Abstract
This work demonstrates that a comprehensive strategy of proteomics identification combined with further validation and detailed functional analysis should be adopted in the field of cancer biomarker discovery. A comparative proteomics approach was utilized to identify differentially expressed proteins in 10 oral squamous carcinoma samples paired with their corresponding normal tissues. A total of 52 significantly and consistently altered proteins were identified with eight of these being reported for the first time in oral squamous carcinoma. Of the eight newly implicated proteins, RACK1 was chosen for detailed analysis. RACK1 was demonstrated to be up-regulated in cancer at both the mRNA and protein levels. Immunohistochemical examination showed that the enhanced expression of RACK1 was correlated with the severity of the epithelial dysplasia as well as clinical stage, lymph node involvement, and recurrence, which are known indicators of a relatively poor prognosis in oral squamous carcinoma patients. RNA interference specifically targeted to silence RACK1 could initiate apoptosis of oral squamous carcinoma cells. Taken together, the results indicate that RACK1 is up-regulated in oral squamous carcinoma, not only being closely related to cell proliferation and apoptosis but also linked to clinical invasiveness and metastasis in carcinogenesis. The observations suggest that RACK1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of oral squamous carcinoma. Further this comprehensive strategy could be used for identifying other differentially expressed proteins that have potential to be candidate biomarkers of oral squamous carcinoma.
Highlights
This work demonstrates that a comprehensive strategy of proteomics identification combined with further validation and detailed functional analysis should be adopted in the field of cancer biomarker discovery
The patients who had advanced clinical stage disease (p ϭ 0.005), metastasis (p Ͻ 0.001), or recurrent lesion (p ϭ 0.01) showed increased RACK1 expression. Because the latter three factors have a great impact on the prognosis of patients, we propose that RACK1 is a good potential predictor of tumor outcome (Table IV)
The results showed that, in Tca8113 cells, siRACK1-231 induced 20% cell apoptosis compared with the controls (2.1% for PBS controls, 2.3% for liposome controls, and 4.7% for the small interfering RNA (siRNA) for negative control (Fig. 6A)
Summary
Tissue Samples—All tissue specimens were obtained from West China Stomatological Hospital, Sichuan University. Tryptic In-gel Digestion—In-gel digestion of proteins was carried out using mass spectrometry grade Trypsin Gold (Promega, Madison, WI) according to the manufacturer’s instructions. Protein Identification and Database Searching—The MS/MS data, “pkl list” (pkl) files acquired by the software ProteinLynx 2.2.5 (Waters), included the mass values and the intensity and the charge of the precursor ions (parent ions with ϩ2 or ϩ3 charge in this study). RNA Interference—Four pairs of RACK1-specific small interfering RNA (siRNA) and the siRNA for negative control were synthesized by Ambion Inc. The cells were grown on a 6-well plate in growth medium until they reached 70% confluence; transfections of siRNA were carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For Western blotting analysis, ϳ20 g of protein were separated by 12% SDS-PAGE, transferred to a PVDF membrane (Millipore), and probed with polyclonal rabbit anti-RACK1 antibody (1:1000; Santa Cruz Biotechnology). Acyl-CoA-binding protein Fatty acid-binding protein, epidermal Carbonic anhydrase 3 Ubiquitin-conjugating enzyme
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