Abstract
Oral squamous cell carcinoma (OSCC) remains a major public health concern due to its late diagnosis and poor survival outcomes. Non-invasive biomarkers capable of detecting early-stage OSCC are urgently needed. Salivary exosomal microRNAs (miRNAs) have emerged as promising molecular indicators owing to their stability, specificity, and ease of collection.
To evaluate the diagnostic performance of salivary exosomal miR-21, miR-31, miR-155, and miR-200c as biomarkers for early OSCC detection.
This prospective case-control study included 140 participants categorized into three groups: OSCC patients (n = 60), individuals with benign oral lesions (n = 40), and healthy controls (n = 40). Saliva samples were collected, and exosomes were isolated and validated using NTA, TEM, and Western blot. Exosomal miRNAs were extracted and quantified via qRT-PCR. Statistical analyses included ANOVA, ROC curve analysis, and multivariate logistic regression.
All four miRNAs were significantly elevated in OSCC patients (p < 0.001). miR-21 showed the highest diagnostic accuracy (AUC = 0.89), while the combined panel achieved an AUC of 0.93, with sensitivity and specificity exceeding 90% and 88%, respectively. Strong correlations between miRNA levels and tumor stage were observed for miR-21 and miR-155.
Salivary exosomal miRNAs exhibit high diagnostic accuracy for OSCC, even in early-stage disease. This non-invasive molecular approach offers a feasible and scalable alternative to traditional diagnostic modalities and holds significant potential for integration into routine screening and surveillance protocols.
References
- Arunkumar, G., Deva Magendhra Rao, A. K., Manikandan, M., Prasanna Srinivasa Rao, H., Subbiah, S., Ilangovan, R., et al. (2018). Dysregulation of miR-200 family microRNAs and epithelial-mesenchymal transition markers in oral squamous cell carcinoma. Oncology Letters, 15(1), 649–657. https://doi.org/10.3892/ol.2017.7296DOI ↗Google Scholar ↗
- Byun, J. S., Lee, H. Y., Tian, J., Moon, J. S., Choi, J., Lee, S. H., et al. (2021). Effect of salivary exosomal miR-25-3p on periodontitis with insulin resistance. Frontiers in Immunology, 12, 775046. https://doi.org/10.3389/fimmu.2021.775046DOI ↗Google Scholar ↗
- Cao, Y., Green, K., Quattlebaum, S., Milam, B., Lu, L., Gao, D., et al. (2018). Methylated genomic loci encoding microRNA as a biomarker panel in tissue and saliva for head and neck squamous cell carcinoma. Clinical Epigenetics, 10, 43. https://doi.org/10.1186/s13148-018-0470-7DOI ↗Google Scholar ↗
- Chen, Y., & Wang, X. (2020). miRDB: An online database for prediction of functional microRNA targets. Nucleic Acids Research, 48(D1), D127–D131. https://doi.org/10.1093/nar/gkz757DOI ↗Google Scholar ↗
- Chien, H. T., Cheng, S. D., Liao, C. T., Wang, H. M., & Huang, S. F. (2019). Amplification of the EGFR and CCND1 are coordinated and play important roles in the progression of oral squamous cell carcinomas. Cancers (Basel), 11(6), 760. https://doi.org/10.3390/cancers11060760DOI ↗Google Scholar ↗
- Farag, A. F., Sabry, D., Hassabou, N. F., & Alaa El-Din, Y. (2021). MicroRNA-134/microRNA-200a derived salivary exosomes are novel diagnostic biomarkers of oral squamous cell carcinoma. Egyptian Dental Journal, 67(1), 367–377. https://doi.org/10.21608/edj.2020.47990.1317DOI ↗Google Scholar ↗
- Faur, C. I., Rotaru, H., Osan, C., Jurj, A., Roman, R. C., Moldovan, M., et al. (2021). Salivary exosomal microRNAs as biomarkers for head and neck cancer detection – A literature review. Maxillofacial Plastic and Reconstructive Surgery, 43(1), 19. https://doi.org/10.1186/s40902-021-00303-9DOI ↗Google Scholar ↗
- Faur, C. I., Roman, R. C., Jurj, A., Raduly, L., Almasan, O., Rotaru, H., et al. (2022). Salivary exosomal microRNA-486-5p and microRNA-10b-5p in oral and oropharyngeal squamous cell carcinoma. Medicina (Lithuania), 58(10), 1478. https://doi.org/10.3390/medicina58101478DOI ↗Google Scholar ↗
- Gai, C., Camussi, F., Broccoletti, R., Gambino, A., Cabras, M., Molinaro, L., et al. (2018). Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma. BMC Cancer, 18, 439. https://doi.org/10.1186/s12885-018-4364-zDOI ↗Google Scholar ↗
- Han, Y., Jia, L., Zheng, Y., & Li, W. (2018). Salivary exosomes: Emerging roles in systemic disease. International Journal of Biological Sciences, 14(6), 633–643. https://doi.org/10.7150/ijbs.25018DOI ↗Google Scholar ↗
- He, L., Ping, F., Fan, Z., Zhang, C., Deng, M., Cheng, B., & Xia, J. (2020). Salivary exosomal miR-24-3p serves as a potential detective biomarker for oral squamous cell carcinoma screening. Biomedicine & Pharmacotherapy, 121, 109553. https://doi.org/10.1016/j.biopha.2019.109553DOI ↗Google Scholar ↗
- Hofmann, L., Abou Kors, T., Ezić, J., Niesler, B., Roth, R., Ludwig, S., et al. (2022). Plasma- and saliva-derived exosomal miRNA profiles reveal diagnostic potential in head and neck cancer. Frontiers in Cell and Developmental Biology, 10, 971596. https://doi.org/10.3389/fcell.2022.971596DOI ↗Google Scholar ↗
- Kaczor-Urbanowicz, K. E., Martin Carreras-Presas, C., Aro, K., Tu, M., Garcia-Godoy, F., & Wong, D. T. (2017). Saliva diagnostics – Current views and directions. Experimental Biology and Medicine, 242(5), 459–472. https://doi.org/10.1177/1535370216681550DOI ↗Google Scholar ↗
- Kang, J. W., Eun, Y. G., & Lee, Y. C. (2021). Diagnostic value of salivary miRNA in head and neck squamous cell cancer: Systematic review and meta-analysis. International Journal of Molecular Sciences, 22(13), 7026. https://doi.org/10.3390/ijms22137026DOI ↗Google Scholar ↗
- Kosaka, N., Iguchi, H., Yoshioka, Y., Takeshita, F., Matsuki, Y., & Ochiya, T. (2010). Secretory mechanisms and intercellular transfer of microRNAs in living cells. Journal of Biological Chemistry, 285(23), 17442–17452. https://doi.org/10.1074/jbc.M110.107821DOI ↗Google Scholar ↗
- Langevin, S., Kuhnell, D., Parry, T., Biesiada, J., Huang, S., Wise-Draper, T., et al. (2017). Comprehensive microRNA-sequencing of exosomes derived from head and neck carcinoma cells in vitro. Oncotarget, 8(47), 82459–82474. https://doi.org/10.18632/oncotarget.19614DOI ↗Google Scholar ↗
- Lu, Y., Ye, L., Jian, X., Yang, D., Zhang, H., Tong, Z., et al. (2022). Integrated microfluidic system for isolating exosome and analyzing protein marker PD-L1. Biosensors and Bioelectronics, 204, 113879. https://doi.org/10.1016/j.bios.2021.113879DOI ↗Google Scholar ↗
- Mohr, A. M., & Mott, J. L. (2015). Overview of microRNA biology. Seminars in Liver Disease, 35(1), 3–11. https://doi.org/10.1055/s-0034-1397344DOI ↗Google Scholar ↗
- Park, N. J., Zhou, H., Elashoff, D., Henson, B. S., Kastratovic, D. A., Abemayor, E., & Wong, D. T. (2009). Salivary microRNA: Discovery, characterization, and clinical utility for oral cancer detection. Clinical Cancer Research, 15(17), 5473–5477. https://doi.org/10.1158/1078-0432.CCR-09-0736DOI ↗Google Scholar ↗
- Patel, A., Patel, S., Patel, P., Mandlik, D., Patel, K., & Tanavde, V. (2022). Salivary exosomal miRNA-1307-5p predicts disease aggressiveness and poor prognosis in oral squamous cell carcinoma. International Journal of Molecular Sciences, 23(18), 10639. https://doi.org/10.3390/ijms231810639DOI ↗Google Scholar ↗
- Patel, A., Patel, P., Mandlik, D., Patel, K., Malaviya, P., Johar, K., et al. (2023). A novel 3-miRNA network regulates tumour progression in oral squamous cell carcinoma. Biomarker Research, 11, 64. https://doi.org/10.1186/s40364-023-00505-5DOI ↗Google Scholar ↗
- Rapado-González, Ó., Majem, B., Muinelo-Romay, L., Álvarez-Castro, A., Santamaría, A., Gil-Moreno, A., et al. (2018). Human salivary microRNAs in cancer. Journal of Cancer, 9(4), 638–649. https://doi.org/10.7150/jca.21180DOI ↗Google Scholar ↗
- Salazar, C., Nagadia, R., Pandit, P., Cooper-White, J., Banerjee, N., Dimitrova, N., et al. (2014). A novel saliva-based microRNA biomarker panel to detect head and neck cancers. Cell Oncology, 37(5), 331–338. https://doi.org/10.1007/s13402-014-0188-2DOI ↗Google Scholar ↗
- Sanesi, L., et al. (2024). Pathways of the main miRs. Archives of Oral Biology, 165, 106012. https://doi.org/10.1016/j.archoralbio.2024.106012DOI ↗Google Scholar ↗
- Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2021). Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide. CA: A Cancer Journal for Clinicians, 71(3), 209–249. https://doi.org/10.3322/caac.21660DOI ↗Google Scholar ↗
- Valadi, H., Ekström, K., Bossios, A., Sjöstrand, M., Lee, J. J., & Lötvall, J. O. (2007). Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange. Nature Cell Biology, 9(6), 654–659. https://doi.org/10.1038/ncb1596DOI ↗Google Scholar ↗
- Wang, J., Yin, K., Lv, X., Yang, Q., Shao, M., Liu, X., & Sun, H. (2019). MicroRNA-24-3p regulates Hodgkin’s lymphoma cell proliferation, migration and invasion by targeting DEDD. Oncology Letters, 17(1), 365–371. https://doi.org/10.3892/ol.2018.9599DOI ↗Google Scholar ↗
- Zahran, F., Ghalwash, D., Shaker, O., Al-Johani, K., & Scully, C. (2015). Salivary microRNAs in oral cancer. Oral Diseases, 21(7), 739–747. https://doi.org/10.1111/odi.12340DOI ↗Google Scholar ↗
- Zanoni, D. K., Patel, S. G., & Shah, J. P. (2019). Changes in the 8th edition of the AJCC staging of head and neck cancer. Current Oncology Reports, 21, 52. https://doi.org/10.1007/s11912-019-0799-xDOI ↗Google Scholar ↗
- Patel, A., et al. (2022). A novel 4-miRNA signature from salivary exosomes regulates multiple signalling pathways and predicts tumour progression in OSCC. bioRxiv. https://doi.org/10.1101/2022.05.31.494114DOI ↗Google Scholar ↗
