Temporal quantitative proteomics by iTRAQ 2D-LC-MS/MS and corresponding mRNA expression analysis identify post-transcriptional modulation of actin-cytoskeleton regulators during TGF-beta-Induced epithelial-mesenchymal transition.

ABSTRACT

To gain insights into how TGF-beta regulates epithelial-mesenchymal transition (EMT), we assessed the time course of proteins and mRNAs during EMT by multiplex iTRAQ labeling and 2D-LC-MS/MS, and by hybridization, respectively. Temporal iTRAQ analysis identified 66 proteins as differentially expressed during EMT, including newly associated proteins calpain, fascin and macrophage-migration inhibitory factor (MIF). Comparing protein and mRNA expression overtime showed that all the 14 up-regulated proteins involved in the actin-cytoskeleton remodeling were accompanied by increases in corresponding mRNA expression. Interestingly, siRNA mediated knockdown of cofilin1 potentiated TGF-beta-induced EMT. Further analysis of cofilin1 and beta-actin revealed an increase in their mRNA stability in response to TGF-beta, contributing to the observed increase in mRNA and protein expression. These results are the first demonstration of post-transcriptional regulation of cytoskeletal remodelling and a key role for cofilin1 during TGF-beta-induced EMT.