Abstract
BackgroundSeveral 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.MethodsOSCC 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.ResultsA 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.ConclusionsIn 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.
Highlights
Several studies in the past have investigated the expression of micro RNAs in saliva as potential biomarkers
MiRNA expression analysis by qRT-PCR array To select differentially expressed micro RNAs (miRNAs), qRT-PCR array analysis was performed on extracellular vesicles (EVs) isolated from five qRT-PCR On the basis of results obtained from the array, we studied miRNAs up-regulated or selectively expressed by patients in a cohort of additional 16 Oral squamous cell carcinoma (OSCC) patients and 6 controls. 500 ng of total RNA was retro-transcribed to cDNA with miScript II RT Kit (Qiagen, Hilden, D) and evaluated for the expression of five miRNAs upregulated in patients compared to healthy controls
No evidence has been reported confirming the involvement of miR-412-3p, miR-512-3p, miR-302b-3p, and miR-517b-3p in OSCC, our work provides new insights about the dysregulation of miRNAs in the tumor environment
Summary
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. Oral squamous cell carcinoma (OSCC) is the most frequent cancer of the head and neck [1, 2]. Extracellular vesicles (EVs) have gained significant attention as a conceivable source of biomarkers. These small membrane-bound vesicles are categorized into 3 different types: exosomes, microvesicles or ectosomes, and apoptotic bodies [6]. Tumor-derived EVs have been identified to influence the tumor environment by promoting cancer progression, survival, invasion, and angiogenesis [7]. As EVs carry biologically active proteins and nucleic acids from the parent cells, tumorderived EVs could act as molecular signatures of cancer cells from which they are derived [7, 8]
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