MicroRNA profiling of a CD133(+) spheroid-forming subpopulation of the OVCAR3 human ovarian cancer cell line

Abstract

Background: Cancer stem cells (CSCs) are thought to be a source of tumor recurrence due to their stem cell-like properties. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has an important role in tumorigenesis. Cluster of differentiation (CD) 133(+) and spheroid formation have been reported to be one of the main features of ovarian CSCs. Therefore, we determined the miRNA expression profile of a CD133(+) spheroid-forming subpopulation of the OVCAR3 human ovarian cancer cell line.

Methods: Initially, we confirmed the enrichment of the OVCAR3 CD133 subpopulation by evaluating in vitro anchorage-independent growth. After obtaining a subpopulation of CD133(+) OVCAR3 cells with > 98% purity via cell sorting, miRNA microarray and real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed to evaluate its miRNA profile.

Results: We found 37 differentially expressed miRNAs in the CD133(+) spheroid-forming subpopulation of OVCAR3 cells, 34 of which were significantly up-regulated, including miR-205, miR-146a, miR-200a, miR-200b, and miR-3, and 3 of which were significantly down-regulated, including miR-1202 and miR-1181.

Conclusions: Our results indicate that dysregulation of miRNA may play a role in the stem cell-like properties of ovarian CSCs.

Figure 1

Representative images of tumor spheres from OVCAR3, TOV112D, and SKOV3 cells in a sphere culture system with serum-free DMEM-F12 (Invitrogen, Carlsbad, Calif., USA) supplemented with 10 ng/mL basic fibroblast growth factor (bFGF) and 20 ng/mL epidermal growth factor (EGF) and plated in an ultra-low attachment plate.

Figure 2

Increased paclitaxel resistance of OVCAR3 and SKOV3 cells in an anchorage-independent culture system compared with those in a conventional adherent culture system.

Figure 3

Flow cytometry analysis with OVCAR3, SKOV3, and TOV-112D were performed before and after anchorage-independent culture at least three times. CD133⁺and CD44⁺populations were enriched in a sphere culture system in TOV112D and OVCAR3 cells; however, CD44⁺populations were initially high in the SKOV3 cell line.

Figure 4

RT-PCR and Western blot analysis showed that a CD133⁺spheroid-forming subpopulation of OVCAR3 over-expresses “stemness” genes, compared to the levels of cancer cells from an adherent culture system as well as those from a CD133^-spheroid-forming subpopulation of OVCAR3 cells.

Figure 5

Unsupervised hierarchical clustering analysis of miRNAs that exhibited a > two-fold increase or decrease in the CD133+ spheroid-forming subpopulation of the OVCAR3 human ovarian cancer cell line.