Proteomics analysis of siRNA-mediated silencing of Wilms' tumor 1 in the MDA-MB-468 breast cancer cell line

Abstract

The Wilms' tumor 1 (WT1) gene encodes a zinc finger which appears to be a transcriptional activator or repressor for many genes involved in cell differentiation, growth and apoptosis. In order to determine the relationship between WT1 and related proteins, WT1 was silenced with small interfering RNA (siRNA) and the protein expression pattern was analyzed by proteomics analysis including one-dimensional gel electrophoresis (1-DE) and LC-MS/MS mass spectrometry. The results revealed that 14 proteins were expressed in WT1-silenced cells (siRNA WT1) and 12 proteins were expressed in the WT1-expressing cells (siRNA neg), respectively. These proteins may be classified by their functions in apoptosis, cell signaling, protein folding, gene expression, redox-regulation, transport, structural and unknown functions. Mitogaligin, an apoptosis-related molecule, was identified when WT1 was silenced while the proteins related to the signaling pathway were detected in both siRNA neg and siRNA WT1 but the type of proteins were different. For example, the IBtK protein and the SH2 domain-containing protein were present in siRNA WT1 conditions, while the platelet-derived growth factor receptor α (PDGFRA) and Rho guanine nucleotide exchange factor 1 (Rho-GEF 1) were expressed in siRNA neg. Of these, Rho-GEF was selected for validation by western blot analysis and demonstrated to be present only in the presence of WT1. In conclusion, WT1 is related to mitogaligin via EGFR and behaves as an anti-apoptotic molecule. Moreover, WT1 may be associated with PDGFRA and Rho-GEF 1 that activates proliferation in MDA-MB-468 cells.