The Molecular Mechanism Underlying Deregulation Of Iron Exporter Ferroportin In Tumors

ObjectiveTo investigate the role of ferroportin in effecting the prostate cancer and breast cancer cell growth, and to reveal the mechanism of ferroportin downregulation in tumor.MethodsThe mRNA and protein expression levels of ferroportin were detected by qRT-PCR and Western blot in tumor specimens from breast cancer patients. The forced expression of human ferroportin plasmid was constructed. The ferroportin overexpression plasmid and ferroportin specific shRNA were transfected into prostate cancer cell line PC3and breast cancer cell line MDA-MB-231respectively, in order to construct the stable high or low expressed tumor cell lines. Cells counting, MTT and BrdU assay were used to evaluate the cell proliferation ability after ferroportin was upregulated or downregulated. Colony formation assay was used to estimate the ability of colony formation of tumor cells. The in vitro experiments were comfirmed through establishing the tumor animal models. To elucidate the mechanism underlying downregulated expression of ferroportin in breast cancer, we predicted that the3.0kb region of human ferroportin promoter through literature search, web-based software and microarray analysis. We focused on Nrf2and MZF-1in our study. We performed Chromatin immunoprecipitation (ChIP) assay with the Abs against human Nrf2and MZF-1to confirme the interaction between-Nrf2、MZF-1and ferroportin promoter. The DNA fragments of the ferroportin promoter with full-or deleted-binding motifs for the Nrf2and MZF-1binding site were cloned into the pGL3Luciferase reporter vector, and the luciferase reporter assay was performed to assess the contribution of every predicted transcriptional binding site. The MZF-1overexpression plasmid and MZF-1specific siRNA were transfected into PC3cell, cell proliferation ability was assessed by cells counting, MTT, BrdU and LIVE viability assay. Online tools were used to predict the potential miRNAs and transcription factors of MZF-1. After miR-492mimic was transfected into PC3cells, cell proliferation ability and MZF-1concentration were detected. pGL3Luciferase reporter plasmid containing the fragment of the MZF-13’UTR was generated to analyze the effect of miR-492on MZF-1. The mRNA and protein expression levels of MZF-1were detected by qRT-PCR and Western blot using AP4and c-Myb specific siRNAs. To validate the role of AP4and c-Myb in transcriptional regulation of MZF-1, ChIP assay and luciferase reporter assay were performed.ResultsThe expression level of ferroportin was greatly decreased in tumors compared to the adjacent tissues, especially in malignant tumors. Low ferroportin in tumors predicted a poor prognosis compared to the high ferroportin level. Upon ferroportin reduction, prostate cancer PC3cells and breast cancer MDA-MB-231cells proliferation and colony formation ability were greatly enhanced compared to the scrambled control. In contrast, compared to the control, increased ferroportin reduced the growth of PC3and MDA-MB-231cells and the capacity of colony fomiation. We confirmed the binding of Nrf2on ferroportin promoter by ChIP. The luciferase reporter assessment determined that the construct bearing a complete Nrf2-binding motif stimulated luciferase activity; in contrast, deletion of this binding motif abolished this enhancement, demonstrating a contributing role of Nrf2in enhancing ferroportin transcription. Meanwhile, ChIP assay demonstrated the MZF-l’s binding to the3sites of ferroportin promoter. The luciferase reporter assay data suggested that the second bind motif (-463/-458) could be crucial in response to MZF-1-regulated ferroportin expression. In parallel to the ferroportin concentration, we observed a substantial decline of Nrf2and MZF-1in tumors compared to adjacent tissues at the mRNA and protein levels, particularly in malignant tumors. Decreased MZF-1promoted the proliferation of PC3cells in vitro; on the contrary, increased MZF-1inhibited the growth of PC3cells compared to the control. Overexpression of miR-492enhanced the PC3cells proliferation ability and reduced the protein expression level of MZF-1. MiR-492downregulated MZF-1by target binding of miR-492on the region of MZF-13’UTR. On the other hand, AP4and c-Myb both transactivated MZF-1expression through binding to the MZF-1promoter. The decrease of MZF-1concentration upon AP4or c-Myb specific siRNAs was observed, supporting the regulation of MZF-1by AP4and c-Myb. ConclusionDownregulation of iron exporter ferroportin promotes the proliferation of PC3cells and MDA-MB-231cells in vitro and in vivo. Nrf2and MZF-1regulate ferroportin transcription. Decreased MZF-1promoted the proliferation of PC3cells. MiR-492inhibits the expression of MZF-1by binding on the region of MZF-13’UTR. Additionally, AP4and c-Myb can transactivate MZF-1expression through binding to the promoter of MZF-1.