Figure 2

Proteomic (2DE-DIGE) profiling identifies novel proteins and pathways modulated by miR-27a in CRC cell lines. (a) miR-27a expression was analysed by quantitative reverse transcriptase-PCR in the indicated CRC cell lines. The histogram reports the fold change with respect to HT29 cells taken as control. U6 RNA was used as calibrator. (b) HCT116 cells were stably transfected with the miR-Zip-27a vector, carrying the miR-27a antisense and the GFP reporter gene, or with the empty vector as control. Cells were isolated for GFP expression and for the miR-27a low-expressing clones (miR27a_KD); **P≤0.01 (two-tailed Student’s t-test). (d) Schematic representation of the 2DE-DIGE/MS procedure from sample preparation and labelling to gel analysis and protein identification. A representative image of a gel loaded with extracts from HCT116 CTRL and miR27a_KD cells is illustrated: about 1200 protein spots were identified, quantified, normalized, and inter-gel matched. Proteins were recognized by fold change of the corresponding spots. (e) The proteins identified by the 2DE-DIGE were classified into nine functional classes, according to their biological activities, as depicted in the pie chart. (f) A three-dimensional view of the 2DE-DIGE quantification is illustrated along with the standard abundance of the spots of interest identified in the gels obtained from HCT116 CTRL and miR27a_KD cells extracts. (g) Western blotting analysis of the selected proteins identified as differentially expressed in the 2DE-DIGE/MS analysis in HCT116 CRTL, miR27a_KD and miR27a_OE cells (HCT116-derived cell line that overexpresses an exogenous miR-27a). The relative protein fold change, obtained by densitometric analysis and normalization to β-Actin, is reported in the corresponding histogram. *P≤0.05; **P≤0.01 (two-tailed Student’s t-test). Data are representative of three independent experiments and error bars represent S.D. of technical replicates (mean±S.D.) in panels (a), (b), (c) and (g)