Identification of cancer stem cells from hepatocellular carcinoma cell lines and their related microRNAs.

The aim of this study was to identify cancer stem cells (CSC) from three hepatocellular carcinoma (HCC) cell lines and to screen for specific microRNAs (miRNAs) regulating CSCs. Side population (SP) phenotype analysis was used. Four factors in the staining process, the incubation time, shaking interval, culture time and Hoechst 33342 concentration were explored, respectively, to define the SP subtype. CSC characteristics of SP cells were verified by sphere-forming assay and tumorigenic ability in NOD/SCID mice. QPCR assay for 370 miRNAs was performed to identify the differential miRNA expression between SP and Non-SP (NSP) cells in the PLC/PRF/5 cell line. The selected miRNAs were tested again in SP and NSP cells from Huh-7 and Hep-3B cell lines by qPCR assay. All four factors influenced SP percentage, when the other three conditions were fixed, the optimal Hoechst 33342 concentrations determined were 11 µg/ml for PLC/PRF/5 cells, 4 µg/ml for Huh-7 and 5 µg/ml for Hep-3B cells. The resultant SP percentage was 0.73±0.12%, 0.49±0.04% and 0.63±0.08%, respectively. The purity of sorted SP cells was >85%. Floating spheres were formed by SP cells from all three cell lines, while NSP cells did not form a single floating sphere. Mice injected with SP cells on the right side formed more tumor masses compared to their counterpart NSP at the same injection dosage; qPCR profiling identified 27 differentially expressed miRNAs in PLC/PRF/5 cells. Subsequent qPCR assay showed that miR-9* and miR-194 were also downregulated in SP cells from Huh-7 and Hep-3B. The present study identified CSCs via SP and sphere-forming assay from three liver cancer cell lines. Altogether, 27 CSC-specific miRNAs were determined in PLC/PRF/5; miR-9* and miR-194 were identified as the common CSC-specific miRNAs across the three HCC cell lines.