MicroRNA-29b-1 is involved in self-renewal and fate decisions of human osteosarcoma 3AB-OS cancer stem cells

Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. Moreover, identification of CSC-related MicroRNAs (miRNAs) would provide information for a better understanding of CSCs. miR-29 family is a class of miRNAs aberrantly expressed in multiple cancers. They are frequently down-regulated in osteosarcoma (OS), the most common form of childhood cancer with a potent metastasizing potential. 3AB-OS CSC, a human pluripotent CSC line by us produced from the human osteosarcoma MG63 cells (1) is a useful model to study CSC origin and roles (2). Previously, we have shown that in 3AB-OS CSCs miR-29b is potently down-regulated (2). Here, after stable transfection of 3AB-OS cells with miR-29b-1, we investigated its role in regulating cell proliferation, sarcosphere-forming ability, clonogenic growth, chemosensitivity, migration and invasive ability of 3AB-OS CSCs, in vitro. We found that miR-29b-1 overexpression consistently reduced both, 3AB-OS CSCs growth in two- and three-dimensional culture systems and their sarcosphere- and colony- forming ability. It also sensitized 3AB-OS cells to chemotherapeutic drug-induced apoptosis. Using publicly available databases, we proceeded to identify potential miR-29b target genes, known to play a role in the above reported functions. Among these targets we analyzed CD133, N-Myc, CCND2, E2F1 and E2F2, Bcl-2 and IAP-2. We even analyzed the most important stemness markers as Oct3/4, Sox2 and Nanog. Real-time RT-PCR and western-blot analyses showed that miR-29b-1 negatively regulated the expression of these markers. Overall, the results show that miR-29b-1 suppresses stemness properties of 3AB-OS CSCs and suggest that developing miR-29b- 1 as a novel therapeutic agent might offer benefits for OS treatment.