| Objective Determine the target gene of miR-592 in colorectal cancer.Methods 1 The expression of FOXO3 A in 20 clinic human CRC and matched adjacent non-tumor tissues was evaluated by an immunohistochemistry(IHC) staining using rabbit anti-FOXO3 A antibody,and immunoblotting assay also be used to detected the expression of FOXO3 A in CRC tissue and matched normal tissue. 2 In order to validate the FOXO3 A mRNA was a target of miR-592, a reporter plasmid containing luciferase with the 3’UTR sequence of human FOXO3 mRNA was generated. The cDNA generated from LOVO RNA was used as templates for amplification of FOXO3 3’UTR fragment by a PCR assay. The results of dual luciferase assay showed relative luciferase activity in the cells transfected with miR-592 mimics, compared with the control miRNA or inhibitor transfected cells.3 Transfect LV-miR-592-inh 和 LV-miR-NC with CRC cell line LS174 T,detect each cell lines the expression of FOXO3 A by Western blotting.Results 1 Our immunohistochemical staining results revealed a strikingly down-regulated expression of FOXO3 A in CRC tumor tissues, as compared to the matched adjacent non-tumor tissues,the result of immunoblotting assay also comfirm that.2 The results of dual luciferase assay showed a significant decrease of relative luciferase activity in the cells transfected with miR-592 mimics, in comparison with the control miRNA or inhibitor transfected cells.3 immunoblotting assay further confirmed that LS174 T CRC cells transduced with LV-miR-592-inh showed a restoration of FOXO3 A protein, as compared with its na?ve and LV-NC transduced parent cells.Conclusion The transcription factor forkhead box protein O3A(FOXO3A) was identified as a potential target of miR-592. |
