MicroRNA expression profiling identifies miR-328 regulates cancer stem cell-like SP cells in colorectal cancer

Side population (SP) cells and their relationship to stem cell-like properties have been insufficiently studied in colorectal cancer (CRC). MicroRNAs (miRNAs) have attracted much attention but their roles in the maintenance of SP phenotype remain unclear.

The SPs from CRC cell lines and primary cell cultures were analysed for stem cell-like properties. MiRNA microarray analysis identified miR-328 as a potential stemness miRNA of SP phenotype. The level of miR-328 expression in clinical samples and its correlation with SP fraction were determined. Gain-of-function and loss-of-function studies were performed to examine its roles in cancer stem-like SP cells. Furthermore, bioinformatics prediction and experimental validation were used to identify miR-328 target genes.

The SP cells sorted from CRC possess cancer stem cell (CSC)-like properties, including self-renewal, differentiation, resistance to chemotherapy, invasive and strong tumour formation ability. MiR-328 expression was significantly reduced in SP cells compared with Non-SP cells (P<0.05). Moreover, miR-328 expression was downregulated in CRC (n=33, P<0.05) and low miR-328 expression tend to correlate with high SP fraction (n=15, r=0.6559, P<0.05, Pearson's correlation). Functional studies indicated that miR-328 expression affects the number of SP cells. In addition, miR-328 overexpression reversed drug resistance and inhibited cell invasion of SP cells. Furthermore, luciferase reporter assay demonstrated that miR-328 directly targets ABCG2 and MMP16 and affects the levels of mRNA and protein expression in SP cells.

These findings indicate that CRC contain cancer stem-like SP cells. MiR-328 has an important role in maintaining cancer stem-like SP phenotype that may be a potential target for effective CRC therapy.