Identification, characterization and microRNA expression profiling of side population cells in human oral squamous...

The present study aimed to evaluate the stem cell markers, characteristics and biological functions of cancer stem‑like side population (SP) cells in human oral cancer. SP cells were isolated from the human oral squamous cell carcinoma Tca8113 cell line by Hoechst 33342 fluorescence dye and flow cytometry. The colony forming and proliferative capability of SP and non‑SP cells were detected using a live‑cell analysis system in vitro. The number of cells expressing stem cell markers was compared between SP cells and non‑SP cells by flow cytometry. Reverse transcription‑quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels of stem cell genes, respectively. Differential expression of microRNAs (miRNAs) in SP and non‑SP cells was determined by microarray hybridization and an miRNA regulation network was produced. With regard to the proliferation capability, SP cells reached 60.0% confluence after 40 h of growth compared with 35.1% confluence for non‑SP cells (P<0.05). The number of colonies in SP cells was 43.1±9.2 compared with 33.0±8.2 of non‑SP cells (P<0.05). The aldehyde dehydrogenase‑1 (ALDH1)‑positive cell number in the SP cells was increased by 10 times compared with the non‑SP cells (P<0.01). The mRNA and protein expression levels of ALDH1, SRY‑box 2, POU class 5 homeobox 1 and Nanog homeobox in SP cells were significantly higher compared with non‑SP cells (P<0.05). Microarray hybridization demonstrated that 21 miRNAs were upregulated and 13 miRNAs were downregulated in SP cells compared with non‑SP cells. SP cells in Tca8113 demonstrated greater capability of proliferation and colony formation compared with non‑SP cells in vitro. Stem cell markers were overexpressed in SP cells compared with non‑SP cells.