 1: Mock HeLa cells without DNA transfected. 2–6: An increasing amount of pCG-pri-miR-7–1 was transfected into HeLa cells. The expression of α-Syn and α-tubulin were detected by western blot 48 h after transfection. Relative α-Syn/α-tubulin levels were normalized to mock. Mean values and SEM from three independent repeats are shown. Statistically significant differences compared to mock were interpreted by SPSS one-way ANOVA, with post hoc LSD test, *P< 0.05, **P< 0.01, ***P< 0.001.