Differential miRNA Expression in Oral Cancer Oncosomes: A Pilot In vitro Study

Abstract

Aims: Exosomes are small membranous secreted vesicles (30-120 nm) believed to function as intercellular messengers delivering their cargo of RNA and protein to target cells. While many cells secrete exosomes, cancer cells have been found to produce higher numbers of exosomes than normal cells. Cancer specific exosomes, also termed oncosomes, transport intercellular bioactive molecules including proteins, lipids, and microRNAs (miRNA), the latter of which are discarded into the extracellular environment via exosomes. These bioactive molecules can modulate oral squamous cell carcinomas (OSCC) disease progression in vivo. To date, only one study had demonstrated the secretion of oncosomes from cultured OSCC cells, therefore the objective of this study is to determine if intact oncosomes can be isolated from oral cancer cells. Study Design: This is an observational laboratory-based study of human oral cancer cell cultures. Place and Duration of Study: This study was conducted in the Department of Biomedical Sciences at the University of Nevada, Las Vegas – School of Dental Medicine between May 2016 and May 2017. Methodology: Using a reagent that binds water and forces less-soluble lipid vesicles out of solution, oncosomes from oral cancer cell cultures (SCC4, SCC9, SCC15, SCC25 and CAL27) were collected by low-speed centrifugation. qRT-PCR was performed on RNA isolated from the culture-derived oncosomes for miR-21, miR-365, miR-155 and miR-133a1; all previously identified from cancers of other tissues. Results: Normal, non-cancerous HGF-1 (human gingival fibroblasts) had low (almost) undetectable expression of miR-21, -133, -155, and -365. Oral cancer cell lines (SCC4, SCC9, SCC15, SCC25 and CAL27) had moderate to high expression of at least one microRNA – although this varied significantly by cell line. Conclusion: Exosomes can be successfully isolated from OSCC conditioned media and miRNAs are detectable through TaqMan microRNA assays, with a unique characteristic expression of the miRNAs in the cell lines examined. Although more investigation is needed, potential correlations between miRNA levels and proliferation rates were also observed.