Comparative proteomics analysis of normal thyroid follicular epithelial cell line Nthy-ori 3-1 before and after transfection with PPFP gene / 肿瘤

ABSTRACT

Objective: To explore the protein component change in normal thyroid follicular epithelial cell line Nthy-ori 3-1 transfected with oncogene PAX8/PPARgamma fusion (PPFP) gene using proteomics technologies in order to find the proteins regulated by PPFP gene, providing clues to looking for follicular thyroid carcinoma (FTC) marker proteins or proteins as drug targets. Methods: The Nthy-ori 3-1PPFP cells, Nthy-ori 3-1vector cells and Nthy-ori 3-1 cells were collected to exract total proteins. The two-dimensional gel electrophoresis (2-DE) maps of total proteins in cells in three groups were created. The 2-DE maps of the three groups were analyzed and compared using PDQuest software to find out differentially expressed protein spots. These differentially expressed candidate proteins were identified by matrix-assisted laser desorption-ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS). Five protein spots [prohibitin, galectin-1, cytokeratin 8, cytokeratin 19 and heat shock protein 27 (HSP27)] which were considered to be more closely related to the occurrence and development of FTC and have a higher MALDI-TOF-MS score and a greater coverage were selected and detected by Western blotting. Results: The 2-DE maps of total proteins in Nthy-ori 3-1PPFP, Nthy-ori 3-1vector and Nthy-ori 3-1 cells were successfully established. Thirty-eight differentially expressed protein spots with a difference in expression more than twice between Nthy-ori 3-1PPFP cells and the Nthy-ori 3-1 and Nthy-ori 3-1vector cells were identified by using PDQuest 2-D Analysis Software. Combination of MALDI-TOF- MS and database search identified a total of 28 differentially expressed protein spots. Of all the identified protein spots, 19 proteins were upregulated and 9 proteins were down-regulated in Nthy-ori 3-1PPFP cells as compared with the Nthy-ori 3-1vector and Nthy-ori 3-1 cells. This result was fully consistent with the results of proteomics. Conclusion: Twenty-eight differentially expressed proteins in Nthy-ori 3-1PPFP cells transfected with PPFP gene are identified. These proteins may be dominated and regulated by PPFP gene. This finding provides a basis for clarifying the molecular mechanism of the occurrence and development of FTC.