Abstract
Background Results of previous studies suggest that NANOG may be an important prognostic biomarker in oral squamous cell carcinoma (OSCC), but there are contradictory results regarding NANOG expression patterns on mRNA and protein levels, and the mechanisms of its regulation are poorly understood. Our aim was to analyze the expression and diagnostic significance of NANOG in OSCC, and the possible mechanisms of its regulation, i.e., protein regulators on mRNA level (OCT4, SOX2, KLF4, AGR2, and NOTCH1), methylation status, copy number variation, and regulatory miRNAs, miR-145, miR-335, miR-150, miR-34a, miR-128, and miR-27a. Methods Our study included 120 patients with OSCC. Expression of NANOG protein and mRNA was analyzed using immunohistochemistry and qPCR. Expression of regulatory factors, miRNAs, and copy number variation was performed using qPCR. Methylation status of NANOG promoter was determined using PCR and Sanger sequencing. Results We detected upregulation of NANOG and OCT4 and downregulation of NOTCH1 and AGR2 mRNA in OSCC with lymph node metastases compared to OSCC without lymph node metastases. We observed a strong positive correlation between mRNAs of NANOG and those of its protein regulators OCT4, SOX2, NOTCH1, AGR2, and KLF4. The expression of NANOG was in positive correlation with the expression of miR-34a. There was also a correlation between T status of OSCC and the expression of miR-335 and miR-150 and a correlation of miR-150 with the N status of T2 OSCC. NANOG promoter methylation and copy number variation were only observed in a small proportion of samples. Conclusions Our findings confirm the diagnostic significance of NANOG in OSCC and provide information on NANOG expression patterns on both mRNA and protein levels. They also suggest that protein regulators and microRNAs might play a crucial role, whereas methylation of its promoter and copy number variation probably have a minor role in the regulation of NANOG expression in OSCC.
Highlights
Despite intensive research, head and neck squamous cell carcinomas still have a very high mortality rate (50%) and an overall 5-year survival of only about 40–50%
Our findings confirm the diagnostic significance of NANOG in oral squamous cell carcinoma (OSCC) and provide information on NANOG expression patterns on both mRNA and protein levels. They suggest that protein regulators and microRNAs might play a crucial role, whereas methylation of its promoter and copy number variation probably have a minor role in the regulation of NANOG expression in OSCC
To understand the regulation of the NANOG in the development and progression of OSCC better, we aimed to characterize the expression of NANOG in correlation with its regulators OCT4, SOX2, KLF4, AGR2, and NOTCH1 and with methylation status [24], copy number variation (CNV), and regulatory miRNAs, miR-145, miR-335, miR-150, miR-34a, miR-128, and miR-27a [25,26,27,28,29,30]. miRNAs were chosen based on miRNA-NANOG interactions in miRTarBase and TarBase v. 8 based on strong evidence validation methods
Summary
Head and neck squamous cell carcinomas still have a very high mortality rate (50%) and an overall 5-year survival of only about 40–50%. Results of previous studies suggest that NANOG may be an important prognostic biomarker in oral squamous cell carcinoma (OSCC), but there are contradictory results regarding NANOG expression patterns on mRNA and protein levels, and the mechanisms of its regulation are poorly understood. Our aim was to analyze the expression and diagnostic significance of NANOG in OSCC, and the possible mechanisms of its regulation, i.e., protein regulators on mRNA level (OCT4, SOX2, KLF4, AGR2, and NOTCH1), methylation status, copy number variation, and regulatory miRNAs, miR-145, miR-335, miR-150, miR34a, miR-128, and miR-27a. Our findings confirm the diagnostic significance of NANOG in OSCC and provide information on NANOG expression patterns on both mRNA and protein levels They suggest that protein regulators and microRNAs might play a crucial role, whereas methylation of its promoter and copy number variation probably have a minor role in the regulation of NANOG expression in OSCC
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