Expression of the Neuron-Related Genes in hNGF-β-Treated PC12 Cells

Human nerve growth factor-β (hNGF-β) plays a major role in the development and survival of the central and peripheral nervous systems; therefore, it can, in principle, be used as a therapeutic agent for the treatment of neurodegenerative diseases. As was shown, hNGF-β stimulates neuronal differentiation in vitro and regulates the expression of neuron-related genes. In our study, expression of the ATP1B1, Clusterin, MAO-A, and DUSP6 genes was evaluated in hNGF-treated PC12 cells. At first, hNGF-β protein was generated in a pET39b/BL21 (DE3) bacterial expression system, and biological activity of this protein was confirmed by differentiation of cultured PC12 cells. These cells were treated by the addition of recombinant hNGF-β protein in a final dose of 50 ng/ml diluted in RPMI 1640. Then total RNA was extracted from harvested PC12 cells at day 7 after treatment and used to analyze the expression levels of the above-mentioned genes by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. It was found that purified hNGF-β protein migrated as a clear band of ~37 kDa in SDS-PAGE. Moreover, differentiation of PC12 cells was observed 7 days after hNGF-β treatment. Expression of the ATP1B1 and DUSP6 genes was upregulated (230 and 193%), respectively after addition of hNGF-β protein. In contrast, the MAO-A and Clusterin genes showed practically no change in their expression, as compared to untreated cells. Generally, our study demonstrated that the treatment of PC12 cells with hNGF-β protein induces neural morphological modifications and differences in the expression of certain neuron-related genes. Changes in the expression of these genes will be applied in an experimental model.