FPN1, Targeted By Mir-17-5p, Regulates Cellular Iron Homeostasis Via activating Nrf2/Mir-17-5p/FPN1 Axis in Preclinical Model of Multiple Myeloma

Abstract

Multiple myeloma (MM) is a hematologic malignancy with lytic bone lessions, including dysregulation of iron homeostasis. Ferroportin (FPN1), the only known cellular iron exporter, has been demonstrated to have a dysregulated expression and to be associated with poor clinical outcome. However, the detailed mechanism of FPN1 in iron transportion is not clear. Our current study showed that miR-17-5p was another new FPN1 microRNA target, which had a negative correlation with FPN1 expression level in MM. We also identified that nuclear factor erythroid 2-related factor 2 (Nrf2) induced the transcription of miR-17-5p, which in turn suppressed the protein level of FPN1. Moreover, chromatin immunopreciptation (ChIP) and luciferase reporter assay revealed that Nrf2 directly inhibited the transcription of FPN1. Here, we investigated the mechanism of unbalanced Nrf2/miR-17-5p/FPN1 axis and CRISPR-mediated knockout of FPN1 function involved in iron metabolism of MM.

To investigate the potential role of miR-17-5p in MM, several public datasets were analyzed, and we identified that miR-17-5p was significantly overexpressed in the progression of MM. We next examined the functional effect on cell proliferation and apoptosis, ARP1 and OCI-MY5 cells were transiently transfected with miR-17-5p mimics or inhibitors. The experiment revealed that miR-17-5p promoted proliferation, cell cycle and apoptosis resistance both in vitro and in vivo. Besides, we explored whether FPN1 could rescue the effect on cells growth by overexpressing FPN1 in MM cells co-transfecting with miR-17-5p mimics or scramble. Our results showed that FPN1 restoration partially induced an inhibitory effect on myeloma cells, confirming that miR-17-5p accumulation favored cell growth and survival by targeting FPN1.

Furthermore, to investigate the regulatory mechanism of FPN1 in iron transportion, several transcription factor targeting predication algorithms were exploited, and we revealed that the FPN1 and miR-17-5p common target gene Nrf2, was significantly relative overexpressed in myeloma. We also analyzed the MM patient public datasets, including paired samples and different stages of disease, and all showed Nrf2 overexpression in relapsed MM with poor prognosis. Luciferase activity analysis verified that FPN1 was significantly inhibited when co-transfected, including truncated bodies of FPN1 promotor sequence. Meanwhile, ChIP-PCR showed that there was an interaction between Nrf2 and FPN1. Additionally, the direct interactions of Nrf2 with the miR-17-5p promoter were also confirmed, suggesting that Nrf2 bound to the miR-17-5p promoter region, thus regulating the transcription of miR-17-5p. These data indicateed that Nrf2 activation could modulate FPN1 levels directly or through miR-17-5p.

To more comprehensively evaluate FPN1 in regulating iron exportation in MM, we introduced single guide (sg) RNA targeting FPN1 into MM cells stably expressing Cas9. Of note, CRISPR-mediated FPN1 knockout promoted the growth and increased intracellular iron levels. In addition, to test the requirement of iron for the growth of myeloma, two sgFPN1 depleted MM cells were treated with iron supplement Fecl3, or the iron chelator deferoxamine (DFO). Fecl3 had significantly increased intracellular iron while the effect treated with DFO was opposite. Besides, MM cell proliferation was significantly inhibited by DFO treatment, further confirming the role of intracellular iron in MM cell growth. We also analyzed the mRNA levels of signature genes involved in maintaining cellular iron homeostasis and the level of reactive oxygen species (ROS) generation during the iron-deficient or iron-abundant state, and our results showed that these genes involved in iron acquisition or iron storage were differentially expressed and influenced intracellular iron availability and ROS levels.

In conclusion, our data suggested that the miR-17-5p/FPN1 axis induced by Nrf2 regulated intracellular liable iron pool, thus providing surplus iron for metabolic processes like DNA synthesis, the proliferation and growth of myeloma cells. Understanding the mechanisms on iron metabolism is important to develop new therapeutic strategies for clinical application to the treatment of MM.

Funding

This study was supported by grants from the National Natural Science Foundation of China (Nos. 81570190; 81670194 and 81529001).