title: miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1.
abstract: BACKGROUND
  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.
  
  
  METHODS
  Tumor 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei China.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Guo
authors.affiliation: Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei China.
authors.forename: Ling
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei China.
authors.forename: Zhengpei
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei China.
authors.forename: Guo
authors.initials: G
authors.lastname: Dai
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Department of Orthopedic Oncology, Peking University Cancer Hospital & Institute, Beijing, China.
authors.forename: Tian
authors.initials: T
authors.lastname: Gao
authors.affiliation: Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei China.
authors.forename: Weichun
authors.initials: W
authors.lastname: Guo
keywords: Cancer stem-like cells; Osteosarcoma; POU5F1; Targeted therapy; microRNA