Novel Disease-Specific Panel of Salivary microRNAs for the Detection of Oral Squamous Cell Carcinoma from Early Invasion to Stage IV Disease

Gintoni, Iphigenia; Vassiliou, Stavros; Kardara Bellou, Myrto; Balakas, Athanasios; Lefantzis, Nikolaos; Papakosta, Veronica; Vlachakis, Dimitrios; Chrousos, George P.; Yapijakis, Christos

doi:10.3390/ijms27115138

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Novel Disease-Specific Panel of Salivary microRNAs for the Detection of Oral Squamous Cell Carcinoma from Early Invasion to Stage IV Disease

by

Iphigenia Gintoni

^1,2,3,*

,

Stavros Vassiliou

³,

Myrto Kardara Bellou

³,

Athanasios Balakas

³,

Nikolaos Lefantzis

³,

Veronica Papakosta

³,

Dimitrios Vlachakis

^2,4

,

George P. Chrousos

²

and

Christos Yapijakis

^1,2,3,*

¹

Unit of Orofacial Genetics, 1st Department of Pediatrics, School of Medicine, National Kapodistrian University of Athens, “Aghia Sophia” Children’s Hospital, 115 27 Athens, Greece

²

Choremion Laboratory, University Research Institute of Maternal and Child Health and Precision Medicine, “Aghia Sophia” Children’s Hospital, 115 27 Athens, Greece

³

Department of Oral and Maxillofacial Surgery, School of Medicine, National Kapodistrian University of Athens, Attikon Hospital, 124 62 Athens, Greece

⁴

Laboratory of Genetics, Department of Biotechnology, School of Applied Biology and Biotechnology, Agricultural University of Athens, 11855 Athens, Greece

^*

Authors to whom correspondence should be addressed.

Int. J. Mol. Sci. 2026, 27(11), 5138; https://doi.org/10.3390/ijms27115138 (registering DOI)

Submission received: 2 March 2026 / Revised: 17 April 2026 / Accepted: 9 May 2026 / Published: 5 June 2026

(This article belongs to the Section Molecular Oncology)

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Abstract

Oral squamous cell carcinoma (OSCC) is characterized by consistently high mortality rates (≤60%) despite therapeutic advances. This is attributable to diagnostic delays arising from the asymptomatic early stages and time-consuming protocols. Hence, the establishment of reliable biomarkers for the routine assessment of the oral mucosa is imperative. MicroRNAs (miRNAs), key epigenetic regulators of gene expression, represent ideal candidates given their characteristic dysregulation across different pathologies. Here, we aimed to identify novel OSCC-specific miRNAs for the saliva-based detection of OSCC from the presymptomatic stage of early invasion. Through a multistep bioinformatic workflow, four miRNAs (miR-20b-5p, miR-484, miR-185-5p and miR-181d-5p) were identified as disease-specific since they simultaneously regulated >65% of a panel encompassing the 15 primarily overexpressed oncogenes in OSCC and a stage-specific panel including the six upregulated genes that genetically define the malignant stages of sequential oral carcinogenesis. The salivary expression of the identified miRNAs was studied in 31 OSCC patients and 31 healthy controls, using quantitative real-time PCR, followed by statistical analysis and an evaluation of the diagnostic accuracy. All studied miRNAs were significantly downregulated in the saliva of OSCC patients compared to controls (miR-484, p < 0.001; miR-181d-5p, p < 0.001; miR-185, p = 0.008; miR-20b, p = 0.026) and exhibited combinatory diagnostic performance of 95.4% (p < 0.001) for OSCC detection. Their expression remained uninfluenced by lifestyle and clinicopathological parameters, including smoking/alcohol, tumor site, grade and disease stage. The proposed 4-miRNA panel exhibits high diagnostic performance for the early, saliva-based detection of OSCC, irrespective of histopathological and lifestyle confounders, highlighting its potential as a robust non-invasive screening tool.

Keywords: oral cancer; OSCC; early invasion; microRNA; miR-20b-5p; miR-181d-5p; miR-185-5p; miR-484; biomarker; saliva; liquid biopsy; early diagnosis; presymptomatic detection

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MDPI and ACS Style

Gintoni, I.; Vassiliou, S.; Kardara Bellou, M.; Balakas, A.; Lefantzis, N.; Papakosta, V.; Vlachakis, D.; Chrousos, G.P.; Yapijakis, C. Novel Disease-Specific Panel of Salivary microRNAs for the Detection of Oral Squamous Cell Carcinoma from Early Invasion to Stage IV Disease. Int. J. Mol. Sci. 2026, 27, 5138. https://doi.org/10.3390/ijms27115138

AMA Style

Gintoni I, Vassiliou S, Kardara Bellou M, Balakas A, Lefantzis N, Papakosta V, Vlachakis D, Chrousos GP, Yapijakis C. Novel Disease-Specific Panel of Salivary microRNAs for the Detection of Oral Squamous Cell Carcinoma from Early Invasion to Stage IV Disease. International Journal of Molecular Sciences. 2026; 27(11):5138. https://doi.org/10.3390/ijms27115138

Chicago/Turabian Style

Gintoni, Iphigenia, Stavros Vassiliou, Myrto Kardara Bellou, Athanasios Balakas, Nikolaos Lefantzis, Veronica Papakosta, Dimitrios Vlachakis, George P. Chrousos, and Christos Yapijakis. 2026. "Novel Disease-Specific Panel of Salivary microRNAs for the Detection of Oral Squamous Cell Carcinoma from Early Invasion to Stage IV Disease" International Journal of Molecular Sciences 27, no. 11: 5138. https://doi.org/10.3390/ijms27115138

APA Style

Gintoni, I., Vassiliou, S., Kardara Bellou, M., Balakas, A., Lefantzis, N., Papakosta, V., Vlachakis, D., Chrousos, G. P., & Yapijakis, C. (2026). Novel Disease-Specific Panel of Salivary microRNAs for the Detection of Oral Squamous Cell Carcinoma from Early Invasion to Stage IV Disease. International Journal of Molecular Sciences, 27(11), 5138. https://doi.org/10.3390/ijms27115138

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Novel Disease-Specific Panel of Salivary microRNAs for the Detection of Oral Squamous Cell Carcinoma from Early Invasion to Stage IV Disease

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