title: Micro-RNA expression in cisplatin resistant germ cell tumor cell lines.
abstract: BACKGROUND
  We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance.
  
  
  METHODS
  Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R) showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP). Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738) known microRNA species of human origin.
  
  
  RESULTS
  Altogether 72 of 738 (9.8%) microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15) of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%). The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79%) and NCCIT-R/NCCIT (64%), and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%). Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold) in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency), as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21) were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up-regulated) and hsa-miR-99a/-100/-145 (up to 10-fold down-regulated).
  
  
  CONCLUSIONS
  Examining almost all known human micro-RNA species confirmed the miR-371-373 cluster as a promising target for explaining cisplatin resistance, potentially by counteracting wild-type P53 induced senescence or linking it with the potency to differentiate. Moreover, we describe for the first time an association of the up-regulation of micro-RNA species such as hsa-miR-512-3p/-515/-517/-518/-525 and down-regulation of hsa-miR-99a/-100/-145 with a cisplatin resistant phenotype in human germ cell tumors. Further functional analyses are warranted to gain insight into their role in drug resistance.
authors.affiliation: Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany. matthias.port@web.de
authors.forename: Matthias
authors.initials: M
authors.lastname: Port
authors.forename: Stephanie
authors.initials: S
authors.lastname: Glaesener
authors.forename: Christian
authors.initials: C
authors.lastname: Ruf
authors.forename: Armin
authors.initials: A
authors.lastname: Riecke
authors.forename: Carsten
authors.initials: C
authors.lastname: Bokemeyer
authors.forename: Viktor
authors.initials: V
authors.lastname: Meineke
authors.forename: Friedemann
authors.initials: F
authors.lastname: Honecker
authors.forename: Michael
authors.initials: M
authors.lastname: Abend
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009373:Neoplasms, Germ Cell and Embryonal / Q000503:physiopathology*
chemical_list: D000970:Antineoplastic Agents; D035683:MicroRNAs; D002945:Cisplatin