title: MicroRNA-433 negatively regulates the expression of thymidylate synthase (TYMS) responsible for 5-fluorouracil sensitivity in HeLa cells.
abstract: BACKGROUND
  Thymidylate synthase (TYMS) is an important folate-dependent enzyme in DNA synthesis and an important target for cancer chemotherapy. High TYMS expression levels in tumors are generally associated with resistance to 5-fluorouracil (5-FU). The cause of the variability in TYMS expression is still not fully understood, however, only a small proportion of the TYMS expression can be explained by TYMS genetic polymorphisms. The purpose of this study is to identify novel microRNAs (miRNAs) which regulate the expression of TYMS and to determine whether miRNAs binding to the 3'-untranslated region (UTR) of TYMS mRNA affect the proliferation of HeLa cells treated with 5-FU.
  
  
  METHODS
  An in silico search was performed to find potential binding sites of miRNAs in TYMS mRNA. The efficacy of predicted miRNAs at the 3'-UTR of TYMS mRNA was evaluated using a dual-luciferase reporter assay. TYMS mRNA and protein expression in HeLa cells was quantified with real-time RT-PCR and Western blotting, respectively. The effects of miR-433 on cell proliferative activity were determined by WST-8 assay.
  
  
  RESULTS
  The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3'-UTR of TYMS mRNA (P < 0.01). The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05). Furthermore, miR-433 increased inhibition of cell proliferation in HeLa cells treated with 5-FU at over 2.0 μM.
  
  
  CONCLUSIONS
  The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells. This is the first report showing that a miRNA regulating TYMS expression has a significant impact on sensitivity to 5-FU treatment.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Gotanda
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hirota
authors.forename: Nozomi
authors.initials: N
authors.lastname: Matsumoto
authors.forename: Ichiro
authors.initials: I
authors.lastname: Ieiri
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005472:Fluorouracil / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; C548883:MIRN433 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D008156:Luciferases; D013940:Thymidylate Synthase; D005472:Fluorouracil
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