Differential MicroRNA Expression of miR-21 and miR-155 within Oral Cancer Extracellular Vesicles in Response to Melatonin

Abstract

Objective: Extracellular vesicles derived from oral cancer cells, which include Exosomes and Oncosomes, are membranous vesicles secreted into the surrounding extracellular environment. These extracellular vesicles can regulate and modulate oral squamous cell carcinoma (OSCC) progression through the horizontal transfer of bioactive molecules including proteins, lipids and microRNA (miRNA). The primary objective of this study was to examine the potential to isolate and evaluate extracellular vesicles (including exosomes) from various oral cancer cell lines and to explore potential differences in miRNA content. Methods: The OSCC cell lines SCC9, SCC25 and CAL27 were cultured in DMEM containing 10% exosome-free fetal bovine serum. Cell-culture conditioned media was collected for exosome and extracellular vesicle isolation after 72 h. Isolation was completed using the Total Exosome Isolation reagent (Invitrogen) and extracellular vesicle RNA was purified using the Total Exosome RNA isolation kit (Invitrogen). Extracellular vesicle miRNA content was evaluated using primers specific for miR-16, -21, -133a and -155. Results: Extracellular vesicles were successfully isolated from all three OSCC cell lines and total extracellular vesicle RNA was isolated. Molecular screening using primers specific for several miRNA revealed differential baseline expression among the different cell lines. The addition of melatonin significantly reduced the expression of miR-155 in all of the OSCC extracellular vesicles. However, miR-21 was significantly increased in each of the three OSCC isolates. No significant changes in miR-133a expression were observed under melatonin administration. Conclusions: Although many studies have documented changes in gene expression among various cancers under melatonin administration, few studies have evaluated these effects on microRNAs. These results may be among the first to evaluate the effects of melatonin on microRNA expression in oral cancers, which suggests the differential modulation of specific microRNAs, such as miR-21, miR-133a and miR-155, may be of significant importance when evaluating the mechanisms and pathways involved in melatonin-associated anti-tumor effects.

Differential exosome miRNA expression in oral cancer stem cells

“…This may be significant as over-expression of miR-155 appears to directly promote cellular proliferation in oral cancers [35][36][37]. These observations may conform with additional evidence that has demonstrated miR-155 may be an indicator of poor prognosis among oral cancers [18,37,38].…”

“…To determine the presence of CSC-specific mRNA biomarkers, RNA was isolated from both ADC and CSC-AiDC-TS using the ABgene Total RNA isolation Reagent kit and the protocol recommended by the manufacturer [18,20]. Analysis of RNA was accomplished using absorbance readings at 260 nm and 280 nm.…”

“…Settings for the thermal cycler were 2 min at 50°C, 10 min at 95°C, followed by 40 cycles that consisted of 15 s at 95°C and 60 s at 60°C. A standard curve was derived from a five-fold serial dilution of cDNA from miR-16, which is used as a positive control for exosomal miRNA and the endogenous reference gene [15,18].…”

“…Recent progress has successfully identified several candidate microRNAs that may be differentially expressed among early-versus late-stage oral cancers, which may provide the basis for this type of differential screening [15,16]. Recent efforts from this group have also contributed to the potential pool of relevant microRNA candidates [17,18]. More specifically, each of these referenced studies has confirmed the expression of miR-21 and miR-155 in the most aggressive and rapidly dividing oral cancer samplesalthough none have specifically screened for the expression of these microRNAs among cancer stem cell isolates.…”

Extracellular Vesicles in Oral Squamous Cell Carcinoma and Oral Potentially Malignant Disorders: A Systematic Review

“…Melatonin has been demonstrated to reduce OSCC tumorigenesis in vivo [74]. Hunsaker et al (2019) [75] studied the effect of the addition of melatonin to culture on exosomal expression of the OSCC-associated microRNAs, miR-21 miR-133a and miR-155, in the OSCC cell lines SSC25, CAL27 and SSC9. Increased expression of both miR-21 [76] and miR-155 [76] have been associated with poor prognosis, whereas miR-133-3p has been reported to inhibit OSCC in vitro [77].…”