Evaluation of anti-Fas ligand-induced apoptosis and neural differentiation of PC12 cells treated with nerve growth factor using small interfering RNA method and sampling by microdialysis

Abstract

The small interfering RNA (siRNA) method is an effective technique for silencing gene expression and is a useful tool for screening the gene functions in drug discovery. Our study found that nerve growth factor (NGF) can increase the cell viability of PC12 cells and that NGF induction up-regulates the expression of Bcl-2 detected by real-time reverse transcription–polymerase chain reaction (RT–PCR). To further investigate the role of Bcl-2 expression in NGF-treated PC12 cells, the plasmid of Bcl-2 siRNA was then transfected into PC12 cells. Moreover, to investigate and continuously monitor the real-time dynamic neurotransmitter release, and to compare with the time course of Bcl-2 expression, a liquid chromatography coupled with electrochemical detection (LC–ED) and with a microdialysis device was used. After 6 h of NGF being added to the PC12 cell culture medium, the dopamine (DA) concentrations were significantly increased ( P < 0.05). This result is simultaneously compatible with the up-regulated messenger RNA (mRNA) expressions of tyrosine hydroxylase (TH), aromatic acid decarboxylase (AADC), and Bcl-2 by RT–PCR. Using the Bcl-2 siRNA method, our data revealed that NGF can inhibit Fas ligand (FasL)-induced apoptosis in PC12 cells through the activation of Bcl-2. The in vitro observation further demonstrated that NGF can stimulate the neurite development in PC12 cells through the activation of Bcl-2. Moreover, the DA concentrations of NGF induction were decreased specifically by Bcl-2 siRNA ( P < 0.05). In sum, our data support that NGF prevents Fas-induced apoptosis, facilitates neural differentiation, promotes dendritic formation, and increases DA release in PC12 cells through activation of Bcl-2.