Non-invasive screening of a microRNA-based dysregulation signature in oral cancer and oral potentially malignant disorders.

INTRODUCTION: We have previously shown that oral swirls are a robust source of microRNA protected by extracellular vesicles, potentially useful to detect oral squamous cell carcinoma (OSCC)-associated molecular aberration. OBJECTIVES: To study a developed dysregulation score and risk classification algorithm based upon a panel of OSCC-associated microRNA in oral swirls from individuals with OSCC and oral potentially malignant disorders (OPMDs). MATERIALS AND METHODS: An OSCC-associated panel of 5 microRNAs (miR-24; miR-21; miR-99a; let-7c; miR-100;) was quantified by qPCR in 190 individuals with and without mucosal abnormalities, including OSCC (n = 53) and OPMDs (n = 74). Each sample was analyzed using a developed dysregulation score (dSCORE) and risk classification algorithm, allocating a LOW- or HIGH-RISK score. The influence of demographic, systemic, oral health and mucosal disease factors on the developed test was analyzed. RESULTS: MicroRNA for analysis can be predictably isolated from oral swirls sourced from individuals with a range of demographic, systemic and oral health findings. Utilizing the presence of HIGH-RISK identified OSCC patients with 86.8% sensitivity and 81.5% specificity. Older age and female gender were associated with higher dSCOREs and higher proportions of HIGH-RISK classification amongst individuals with no mucosal abnormalities. The dSCOREs for all subgroups of OPMDs were significantly different from the OSCC group. CONCLUSION: This is the first comparison of microRNA sourced from oral swirls from individuals with OPMDs with individuals with and without OSCC. A HIGH-RISK dysregulation signature was found to be accurate in indicating the presence of OSCC and exampled to parallel malignant transformation.

Oral swirl samples - a robust source of microRNA protected by extracellular vesicles.

BACKGROUND: MicroRNAs are small non-coding RNAs which are dysregulated in disease states, such as oral cancer. Extracellular vesicles, a potential source of microRNA, are found in saliva. OBJECTIVE: To demonstrate that a quantifiable amount of microRNA can be isolated from oral swirl samples. Additionally, we hypothesized that extracellular vesicles may protect contained microRNA from degradation in these samples. METHOD: A polyethylene glycol-based precipitation was used for extracellular vesicle enrichment of oral swirl samples. Comparison was made between samples treated with and without RNase. Further, samples from three subjects were exposed to a range of conditions over 7 days and assessed for presence of microRNA by reverse-transcription quantitative PCR. Extracellular vesicles from samples were identified under transmission electron microscopy. RESULTS: An adequate quantity of microRNA for qPCR analysis was extractable from samples despite exposure to conditions under which degradation of RNA would be expected. CONCLUSION: A technique was developed to isolate an adequate quantity of microRNA for analysis from oral swirl samples. Extracellular vesicle-associated microRNA may be protected from degradation. This technique moves towards chairside application of translational microRNA research in the field of oral cancer prognostics.

Packaging of prions into exosomes is associated with a novel pathway of PrP processing.

Prion diseases are fatal, transmissible neurodegenerative disorders associated with conversion of the host-encoded prion protein (PrP(C)) into an abnormal pathogenic isoform (PrP(Sc)). Following exposure to the infectious agent (PrP(Sc)) in acquired disease, infection is propagated in lymphoid tissues prior to neuroinvasion and spread within the central nervous system. The mechanism of prion dissemination is perplexing due to the lack of plausible PrP(Sc)-containing mobile cells that could account for prion spread between infected and uninfected tissues. Evidence exists to demonstrate that the culture media of prion-infected neuronal cells contain PrP(Sc) and infectivity but the nature of the infectivity remains unknown. In this study we have identified PrP(C) and PrP(Sc) in association with endogenously expressing PrP neuronal cell-derived exosomes. The exosomes from our prion-infected neuronal cell line were efficient initiators of prion propagation in uninfected recipient cells and to non-neuronal cells. Moreover, our neuronal cell line was susceptible to infection by non-neuronal cell-derived exosome PrP(Sc). Importantly, these exosomes produced prion disease when inoculated into mice. Exosome-associated PrP is packaged via a novel processing pathway that involves the N-terminal modification of PrP and selection of distinct PrP glycoforms for incorporation into these vesicles. These data extend our understanding of the relationship between PrP and exosomes by showing that exosomes can establish infection in both neighbouring and distant cell types and highlight the potential contribution of differentially processed forms of PrP in disease distribution. These data suggest that exosomes represent a potent pool of prion infectivity and provide a mechanism for studying prion spread and PrP processing in cells endogenously expressing PrP.