Abstract
BackgroundOral squamous cell carcinomas (OSCC) secrete exosomes into the surrounding extracellular environment to promote the horizontal transfer of bioactive molecules including microRNA (miRNA). The primary objective of this study was to explore potential differences in miRNA content between OSCC and OSCC stem cells.MethodsThe OSCC cell lines SCC4, SCC15, SCC25 and CAL27 were used in these studies. The corresponding OSCC stem cells that demonstrated phenotypic adhesion independent tumor spheres (AiTS) were manually isolated. All cells were cultured in DMEM containing 10% exosome-free fetal bovine serum. Exosomes were isolated using Total Exosome Isolation reagent (Invitrogen) and RNA was purified using Total Exosome RNA isolation kit (Invitrogen). Exosome miRNA content was evaluated using miRNA Advanced Taqman Assays for miR-21, − 155, − 133, − 34, − 31, − 32, and − 365. The fold change of miRNA content was calculated using the comparative CT (ΔΔCT) method using miR-16 as an endogenous control.ResultsAfter successful cell cultures were established, AiTS (cancer stem cells) were manually separated and confirmed using CD133 and Sox-2 biomarkers. Exosomes and extracellular vesicles were successfully isolated from all cell lines and AiTS isolates for miRNA screening. All isolates exhibited miR-16 expression (positive control), but none contained mir-31, − 32, or 133a. Differential expression of miR-21, miR-34 and miR-155 were observed with patterns observed among the cancer cell lines which were distinct from the corresponding AiTS isolates.ConclusionsExosomes isolated from these different OSCC stem cell populations displayed nearly consistent downregulation/loss of miR-21 and miR-34 suggesting the possibility of a unique miRNA profile characteristic of oral cancer stem cells. These findings highlight the need to investigate the comprehensive functions of miR-21 and miR-34 in tumor progression and continued research to refine a miRNA profile that could aid in distinguishing tumors with poor prognosis.
Highlights
Oral squamous cell carcinomas (OSCC) secrete exosomes into the surrounding extracellular environment to promote the horizontal transfer of bioactive molecules including microRNA
Among the most promising oral cancer biomarkers are circulating microRNAs, which are small, non-coding Ribonucleic acid (RNA) that are secreted by cancers into the extracellular environment in small vesicles known as exosomes or oncosomes [7,8,9]
As cultures were maintained over time, adhesion-independent tumor spheres (AiTS) developed, which were manually isolated and transferred to separate cell culture flasks for SCC4 (Fig. 1a), SCC15 (Fig. 1b), SCC25 (Fig. 1c) and CAL27 (Fig. 1d) cells
Summary
Oral squamous cell carcinomas (OSCC) secrete exosomes into the surrounding extracellular environment to promote the horizontal transfer of bioactive molecules including microRNA (miRNA). Recent evidence has demonstrated that a sub-population of oral cancers may be cancer stem cells (CSC), which exhibit cancerous and stem cell phenotypes – including chemotherapeutic and radiation resistance [3, 4] Based upon these phenotypes, recent efforts have focused on identifying specific biomarkers to identify tumors with sub-populations of CSC [5, 6]. Among the most promising oral cancer biomarkers are circulating microRNAs, which are small, non-coding RNA that are secreted by cancers (and cancer stem cells) into the extracellular environment in small vesicles known as exosomes or oncosomes [7,8,9]. Many of the hundreds of previously identified microRNAs may be expressed in both cancers and cancer stem cells – many thousands of microRNAs have yet to be evaluated [12,13,14]
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