Theranostics 2021; 11(6):2987-2999. doi:10.7150/thno.45157

Research Paper

Genome-wide study of salivary miRNAs identifies miR-423-5p as promising diagnostic and prognostic biomarker in oral squamous cell carcinoma

Chiara Romani1,2,*,✉, Elisa Salviato3,*, Alberto Paderno4,*, Laura Zanotti2, Antonella Ravaggi2,5,6, Alberto Deganello4,7, Giulia Berretti4, Tommaso Gualtieri4, Sergio Marchini8, Maurizio D'Incalci8, Davide Mattavelli4,7, Cesare Piazza9,10, Paolo Bossi7,11, Chiara Romualdi3,12#, Piero Nicolai13,#, Eliana Bignotti2,5#

1. Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
2. 'Angelo Nocivelli' Institute of Molecular Medicine, University of Brescia and ASST Spedali Civili di Brescia, Brescia, Italy
3. Department of Biology, University of Padua, via U. Bassi 58/B Padua 35121, Italy
4. Unit of Otorhinolaryngology - Head and Neck Surgery, ASST Spedali Civili di Brescia, Brescia, Italy
5. Division of Obstetrics and Gynecology, ASST Spedali Civili di Brescia, Brescia, Italy
6. Department of Clinical and Experimental Sciences, Division of Obstetrics and Gynecology University of Brescia, Brescia, Italy
7. Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Brescia, Italy
8. Department of Oncology, IRCCS, "Mario Negri" Institute for Pharmacological Research, Milan, Italy
9. Department of Otorhinolaryngology, Maxillofacial, and Thyroid Surgery, Fondazione IRCCS, National Cancer Institute of Milan, Milan, Italy
10. Department of Oncology and Hematoncology, University of Milan, Milan, Italy
11. Medical Oncology, ASST Spedali Civili di Brescia, Brescia, Italy
12. CRIBI Biotechnology Center, Viale G Colombo 3, Padua 35131, Italy
13. Section of Otorhinolaryngology - Head & Neck Surgery, Department of Neurosciences, University of Padua, Italy
* CR, ES and AP equally contributed.
# CR, PN and EB are co-last authors.

This is an open access article distributed under the terms of the Creative Commons Attribution License ( See for full terms and conditions.
Romani C, Salviato E, Paderno A, Zanotti L, Ravaggi A, Deganello A, Berretti G, Gualtieri T, Marchini S, D'Incalci M, Mattavelli D, Piazza C, Bossi P, Romualdi C, Nicolai P, Bignotti E. Genome-wide study of salivary miRNAs identifies miR-423-5p as promising diagnostic and prognostic biomarker in oral squamous cell carcinoma. Theranostics 2021; 11(6):2987-2999. doi:10.7150/thno.45157. Available from

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Survival rates of oral squamous cell carcinoma (OSCC) remained substantially unchanged over the last decades; thus, additional prognostic tools are strongly needed. Salivary miRNAs have emerged as excellent non-invasive cancer biomarker candidates, but their association with OSCC prognosis has not been investigated yet. In this study, we analyzed global salivary miRNA expression in OSCC patients and healthy controls, with the aim to define its diagnostic and prognostic potential.

Methods: Saliva was collected from patients with newly diagnosed untreated primary OSCC and healthy controls. Global profiling of salivary miRNAs was carried out through a microarray approach, while signature validation was performed by quantitative real-time PCR (RT-qPCR). A stringent statistical approach for microarray and RT-qPCR data normalization was applied. The diagnostic performance of miRNAs and their correlation with OSCC prognosis were comprehensively analyzed.

Results: In total, 25 miRNAs emerged as differentially expressed between OSCC patients and healthy controls and, among them, seven were significantly associated with disease-free survival (DFS). miR-106b-5p, miR-423-5p and miR-193b-3p were expressed at high levels in saliva of OSCC patients and their combination displays the best diagnostic performance (ROC - AUC = 0.98). Moreover, high expression of miR-423-5p was an independent predictor of poor DFS, when included in multivariate survival analysis with the number of positive lymph nodes - the only significant clinical prognosticator. Finally, we observed a significant decrease in miR-423-5p expression in matched post-operative saliva samples, suggesting its potential cancer-specific origin.

Conclusion: Salivary miRNAs identified in our cohort of patients show to be accurate in OSCC detection and to effectively stratify patients according to their likelihood of relapse. These results, if validated in an independent set of patients, could be particularly promising for screening/follow-up of high-risk populations and useful for preoperative prognostic assessment.

Keywords: oral squamous cell carcinoma, salivary miRNA, microarray, diagnostic tumor marker, prognostic biomarker