miR-365 (microRNA): Potential Biomarker in Oral Squamous Cell Carcinoma Exosomes and Extracellular Vesicles

Abstract

Introduction: miR-365 is a non-coding microRNA that regulates transcription and has been demonstrated to promote oncogenesis and metastasis in some cancers, while suppressing these effects in others. Many microRNAs are produced and then exported extracellularly in exosomes, which are small extracellular vesicles ranging from 30 to 100 nm that are found in eukaryotic fluids and facilitate many cellular functions. Exosomes and extracellular vesicles are produced by many cell types, including oral cancer cells—although no study to date has evaluated miR-365 and oral cancer exosomes or extracellular vesicles. Based on this information, our research question was to evaluate whether oral cancers produce exosomes or extracellular vesicles containing miR-365. Materials and Methods: Two commercially available oral cancer cell lines (SCC25 and CAL27) and a normal oral keratinocyte (OKF4) were grown in serum-free media, supplemented with exosome-depleted fetal bovine serum. Extracellular vesicles and exosomes were then isolated using the Invitrogen total exosome RNA and protein isolation kit for processing using the hsa-miR-365a-5p microRNA qPCR assay kit. Results: RNA was successfully isolated from the exosome-depleted supernatant from each cell line—SCC9, SCC15, SCC25, and CAL27 (oral squamous cell carcinomas) and OKF4 (oral epithelial cell line). Relative concentrations of RNA were similar among each cell line, which were not significantly different, p = 0.233. RNA quality was established by A260:A280 absorbance using a NanoDrop, revealing purity ranging 1.73–1.86. Expression of miR-16 was used to confirm the presence of microRNA from the extracted exosomes and extracellular vesicles. The presence of miR-365 was then confirmed and normalized to miR-16 expression, which demonstrated an increased level of miR-365 in both CAL27 and SCC25. In addition, the normalized relative quantity (RQ) for miR-365 exhibited greater variation among SCC25 (1.382–4.363) than CAL27 cells (1.248–1.536). Conclusions: These results confirm that miR-365 is not only expressed in oral cancer cell lines, but also is subsequently exported into exosomes and extracellular vesicles derived from these cultures. These data may help to contextualize the potential for this microRNA to contribute to the phenotypes and behaviors of oral cancers that express this microRNA. Future research will begin to investigate these potential mechanisms and pathways and to determine if miR-365 may be useful as an oral cancer biomarker for salivary or liquid biopsies.