title: Heat shock protein 90B1 plays an oncogenic role and is a target of microRNA-223 in human osteosarcoma.
abstract: OBJECTIVE
  Over the past decade, heat shock protein 90 (Hsp90) has emerged as a potential therapeutic target for cancer. However, the molecular mechanisms of down-regulation Hsp90 expression in osteosarcoma are incompletely understood. To develop potential therapy targeting Heat shock protein 90B1 (Hsp90B1), we studied the roles of miR- 223 in the proliferation and apoptosis of human osteosarcoma.
  
  
  METHODS
  pcDNA3.1(+)- miR-223 plasmid vectors were constructed and transfected into MG63 cells. Co-transfection of miR-223 expression vector with pMIR-Hsp90B1 (The recombined vector of pMIR-GLOTM luciferase vector containing Hsp90B1-3'UTR) led to the reduced activity of luciferase in a dual-luciferase reporter gene assay, suggesting that Hsp90B1 is a target gene of miR-223. Expression of HSP90B1 was detected by RT-PCR and western blotting analysis. Cell proliferation was determined using the MTT assay. Cell-cycle distribution and apoptosis were examined by flow cytometry. PI3K, p-Akt, Akt, mTOR, Bcl-2 and Bid were also detected by western blotting analysis. After a mouse xenograft model of human MG63 tumors was constructed, tumor growth, microvessel density and proliferation in each group was determined.
  
  
  RESULTS
  The pcDNA3.1(+)-miR-223 vector efficiently suppressed the expression of HSP90B1, while silencing miR-223 increased expression of Hsp90B1. Furthermore, overexpression of miR-223 results in significant inhibition of cell growth on culture plates. Moreover, cancer cells showed significant G0/G1 arrest and increased apoptosis due to gene silencing. Protein levels of PI3k, p-Akt, mTOR, and Bcl-2 were decreased, whereas Bid levels were increased. Microvessel density as assessed by CD34 levels and cell growth by PCNA levels decreased according to immunohistochemical analysis.
  
  
  CONCLUSIONS
  Hsp90B1 is a direct target of miR-223 and miR- 223 may have a tumor suppressor function in osteosarcoma through the PI3K/Akt/mTOR pathway and could be used in anticancer therapies in osteosarcoma.
authors.affiliation: Department of Orthopaedics, Shanghai Tenth People's Hospital, Tongji University School of Medicine, 301 Yanchang Rd, Shanghai 200072, China.
authors.forename: Guodong
authors.initials: G
authors.lastname: Li
authors.forename: Ming
authors.initials: M
authors.lastname: Cai
authors.forename: Dong
authors.initials: D
authors.lastname: Fu
authors.forename: Kai
authors.initials: K
authors.lastname: Chen
authors.forename: Mengxiong
authors.initials: M
authors.lastname: Sun
authors.forename: Zhengdong
authors.initials: Z
authors.lastname: Cai
authors.forename: Biao
authors.initials: B
authors.lastname: Cheng
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D001665:Binding Sites; D001859:Bone Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000502:physiology*; D009368:Neoplasm Transplantation; D009857:Oncogenes; D012516:Osteosarcoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D047368:Tumor Burden
chemical_list: D020413:3' Untranslated Regions; D008562:Membrane Glycoproteins; D035683:MicroRNAs; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; C525067:MIR223, human; C053387:endoplasmin; C546842:MTOR protein, human