Oral squamous cell carcinoma (OSCC) develops from normal epithelium via squamous intraepithelial lesions (SILs) as result of a multistep process, characterized by accumulation of altered gene expression, including NANOG. NANOG is a transcription factor, playing crucial role in embryonal development, later it can contribute to cancer development. We analysed diagnostic significance and regulation of NANOG on protein, RNA and DNA levels in oral SILs and OSCC.
Our study included 367 biopsy tissue samples from 172 patients with oral SCC and SILs, and 30 samples of normal oral mucosa. Immunohistochemistry, qPCR and Sanger sequencing were used. At mRNA level, NANOG was expressed in all samples, expression was low in normal mucosa and intensive in high grade SILs, in OSCC it was decreased. At protein level, NANOG was not found in normal mucosa, it was re-expressed in cancerogenesis, ranging from mild staining in low grade SILS to strong staining in high grade SILs and OSCC. Regulatory miRNAs and lncRNAs correlated with expression of NANOG, whereas copy number variation and promoter methylation were not changed.
Our results suggest that expression of NANOG protein is silenced before birth and re-expressed in high grade SILs and OSCC and could be thus used as a diagnostic marker for classifying SILs. The expression of NANOG mRNA was different than the expression of NANOG protein, indicating an important role of post-transcription regulation, with miRNA and lncRNA as the key regulatory mechanisms beside protein regulators. Methylation of its promoter and copy number variation probably have a minor role in the regulation of NANOG expression. We also observed that morphologically normal mucosa adjacent to cancer showed an altered expression of NANOG and its regulators, in contrast to morphologically normal mucosa of healthy persons. Our results thus contributed new insights into the role of NANOG in the development of OSCC.