Methods: Whole stimulated saliva was used. Normal saliva from two donors and pre- and post-treatment saliva from three HNSCC patients were analyzed by iTRAQ (isotope tag for relative and absolute quantitation) proteomic analysis. One putative biomarker, DMBT1 (deleted in malignant brain tumors 1), was validated by immunoblot analysis on 12 paired saliva samples. DMBT1 expression in HNSCC cell lines was investigated by Q-RT-PCR and immunoblot analysis. Silencing of DMBT1 was investigated by modulation of EZH2 by siRNA.
Results: : DMBT1 in saliva was upregulated in normal and post-treatment versus pre-treatment samples. On immunoblot analysis, DMBT1 was upregulated in 9/12 patients at 6 months post-treatment and in 11/12 patients at 12 months post-treatment. In HNSCC cell lines, low expression of DMBT1 was observed. In HNSCC, siRNA-mediated knockdown of EZH2, a histone methyl transferase, increased DMBT1 expression.
Conclusion: This study, based on a small group of patients, suggests that DMBT1 is downregulated in the saliva of HNSCC patients and is upregulated after treatment. DMBT1 can be detected by immunoblot analysis in saliva. Furthermore, in vitro studies show that DMBT1 gene expression in cancer cells is silenced by a histone methyltransferase.
Keywords: Diagnosis, Oral biology, Oral mucosa, Pathology and Saliva