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Regulatory effect of miR-181a on expression of c-fos in cochlear hair cells

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Author:
No author available
Journal Title:
Chinese Journal of Industrial Hygiene and Occupational Diseases
Issue:
10
DOI:
10.3760/cma.j.issn.1001-9391.2012.10.006
Key Word:
c-fos;miRNA;耳蜗毛细胞;靶基因;c-fos;miRNA;inner ear hair cells;target genes

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 Corti 1 (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-181 a 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-181 d 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.Conclusion 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-181 a.Bioinformatic prediction might produce false-positive results.

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