Differential exosome miRNA expression in oral cancer stem cells

Abstract

Background: Oral 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.Methods: The 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. Results: After 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. Conclusions: Exosomes 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.

Cancer stem cell secretome in the tumor microenvironment: a key point for an effective personalized cancer treatment

“…Moreover, in metastatic breast cancer cells, exosome-secreted miR-10b, miR-105 and miR-9 are related to enhanced invasiveness through increased endothelial cell migration, angiogenesis and vascular permeability [43]. The miR-200 family found in extracellular vesicles of breast CSCs was related to their metastatic capacity [44]. In glioblastoma, miR-10b is upregulated in CSCs and its inhibition strongly reduces CSCs proliferation and metastasis.…”

“…Finally, exosomes isolated from oral CSCs displayed nearly consistent downregulation of miR-34 and the up-regulation of miR-21 and miR-155 was related to increased CSC proliferation and migration; therefore, they may be consider indicators of poor prognosis [ 184 ]. However, miR-155 overexpression has been correlated with better prognosis and lower metastatic capacity [ 185 , 186 ] and miR-21 appears down-regulated in CSCs [ 44 ]. These differences could be due CSC and inter-patient heterogeneity and to the dual role played by the same miRNA depending on cell state.…”

Characterization of Dental Pulp Stem Cell Responses to Functional Biomaterials Including Mineralized Trioxide Aggregates

“…RNA was isolated from each DPSC using the ABgene Total RNA isolation reagent kit and protocol recommended by ThermoFisher (Fisher Scientific; Fair Lawn, NJ, USA), as previously described [ 24 , 25 ]. All samples were screened for purity using the NanoDrop spectrophotometer (Fisher Scientific; Fair Lawn, NJ, USA) absorbance readings at A260 and A280 nm.…”

“…RNA obtained from each DPSC isolate was subsequently screened for the presence of CD90 and CD105 and the absence of CD45, according to the International Society for Cellular Therapy (ISCT) criteria for stem cells [ 26 ] using RT-PCR on 1 ug of total RNA with the ABgene Reverse-iT One-Step RT-PCR kit (ThermoFisher Scientific; Fair Lawn, NJ) and a Mastercycler gradient thermocycler (Eppendorf; Hamburg, Germany) that included an initial reverse transcription at 47 °C for 30 min, followed by 30, cycles of PCR with annealing for 30 sec at the appropriate temperature for each primer set, and final extension at 60 °C for one minute, as previously described [ 24 , 25 , 26 ]. The PCR positive cellular RNA control was glyceraldehyde 3-phosphate dehydrogenase (GAPDH).…”

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

“…In fact, preliminary studies from this group screened for the cytoplasmic expression of miR-365 among several oral cancer cell lines, some of which were previously tested (Cal27, Scc-9, and Scc-25) and others that had never been evaluated (Scc-4 and Scc-15), and found differential results [ 16 ] However, no studies to date have evaluated miR-365 expression among extracellular vesicles and exosomes derived from these specific cell lines. In addition, another related study from this group isolated exosomes and extracellular vesicles from oral cancer stem cells to screen for the expression of several microRNAs (miR-21, -31, -32, -34, -133, -155, and -365)—although only the expression of miR-21, -34, and -155 were observed among the exosomes specific to oral cancer stem cells [ 17 ].…”