Biochemical and Biophysical Research Communications
Small interfering RNA targeting the PINK1 induces apoptosis in dopaminergic cells SH-SY5Y
Section snippets
Materials and methods
Cell culture, siRNA transfection, and RNA extraction. Human dopaminergic cell line SH-SY5Y was maintained in DMEM, supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells from passages 10 to 20 were used and they were seeded at 40% confluency into 30 mm six-well dish. The required amount of PINK1 siRNA (Ambion) and 5 μl lipofectamine 2000 (Invitrogen) were diluted into a final volume of 100 μl in Opti-MEM I (Gibco), respectively, and gently mixed and
Results
Real-time RT-PCR analysis showed a dose-dependent reduction of PINK1 mRNA amplification following transfection with 5–160 nM PINK1 siRNA. At 80–160 nM PINK1 siRNA significantly reduced PINK1 mRNA as much as 87% (Fig. 1A). The inhibitory effects lasted up to 4 days following a single dose of siRNA treatment and there was no significant difference between 80 and 160 nM siRNA treatment for 24 h (13.3 ± 5.2 and 13.4 ± 4.3 of control #3 siRNA treated group, respectively, p = 0.974). The protein levels of PINK1
Discussion
Parkinson disease (PD) is a progressive neurodegenerative disorder, associated with the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Current hypothesis suggests that neurodegeneration in PD is associated with a cascade of events that involve genetic factors, oxidative stress, mitochondrial abnormalities, excitatory amino acids, and a rise in intracytoplasmic free calcium. At least five genes have been identified as causal genes for PD: α-synuclein (PARK1) [9],
Acknowledgments
This work was supported by Grants NS 043567 and NS 40370 from the National Institute of Neurological Disorders and Stroke. The authors thank Dr. Junjiang Fu (Department of Molecular and Cellular Biology), Dr. Jonathan Lu and Dr Max Huang (Departments of Medicine, Baylor College of Medicine) for their technical assistance in this study.
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