MYCN RNA interference decreases anti-apoptotic gene expression and proliferation of neuroblastomas

Abstract

Abstract Introduction: MYCN amplification is the most reliable prognostic predictor for neuroblastomas (NB); dysregulation of MYCN expression is critical for NB pathogenesis. Neuroblastomas with MYCN amplification are often resistant to chemotherapeutic drugs and irradiation-induced apoptosis. This process, in part, involves alterations in the regulation of Bcl-2 proteins. Therefore, the purpose of this study was to determine whether MYCN gene silencing alters expression of anti-apoptotic Bcl-2 family proteins and, ultimately, decreases NB proliferation. Methods: Human neuroblastoma cell lines, BE(2)-C, IMR-32, Lan-1, were transiently transfected with small interference (si)RNA against MYCN or control vector (GFP siRNA) over a time-course. Proteins were extracted to determine expression of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1) by Western blot. To assess changes in cell proliferation, colorimetric bromodeoxyuridine (BrdU) immunoassays were performed. Results: Transfection with MYCN siRNA significantly decreased MYCN protein expression in a time-dependent manner in all three NB cell lines. MYCN siRNA also decreased expression of Bcl-2 family proteins (Bcl-2, Bcl-xL and Mcl-1) and significantly decreased DNA synthesis, as measured by BrdU incorporation, in all NB cell lines. Conclusions: Our data demonstrate that MYCN gene silencing down-regulates Bcl-2 family protein expression and subsequent DNA synthesis in NB. Selective targeting of MYCN by RNA interference may be a useful adjunct in the treatment of NB with high MYCN amplification.