Abstract
BackgroundEvidence is accumulating to link cancer stem cells to the pathogenesis and progression of osteosarcoma. The aim of this study is to investigate the role of miR-335 in osteosarcoma stem cells.MethodsTumor spheroid culture and flow cytometry were applied to screen out osteosarcoma stem cells. Real-time quantitative PCR was used to detect the expression level of miR-335 in MG63, U2OS and 143B osteosarcoma stem cells. The relationship of miR-335 expression with osteosarcoma stem cells was then analyzed. Transwell assay and transplantation assay were performed to elucidate biological effects of miR-335 on cell invasion and vivo tumor formation. Western Blot and luciferase assays were executed to investigate the regulation of POU5F1 by miR-335.ResultsThe expression of miR-335 in osteosarcoma stem cells was lower than their differentiated counterparts. Cells expressing miR-335 possessed decreased stem cell-like properties. Gain or loss of function assays were applied to find that miR-335 antagonist promoted stem cell-like properties as well as invasion. Luciferase report and transfection assay showed that POU5F1 was downregulated by miR-335. Pre-miR-335 resulted in tumor enhanced sensitivity to traditional chemotherapy, whereas anti-miR-335 promoted chemoresistance. Finally, the inhibitory effect of miR-335 on in vivo tumor formation showed that combination of pre-miR-335 with cisplatin further reduced the tumor size, and miR-335 brought down the sphere formation capacity induced by cisplatin.ConclusionsThe current study demonstrates that miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1, and miR-335 could target CSCs to synergize with traditional chemotherapeutic agents to overcome osteosarcoma.
Highlights
Evidence is accumulating to link cancer stem cells to the pathogenesis and progression of osteosarcoma
Expression of miR‐335 is downregulated in osteosarcoma stem cells Because CD117 and Stro-1 double positive cells are thought to be enriched for osteosarcoma Cancer stem cells (CSCs), we compared miR-335 expression in double positive (DP) and double negative (DN) cell populations, and showed that the expression of miR-335 decreased significantly in DP cells in all three cell lines (Fig. 1a)
CSCs are capable of forming spheres when cultured without serum and in an anchorage-independent manner, so we examined miR-335 expression in cells cultured under sphereforming conditions
Summary
Evidence is accumulating to link cancer stem cells to the pathogenesis and progression of osteosarcoma. The aim of this study is to investigate the role of miR-335 in osteosarcoma stem cells. Cancer stem cells (CSCs) are defined as cells with a self-renewal ability that can generate heterogeneous cancer cells, which play a key role in recurrence, metastasis, and drug resistance [3]. Several studies have confirmed the existence of osteosarcoma stem cells. Gibbs first reported that stem-like cells exist in osteosarcoma, which are capable of forming self-renewable tumor spheres. Adhikari et al have identified mouse and human osteosarcoma stem cells using mesenchymal stem cell markers CD-117 and Stro-1. These markers were preferentially expressed in spheres and doxorubicin-resistant cells
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