Regulation of miR-132 on Target Gene FOXA1 in Esophageal Carcinoma Cell Line KYSE150

ObjectiveTo construct an eukaryotic expression vector of miR-132 and FOXA1，to verify the regulation effect of miR-132 on the target gene FOXA1 in human esophageal carcinoma cell KYSE150. MethodsAccording to sequence of the mature miR-132 with its flank sequences，PCR primers were designed and miR-132 precursor sequence was amplified from genomic DNA of EC9706 cell. PCR product was cloned into pMD18-T Simple vector and then subcloned into pCDNA3.1(+) between BamHⅠand EcoRⅠ. Human esophageal carcinoma cell line KYSE150 was transfected by pCDNA3.1(+) and pCDNA3.1-miR-132,miR-132 were detected by real-time PCR. FOXA1 was predicted by using bioinformatics. The 3′UTR of candidate target gene FOXA1 was cloned into the downstream of pMIR vector,mutation expression vector pMIR-FOXA1-Mut was also constructed. Co-transfection of both vectors were performed in KYSE150 cells and dual-luciferase reporter assay was analyzed. Western blot was used to detect the expression of FOXA1 protein in KYSE150 cell line transfected with pCDNA3.1(+)-miR-132 and pCDNA3.1(+) respectively. ResultsThe miR-132 expression vector has been constructed successfully and it can effectively express mature miR-132 in esophageal carcinoma cell KYSE150. FOXA1 was selected as a candidate of target genes which has 7 base pairs completely matched to miR-132 in the 3′UTR seed region. Recombined vector pMIR-FOXA1 fused FOXA1 3′UTR and mutation expression vector pMIR-FOXA1-Mut were constructed. Dual-luciferase reporter assay showed that the luciferase activity decreased in the group co-transfected with pCDNA3.1(+)-miR-132 and pMIR-FOXA1. Overexpression of exogenous miR-132 in KYSE150 cell line can significantly suppress the expression of FOXA1 protein. ConclusionmiR-132 can suppress FOXA1 gene expression at the post-transcriptional level by targeting to the specific sequence of FOXA1 gene 3′UTR,FOXA1 is a target gene of miR-132.