Methods: Proteomics (HPLC-MS/MS) was used to identify changes in protein constituency and subsequent function in lung adenocarcinoma cells following silencing of the RAGE gene. Proteins were identified at 99% confidence with XCorr score cut-offs as determined by reversed database search. The protein and peptide identification results were identified using Scaffold v 4.3.4, a program that relies on search engine results (e.g. Sequest, X!Tandem, MASCOT) and uses Bayesian statistics to reliably identify spectral counts. Four biological replicates were performed for each condition, and the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification system was used to classify differentially expressed proteins and segregate them according to cellular process and functions. State-of-the-art biochemical and histology assays were used to verify cellular and molecular physiological functions. Single comparisons were performed using Student's t-test. Bonferroni's correction was also used for multiple testing in the PANTHER Overrepresentation Test.
Results: HPLC-MS/MS identify over 100 differentially expressed proteins following gene silencing of RAGE. PANTHER classification identified changes cellular processes including proteins representative of immune and metabolism responses. Biochemical assays demonstrate that RAGE gene silencing attenuates the ability of the lung adenocarcinoma cell to a pro-growth/pro-inflammatory signaling event, suggesting a potential role in the evolution of carcinogenesis in lung epithelial cells.
Conclusion: RAGE likely plays an important role in the evolution of lung epithelial cells to adenocarcinoma, and RAGE likely plays a significant role in the extent to which lung adenocarcinoma cells may respond to stimuli. Collectively, these data suggest that RAGE may be a potential therapeutic target.