title: [Regulatory effect of miR-181a on expression of c-fos in cochlear hair cells].
abstract: OBJECTIVE
  To investigate the regulatory effect of miR-181a with abnormal expression on the expression of c-fos in cochlear hair cells undergoing oxidative damage.
  
  
  METHODS
  House Ear Institute-Organ of Corti1 (HEI-CO1) cells were assigned to 50, 100, and 200 µmol/L tert-butyl hydroperoxide (t-BHP) exposure groups and control group. The HEI-CO1 cells in the exposure groups were exposed to 50, 100, or 200 µmol/L t-BHP for 12 h. Then, total RNA and total protein were extracted from the HEI-CO1 cells, and the expression of miR-181a/-181d was measured by qPCR. The miR-181a with abnormal expression was selected as the subject of study. The putative miR-181a target sequence in the 3' untranslated region (3'-UTR) of c-fos was predicted by searching on a bioinformatics website. The HEI-CO1 cells were transfected with miR-181a mimics by lipofection, and the transfection efficiency was measured by qPCR. The mRNA and protein expression of c-fos was measured by qPCR and Western blot. The pGL3-c-fos-3'UTR-WT plasmid was constructed, and the luciferase activity of the plasmid in the case of high miR-181a expression was measured using the Dual-Luciferase Reporter Assay System.
  
  
  RESULTS
  Compared with those in the control group, the expression of miR-181a in 100 and 200 µmol/L t-BHP exposure groups was significantly decreased, with expression ratios of 0.744 and 0.766 (P < 0.01), while the expression of miR-181d in 50 µmol/L t-BHP exposure group was significantly increased, with an expression ratio of 1.29 (P < 0.01). There was no significant difference in miR-181a expression between the 100 and 200 µmol/L t-BHP exposure groups (P > 0.05). The predication results revealed that c-fos was regulated by miR-181a in humans and mice, with complete complementarity to the seed region of miR-181a, and there was high degree of target sequence conservation across species. The expression of miR-181a in the HEI-OC1 cells transfected with miR-181a mimics was elevated 892.979 times at 24 hours after transfection. As compared with those of controls, the mRNA and protein expression levels of c-fos in the transfected HEI-OC1 cells were significantly increased (P < 0.05 and P < 0.01). The luciferase activity of pGL3-c-fos-3'UTR-WT plasmid was not suppressed but increased in the case of high miR-181a expression.
  
  
  CONCLUSIONS
  miR-181a has no direct inhibitory effect on the mRNA and protein expression of c-fos, which may not be the target gene of miR-181a. Bioinformatic prediction might produce false-positive results.
authors.affiliation: School of public health, SUN Yat-sen University, Guangzhou 510080, China.
authors.forename: Li-mei
authors.initials: LM
authors.lastname: Chen
authors.forename: Zhi
authors.initials: Z
authors.lastname: Wang
authors.forename: Yao
authors.initials: Y
authors.lastname: Guo
authors.forename: Yi-min
authors.initials: YM
authors.lastname: Liu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D006198:Hair Cells, Auditory / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D020122:tert-Butylhydroperoxide / Q000633:toxicity*
chemical_list: D035683:MicroRNAs; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; C529100:mirn181 microRNA, mouse; D020122:tert-Butylhydroperoxide