Brusatol, a Nrf2 inhibitor, was purchased from Rongbai biological technology, Co

Brusatol, a Nrf2 inhibitor, was purchased from Rongbai biological technology, Co., Ltd. transcription through promoter binding and suppressed miR-17-5p (which also increased FPN1 expression). Nrf2-mediated FPN1 downregulation promoted intracellular iron accumulation and reactive oxygen species. Our study links FPN1 transcriptional and post-transcriptional regulation with MM cell growth and survival, and validates the prognostic value of FPN1 and its utility as a novel therapeutic Caspase-3/7 Inhibitor I target in MM. gene. d Diagram of FPN1 3-UTR wild-type and mutant reporter constructs. e Luciferase reporter assays were performed in HEK293T cell with co-transfection of indicated wild-type or mutant 3-UTR constructs Caspase-3/7 Inhibitor I and miR-17-5p mimic. f, h Cell proliferation and colony formation were assessed in myeloma cells transfected with miR-17-5p mimic or miR-NC, with or without plasmid pCDH-FPN1 vector. g FPN1 expression was identified in FPN1 overexpressing multiple myeloma cells by western blot. i Tumor samples were collected and images were captured using a digital camera. j The indicated cells were injected into the upper flank region of nude mice (luciferase gene in the psiCHECK2 vector. HEK293T cells were co-transfected with vectors harboring the wild-type or mutant FPN1 3-UTR (Fig. ?(Fig.1d)1d) and the miR-17-5p mimic. Luciferase Nt5e activity markedly decreased (0.516??0.030, luciferase gene, respectively, in the psiCHECK2 vector to mimic endogenous FPN1 mRNA expression. miR-17-5p overexpression decreased FPN1 3UTR luciferase activity to 30.8% (0.083) and 27.9% (0.122) of the control in ARP1 and OCI-MY5 cells, and FPN1-5UTR-LUC-3UTR activity to 52.3% (0.047) and 27.8% (0.072) of the control respectively (Fig. ?(Fig.4n,4n, ?,o).o). miR-17-5p overexpression significantly increased FPN1 5UTR-luciferase activity in both cells. Conversely, miR-17-5p knockdown in myeloma cells increased FPN1 3-UTR (1.299??0.117 and 2.382??0.377, em p /em ? ?0.01) and FPN1-5’UTR-LUC-3’UTR (1.613??0.082 and 1.462??0.071, em p /em ? ?0.01) luciferase activities, versus the inhibitor control (Fig. 4, p). After miR-17-5p inhibition, 5-UTR of FPN1 Caspase-3/7 Inhibitor I activity decreased in both ARP-1 (0.464??0.141, em p /em ?=?0.002) and OCI-MY5 (0.661??0.232, em p /em ? ?0.01) cells. Low FPN1 conferred MM cell growth and survival Decreased FPN1 expression in MM-patient samples correlated with short event-free survival (EFS) and inferior OS, with poor patient outcomes in clinical trials35. Here, we introduced a single guideline (sg) RNA targeting FPN1 into MM cell lines stably expressing em Cas9 /em , and FPN1-protein levels were verified by western blotting (Fig. ?(Fig.5a).5a). CRISPR-mediated FPN1 knockout promoted cell growth (Fig. ?(Fig.5b)5b) and increased MM-cell Caspase-3/7 Inhibitor I colony formation (Fig. ?(Fig.5c5c). Open in a separate window Fig. 5 FPN1 regulates multiple myeloma cell intracellular iron and ROS.a ARP1 and OCI-MY5 cells expressing Cas9 were transduced with sgRNA against FPN1 (sgFPN1) or control sgRNA (sgCtrl). Western blot confirmed FPN1 knockout. b FPN1-sgRNA MM cell lines ARP1, OCI-MY5 as well as their controls were analyzed using a Cell Counting kit-8 assay for consecutive 5 days for cell growth. c Clonogenic ability of myeloma cells was examined after FPN1 knockout. d, i The intracellular iron was measured with or without different reagents (50?M DFO and 5?M brusatol) in indicated cells using QuantiChromTM Iron Assay Kit. e, f Two myeloma cells were treated with deferoxamine (DFO) and FeCl3 at indicated concentration for 24, 48, and 72?h, and viability was then analyzed. g FPN1-sgRNA MM cell lines ARP1, OCI-MY5 were treated for 48?h with varying concentrations of DFO and/or brusatol. h ARP1 and OCI-LY5 FPN1-knockout cells with different treatment (50?M DFO and 5?M brusatol) for 48?h in indicated cells and stained with H2DCFDA; then the level of ROS was detected by flow cytometry and data are presented as the mean fluorescence intensity. j Bioinformatics analysis indicated that most interact proteins of FPN1 are involved in iron metabolism. k qRT-PCR Caspase-3/7 Inhibitor I was used to examine expression of genes related to iron metabolism. Data are expressed as the means??standard deviation ( em n /em ?=?3). * em p /em ? ?0.05, ** em p /em ? ?0.01, n.s., not significant, compared to the vehicle control group; Students em t /em -test DFO was used to examine the role of iron in the contributions of FPN1 to cell growth and transformation. DFO reduced myeloma cell viability in a time-dependent manner, but not in a dose-dependent manner (Fig. ?(Fig.5f).5f). FeCl3 showed minimal cytotoxicity in both cell lines, at concentrations up to 600?M (Fig. ?(Fig.5e5e). Both Nrf2-mediated transcriptional regulation and miR-17-5p-mediated FPN 3-UTR regulation altered cellular iron and ROS levels Knocking out FPN1 in ARP1 and OCI-MY5 cells.