Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr 18;18(1):439.
doi: 10.1186/s12885-018-4364-z.

Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma

Chiara Gai  1 , Francesco Camussi  2 , Roberto Broccoletti  2 , Alessio Gambino  2 , Marco Cabras  2 , Luca Molinaro  1 , Stefano Carossa  2 , Giovanni Camussi  1 , Paolo G Arduino  3
Affiliations

Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma

Chiara Gai et al. BMC Cancer. .

Abstract

Background: Several studies in the past have investigated the expression of micro RNAs (miRNAs) in saliva as potential biomarkers. Since miRNAs associated with extracellular vesicles (EVs) are known to be protected from enzymatic degradation, we evaluated whether salivary EVs from patients with oral squamous cell carcinoma (OSCC) were enriched with specific subsets of miRNAs.

Methods: OSCC patients and controls were matched with regards to age, gender and risk factors. Total RNA was extracted from salivary EVs and the differential expression of miRNAs was evaluated by qRT-PCR array and qRT-PCR. The discrimination power of up-regulated miRNAs as biomarkers in OSCC patients versus controls was evaluated by the Receiver Operating Characteristic (ROC) curves.

Results: A preliminary qRT-PCR array was performed on samples from 5 OSCC patients and 5 healthy controls whereby a subset of miRNAs were identified that were differentially expressed. On the basis of these results, a cohort of additional 16 patients and 6 controls were analyzed to further confirm the miRNAs that were up-regulated or selectively expressed in the previous pilot study. The following miRNAs: miR-302b-3p and miR-517b-3p were expressed only in EVs from OSCC patients and miR-512-3p and miR-412-3p were up-regulated in salivary EVs from OSCC patients compared to controls with the ROC curve showing a good discrimination power for OSCC diagnosis. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis suggested the possible involvement of the miRNAs identified in pathways activated in OSCC.

Conclusions: In this work, we suggest that salivary EVs isolated by a simple charge-based precipitation technique can be exploited as a non-invasive source of miRNAs for OSCC diagnosis. Moreover, we have identified a subset of miRNAs selectively enriched in EVs of OSCC patients that could be potential biomarkers.

Keywords: Extracellular vesicles (EVs); Oral squamous cell carcinoma (OSCC); miR-27a-3p (miR-27a); miR-302b-3p (miR-302b); miR-494; miR-517b-3p (miR-517b); miRNA-412-3p (miR-412); miRNA-512-3p (miR-512); microRNA (miRNA).

PubMed Disclaimer

Conflict of interest statement

Ethics approval and consent to participate

The study was approved by local ethical committee “A.O.U. Città della Salute e della Scienza di Torino”, Turin, Italy (n° 310/2015) and all patients gave written informed consent to participate.

Competing interests

GC is named as inventors in a related patent application. All other authors declare no conflict of interest.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Characterization of salivary EVs. (a) Representative NanoSight image of isolated EVs showing particle size (nm)/concentration (10^8 particles/ml) of a representative control (left) and a representative OSCC patient (right). (b) Representative transmission electron microscopy image of purified EVs negatively stained with NanoVan (JEOL Jem-1010 electron microscope, black line = 200 nm) of a control (left) and a patient (right). (c) Representative western blots confirming the expression of the exosome markers: CD63, CD9, Tsg101, and Alix, on salivary EVs from a control (left) and a OSCC patient (right). (d) Representative profiles of RNA isolated from EVs of a healthy control (left) and a patient (right). The graphs show fluorescence intensity [FU]/nucleotide length [nt] and were obtained through bioanalyzer analysis. Four experiments were performed with similar results
Fig. 2
Fig. 2
miRNA relative expression detected by qRT-PCR in salivary EV samples from OSCC patients compared to normal subjects. (a) Expression levels of miRNAs that were significantly up-regulated in patients. (b) Expression levels of miRNAs that were exclusively expressed by OSCC patients. The bars represent mean relative expression (2^-∆∆Ct) of control and patient groups ± SEM, p value (two-sided Mann-Whitney test) are reported. ROC curve describing predictive potency of the up-regulated miRNAs as a diagnostic test. The curves represent specificity versus sensitivity of miR-512-3p (c), miR-412-3p (d). Data are derived from miRNAs’ expression levels (RQ) of OSCC patients and controls. The big gray dots indicate the optimal threshold value of sensitivity and specificity determined by the maximum Youden’s index (sensitivity+specificity-1)
Fig. 3
Fig. 3
KEGG pathway enrichment analysis for up-regulated miRNAs (miR-512-3p, miR-412-3p, miR-27a-3p, miR-494-3p) or miRNAs only expressed in salivary EV from OSCC patients (miR-302b-3p, miR-517b-3p). (a) The graph shows significantly enriched biological pathways labeled with their respective p values. X axis shows the number of miRNAs involved in each pathway. (b) The graph shows the number of target genes for each enriched pathway. (c) The bars represent the number of target genes for each miRNA in each pathway and the respective p value. The legend (lower-right) shows the color assigned to each pathway and is valid for all the figures (a-c). The p value was calculated with FDR correction and threshold was set as 0.05
See this image and copyright information in PMC

References

    1. Moore S, Johnson N, Pierce A. The epidemiology of mouth cancer: a review of global incidence. Oral Dis. 2000;6(2):65–74. doi: 10.1111/j.1601-0825.2000.tb00104.x. - DOI - PubMed
    1. Bagan J, Sarrion G, Jimenez Y. Oral cancer: clinical features. Oral Oncol. 2010;46(6):414–417. doi: 10.1016/j.oraloncology.2010.03.009. - DOI - PubMed
    1. Arduino PG, Carrozzo M, Chiecchio A, Broccoletti R, Tirone F, Borra E, et al. Clinical and histopathologic independent prognostic factors in oral squamous cell carcinoma: a retrospective study of 334 cases. J Oral Maxillofac Surg. 2008;66(8):1570–1579. doi: 10.1016/j.joms.2007.12.024. - DOI - PubMed
    1. Woolgar JA. Histopathological prognosticators in oral and oropharyngeal squamous cell carcinoma. Oral Oncol. 2006;42(3):229–239. doi: 10.1016/j.oraloncology.2005.05.008. - DOI - PubMed
    1. Zanaruddin SN, Saleh A, Yang YH, Hamid S, Mustafa WM, Khairul Bariah AA, et al. Four proteins signature accurately predicts lymph node metastasis and survival in oral squamous cell carcinoma. Hum Pathol. 2013;44(3):417–426. doi: 10.1016/j.humpath.2012.06.007. - DOI - PubMed

Publication types

MeSH terms