Kodai Nakamura

MicroRNAs (miRNAs) have attracted attention as non-invasive cancer biomarkers in various cancers; however, they have not been adequately investigated in oral squamous cell carcinoma (OSCC). This study investigated the diagnostic performance of serum-derived miRNAs at initial diagnosis for primary neck lymph node metastasis and the predictive performance for late neck lymph node metastasis based on long-term (up to approximately 8 years) follow-up of patients with OSCC. The expression of miRNAs in 40 patients with OSCC was quantified using real-time PCR (qPCR), and a comprehensive statistical analysis of the correlation of miRNA expression for primary and late neck lymph node metastases was performed. For the diagnosis of primary neck lymph node metastases, miR-423 and miR-125 were accurate. The miRNA index for primary metastasis diagnosis (miR-PM) calculated by regression analysis showed high diagnostic accuracy. The miR-5100 was useful for predicting late neck lymph node metastases. The miRNA index for late metastasis prediction (miR-LM) calculated using regression analysis showed high prediction accuracy. MiRNAs were useful for diagnosing primary neck lymph node metastases in OSCC and predicting late neck lymph node metastases. It may help to consider individualized treatment, including follow-up, surgical methods, and postoperative management.

A lack of reliable biomarkers for oral squamous cell carcinoma (OSCC) poses a major clinical issue. The sensitivity and specificity of classical serum tumor markers, such as the squamous cell carcinoma antigen (SCC-Ag), are quite poor, especially for early detection. This study aimed to identify specific serum miRNAs potentially serving as OSCC biomarkers. The expression levels of candidate miRNAs in serum samples from 40 OSCC patients and 40 healthy controls were quantitatively analyzed via microarray and reverse transcription PCR (RT-PCR) analyses. To enhance the accuracy of detection, we used Fisher’s linear discriminant analysis to establish a diagnostic model that incorporated a combination of selected miRNAs. Consequently, miR-19a and miR-20a were significantly upregulated in the patient group (p = 0.014 and 0.036, respectively), whereas miR-5100 was downregulated (p = 0.001). We found that a combination of six miRNAs (miR-24, miR-20a, miR-122, miR-150, miR-4419a, and miR-5100) could distinguish between OSCC and the control group with a higher degree of accuracy (Area Under the Curve, AUC: 0.844, sensitivity: 55%, and specificity: 92.5%). Furthermore, compared to serum SCC antigen, the 6-miRNA panel could accurately detect the presence of OSCC. The present specific miRNAs panel may serve as a novel candidate biomarker of oral cancer.

Recent studies suggest that epithelial–mesenchymal transition (EMT) correlates with cancer metastasis. In addition, there is growing evidence of the association of EMT with cancer stem cells (CSCs). Recently, we showed that the T-box transcription factor BRACHYURY could be a strong regulator of EMT and the CSC phenotype, which were effectively suppressed by a BRACHYURY knockdown in an adenoid cystic carcinoma cell line. In this study, we further tested whether BRACHYURY is a regulator of cancer stemness by means of forced expression of BRACHYURY in oral cancer cell lines. BRACHYURY, SOX2, or both were stably transfected into oral carcinoma cell lines. We analysed these transfectants with respect to self-renewal phenotypes using a sphere-formation assay, and we assessed the expression levels of EMT markers and stem cell markers using real-time reverse transcription-polymerase chain reaction (RT-PCR). Cell migration and invasiveness in vitro were evaluated using a wound healing assay and a tumour cell dissemination assay, respectively. Forced expression of BRACHYURY or SOX2 slightly increased expression of EMT and stem cell markers and the self-renewal phenotype. The expression levels, however, were much lower compared to those of cancer stem cell-like cells. Forced co-expression of BRACHYURY and SOX2 strongly upregulated EMT and stem cell markers and the self-renewal phenotype. Cell migration and invasiveness in vitro were also remarkably enhanced. These synergistic effects increased expression levels of FIBRONECTIN, SNAIL, SLUG, ZEB1, and TGF-β2. In particular, the effects on FIBRONECTIN and TGF-β2 were significant. We found that BRACHYURY and SOX2 synergistically promote cancer stemness in oral cancer cells. This finding points to the importance of gene or protein networks associated with BRACHYURY and SOX2 in the development and maintenance of the CSC phenotype.