Relationship among ETV1 genetic polymorphisms, PFS, and microRNA in gastrointestinal stromal tumors.

e22513 Background: In order to evaluate the pharmacokinetic and pharmacogenomic determinants for prognosis of gastrointestinal stromal tumor (GIST). It is necessary to explore a biological predictors to predict and optimal therapeutic strategy. But there were few studied conducted to explore the germline mutation and its mechanisms. Methods: A total of 75 GIST patients treated with Imatinib were enrolled. 35 SNPs (single nucleotide polymorphisms) in KIT/ PDGFRA/ PDGFRB/ ETV1/ FLT1/ MAPK1 et. al were detected using Agena Massarray matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) platform. COX regression analyses were performed to evaluate the key factors of PFS. The luciferase reporter system of rs3735343 wild type and mutation were established. The GIST-T1 cell line was used to evaluate the biology effect of rs3735343. This study was approved by the ethical committee of Sun Yat-Sen University Cancer Center. Results: Several factors influence the PFS, including KIT somatic mutation, tumor size and germline mutation ABCC4 rs4148551, ETV1 rs3735343, FLT1 rs3751397, KIT rs3822214. And COX regression showed that rs3735343 and tumor size are associated with PFS (P = 0.009 and 0.032, Risk Ratio = 8.995 and 4.173). And we found that rs3735343 mutation type can regulate ETV1 3’UTR and protein expression level through miR-4311 in vitro. Conclusions: The primary determinants of PFS in this somatic and germline mutation model suggested new biomarkers and different mechanisms involved in prognosis. This is the first report showed that ETV1 genetic polymorphisms may influence the prognosis through miRNA-4311. Targeting ETV1, miRNA-4311 or their relative pathway might be a new therapy strategy.

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Homeobox-A13 acts as a functional prognostic and diagnostic biomarker via regulating P53 and Wnt signaling pathways in lung cancer

Cancer Biomarkers ◽

10.3233/cbm-200540 ◽

2021 ◽

pp. 1-16

Author(s):

Yang Wang ◽

Bo He ◽

Yan Dong ◽

Gong-Jin He ◽

Xiao-Wei Qi ◽

...

Keyword(s):

Lung Cancer ◽

Signaling Pathways ◽

Cox Regression ◽

Prognostic Significance ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Clinical Feature ◽

Diagnostic Biomarker

BACKGROUND: The prognosis of lung cancer patients is poor without useful prognostic and diagnostic biomarker. To search for novel prognostic and diagnostic markers, we previously found homeobox-A13 (HOXA13) as a promising candidate in lung cancer. OBJECTIVE: To determine the precisely clinical feature, prognostic and diagnostic value, possible role and mechanism of HOXA13. METHODS: Gene-expression was explored by real-time quantitative-PCR, western-blot and tissue-microarray. The associations were analyzed by Chi-square test, Kaplan-Meier and Cox-regression. The roles and mechanisms were evaluated by MTS, EdU, transwell, xenograft tumor and luciferase-reporter assays. RESULTS: HOXA13 expression is increased in tumors, and correlated with age of patients. HOXA13 expression is associated with unfavorable overall survival and relapse-free survival of patients in four cohorts. Interestingly, HOXA13 has different prognostic significance in adenocarcinoma (ADC) and squamous-cell carcinoma (SCC), and is a sex- and smoke-related prognostic factor only in ADC. Importantly, HOXA13 can serve as a diagnostic biomarker for lung cancer, especially for SCC. HOXA13 can promote cancer-cell proliferation, migration and invasion in vitro, and facilitate tumorigenicity and tumor metastasis in vivo. HOXA13 acts the oncogenic roles on tumor growth and metastasis by regulating P53 and Wnt/β-catenin signaling activities in lung cancer. CONCLUSIONS: HOXA13 is a new prognostic and diagnostic biomarker associated with P53 and Wnt/β-catenin signaling pathways.

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The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

Bioscience Reports ◽

10.1042/bsr20120043 ◽

2012 ◽

Vol 32 (6) ◽

pp. 531-537 ◽

Cited By ~ 16

Author(s):

Albert Braeuning ◽

Silvia Vetter

Keyword(s):

Photon Emission ◽

Gene Promoter ◽

Firefly Luciferase ◽

Nuclear Factor Κb ◽

Signalling Pathway ◽

Luciferase Reporter ◽

Nuclear Factor ◽

Reporter System ◽

Reporter Assays

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

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Abstract 1384: Osteogenic Transcription Factor Runx2 Regulates Expression of RANKL during VSMC Calcification

Circulation ◽

10.1161/circ.118.suppl_18.s_318 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Chang Hyun Byon ◽

Jay McDonald ◽

Yabing Chen

Keyword(s):

Oxidative Stress ◽

Transcription Factor ◽

Molecular Mechanism ◽

Luciferase Reporter ◽

Rankl Expression ◽

Reporter System ◽

Mobility Shift ◽

Atherosclerotic Lesions ◽

Flanking Region

The expression of receptor activator of nuclear factor κ B (RANKL) is up-regulated in calcified atherosclerotic lesions, whereas it is frequently undetectable in normal vessels. The underlying molecular mechanism of increased expression of RANKL in calcified vessels is not known. We have previously demonstrated that oxidative stress induces calcification of vascular smooth muscle cells (VSMC) in vitro . Therefore, we determined whether oxidative stress regulates RANKL expression in VSMC and the underlying molecular mechanism. Consistent with previous observations in vivo , we found that the expression of RANKL in VSMC isolated from mouse. However, hydrogen peroxide (H 2 O 2 ), which induces VSMC calcification, induced a 33-fold increase in the transcripts of RANKL as determined by real-time PCR. Increased expression of RANKL protein was further confirmed by ELISA. Using flow cytometry, we demonstrated that membrane-bound RANKL was increased by oxidative stress. To characterize the molecular mechanism underlying H 2 O 2 -induced RANKL expression, we employed the luciferase reporter system with a series of deletion mutants of the RANKL 5′-flanking region. The H 2 O 2 responsive region is located between −200 to −400 in the 5′-flanking region of RANKL gene. Analyses of the sequence of this region identified multiple binding sites for the key osteogenic transcription factor, Runx2, which we previously reported to be an essential regulator of VSMC calcification. Electrophoretic mobility shift analyses demonstrated increased binding of Runx2 on the RANKL promoter sequence in nuclear extracts from VSMC exposed to H 2 O 2 . To further determine the role of Runx2 in regulating RANKL expression, we generated stable Runx2 knockdown VSMC with the use of lentivirus-carrying shRNA for Runx2 gene. H 2 O 2 -induced RANKL expression was abrogated in VSMC with Runx2 knockdown. In addition, adenovirus-mediated overexpression of Runx2 in VSMC induced the expression of RANKL. In summary, we have demonstrated that H 2 O 2 induces the expression of RANKL in VSMC, which is regulated by the osteogenic transcription factor Runx2. These observations provide novel molecular insights into the regulation of RANKL and its role on the pathogenesis of calcified atherosclerotic lesions.

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Coxsackievirus B3 Responds to Polyamine Depletion via Enhancement of 2A and 3C Protease Activity

Viruses ◽

10.3390/v11050403 ◽

2019 ◽

Vol 11 (5) ◽

pp. 403 ◽

Cited By ~ 8

Author(s):

Courtney N. Dial ◽

Patrick M. Tate ◽

Thomas M. Kicmal ◽

Bryan C. Mounce

Keyword(s):

Protease Activity ◽

Rna Virus ◽

Coxsackievirus B3 ◽

Eukaryotic Cells ◽

Luciferase Reporter ◽

Reporter System ◽

3C Protease ◽

Picornavirus Infection

Polyamines are small positively-charged molecules abundant in eukaryotic cells that are crucial to RNA virus replication. In eukaryotic cells, polyamines facilitate processes such as transcription, translation, and DNA replication, and viruses similarly rely on polyamines to facilitate transcription and translation. Whether polyamines function at additional stages in viral replication remains poorly understood. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to polyamine depletion both in vitro and in vivo; however, precisely how polyamine function in picornavirus infection has not been described. Here, we describe CVB3 mutants that arise with passage in polyamine-depleted conditions. We observe mutations in the 2A and 3C proteases, and we find that these mutant proteases confer resistance to polyamine depletion. Using a split luciferase reporter system to measure protease activity, we determined that polyamines facilitate viral protease activity. We further observe that the 2A and 3C protease mutations enhance reporter protease activity in polyamine-depleted conditions. Finally, we find that these mutations promote cleavage of cellular eIF4G during infection of polyamine-depleted cells. In sum, our results suggest that polyamines are crucial to protease function during picornavirus infection. Further, these data highlight viral proteases as potential antiviral targets and highlight how CVB3 may overcome polyamine-depleting antiviral therapies.

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Identification and Effects of Novel Promoter Region Haplotypes in the Human Equilibrative Nucleoside Transporter, hENT1.

Blood ◽

10.1182/blood.v104.11.2083.2083 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2083-2083

Author(s):

Scott N. Myers ◽

Rakesh K. Goyal ◽

Jennifer D. Roy ◽

Robert E. Ferrell

Keyword(s):

De Novo ◽

Remission Rate ◽

Leukemic Cells ◽

Luciferase Reporter ◽

Luciferase Expression ◽

Nucleoside Transporter ◽

Nucleotide Polymorphisms ◽

Flanking Sequence ◽

Equilibrative Nucleoside Transporter

Abstract Front-line induction chemotherapy regimens containing cytosine arabinoside (Ara-C) and anthracyclines result in 80% complete remission rate in childhood acute myeloid leukemia (AML) but their cure rate is about 35 – 50%, one of the lowest of all childhood cancers. Understanding the factors that contribute to emergence of chemoresistant leukemic cells is crucial to improving treatment outcome in children with AML. We are interested in studying the role of variation in Ara-C transport and biotransformation pathway genes in the efficacy and toxicity of treatment of childhood AML. To permeate the cell membrane, Ara-C is mainly dependent on human equilibrative nucleoside transporter 1 (hENT1; SLC29A1; gene localized to 6p21.1). Several studies have suggested an important role for altered levels of hENT1 in the chemosensitivity of AML blasts to Ara-C (Galmarini et al. Leukemia2001; 15(6):87; Gati et al. Leuk Lymphoma1998; 32(1–2):45). Osato and colleagues identified two single nucleotide polymorphisms (SNPs) in the hENT1 coding sequence that led to missense changes, but their in vitro analysis did not detect differences in the activity of variant alleles in a yeast transfection system (Osato et al. Pharmacogenetics2003;13(5):297). To identify variation in hENT1 that might influence its expression, we sequenced 1.6Kb of the proximal 5′-flanking sequence of the gene in 42 unrelated individuals and identified three SNPs at positions C-1345G, G-1050A, and G-706C. TRANSFAC analysis (www.genomatix.de) predicted that two of these (C-1345G & G-706C) would alter consensus transcription factor binding site sequences. We cloned four naturally occurring haplotypes (CGG, CAG, CGC, and GAG) using the TOPO-TA cloning kit, then transfected Cos-1 cells using the Lipofectamine 2000 protocol. Gene expression was assayed using the Promega Dual-Luciferase Reporter Assay System and read on a Molecular Devices HT Analyzer. Luciferase activity was measured at 24 and 48 hours after transfection for six replicates of every condition during three separate transfections. To correct for differences in transfection efficiencies, experimental (Photinus pyralis) luciferase activities were normalized by co-transfection with control (Renilla reniformis) luciferase plasmid. Compared to the wild type CGG haplotype, variant haplotypes CAG, CGC, and GAG drive luciferase expression at approximately 2x (p <0.0001), 1.4x (p <0.001) and 1.2x (p =0.08), respectively. This leads to the hypothesis that individuals carrying CAG or CGC haplotypes (17% of the population) exhibit higher levels of hENT1 expression and are more sensitive to Ara-C exposure. Experiments are underway to quantify gene transcripts in people of known hENT1 haplotypes. We also plan to genotype a large cohort of children with de novo AML for these three SNPs in hENT1 and correlate clinical outcomes in individuals carrying the low- versus the high-expressing haplotypes.

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microRNA-27b inhibits cell proliferation and invasion in bladder cancer by targeting engrailed-2

Bioscience Reports ◽

10.1042/bsr20201000 ◽

2020 ◽

Author(s):

Yunfei Li ◽

Qilin Duan ◽

Lu Gan ◽

Wei Li ◽

Jianggen Yang ◽

...

Keyword(s):

Bladder Cancer ◽

Malignant Tumour ◽

Expression Level ◽

Luciferase Reporter ◽

Reporter System ◽

Invasion And Migration ◽

Great Heterogeneity ◽

And Migration

Background: Bladder cancer is considered a malignant tumour characterised by great heterogeneity. Engrailed-2 may be a gene implicated in bladder cancer. Bioinformatics analysis found base pair complementation between microRNA-27b and engrailed-2. This study aimed to investigate the reciprocal association between microRNA-27b and engrailed-2 in bladder cancer. Methods: The microRNA-27b and the proteins of engrailed-2 in the tissues and cells of the bladder were detected. The processes of apoptosis, proliferation, invasion, and migration of tumour cells were evaluated. The co-action between microRNA-27b and engrailed-2 was detected by a luciferase reporter system. Finally, the interaction between microRNA-27b and engrailed-2 was further verified in vivo. Results: The study found that the expression level of microRNA-27b is lower in bladder cancer tissues and cells than that in neighbouring ordinary tissues, whereas the opposite outcome was observed regarding the expression level of engrailed-2. Furthermore, microRNA-27b expression level is not significantly linked to the age of patients with bladder cancer; however, it is significantly associated with the clinicopathological grade of bladder cancer. Notably, engrailed-2 is negatively regulated by microRNA-27b. Transfection with microRNA-27b was associated with a significant reduction in the activity of bladder cancer cells and promoted apoptosis, while engrailed-2 restoration effectively reversed the above effects of microRNA-27b on bladder cancer in vitro and in vivo. Conclusions: In conclusion, engrailed-2 is engaged in the development and process of bladder cancer through the negative mediation of microRNA-27b; additionally, microRNA-27b/engrailed-2 could form a signalling pathway with a significant effect on the process of bladder cancer.

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Long Non-Coding RNA AC022306.2 Promotes Cell Proliferation, Migration, and Invasion by Mediating GALK2 in Epithelial Ovarian Cancer

10.21203/rs.3.rs-1083813/v1 ◽

2021 ◽

Author(s):

Xin Liu ◽

Zhenghao Huang ◽

Honglei Qin ◽

Jingwen Chen ◽

Yang Zhao

Keyword(s):

Ovarian Cancer ◽

Figo Stage ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Reporter System ◽

Non Coding Rna ◽

Tumor Tissues ◽

Long Non Coding Rna

Abstract BackgroundLong non-coding RNA (LncRNA) has been exhibited to exert significant function among human cancers. AC022306.2, as a newly discovered lncRNA, has an unclear function in ovarian cancer (OC). This study aims to uncover the functional role of AC022306.2 in OC and discover its possible mechanism. MethodsThe expression of AC022306.2 and Galactokinase 2 (GALK2) in OC tissues and adjacent non-tumor tissues was detected via qRT-PCR. The CCK-8 assay, cell clonogenesis assay, scratch healing assay and trans-well assay were used to reveal the function of AC022306.2 and GALK2 in ovarian cancer cell lines. Mice xenografts experiment was performed. Bioinformatics predicted the microRNA (miRNA) that bond with AC022306.2 and GALK2, and dual luciferase reporter system confirmed it. Rescue experiments of miRNA mimics and siGALK2 transfection on the basis of AC022306.2 over-expression were carried out to uncover the mechanism by which AC022306.2 played cancer-promoting roles in ovarian cancer.ResultsIt was found that AC022306.2 was up-regulated in EOC tissues compared with adjacent non-tumor tissues. The elevated expression of AC022306.2 was related to the FIGO stage of OC. Functional experiments showed that AC022306.2 overexpression accelerated proliferation and aggression of OC cells in vitro and accelerated tumor growth in vivo. We also found that GALK2 was up-regulated in OC tissues. The expression of GALK2 mRNA in OC tissue was positively associated with the expression of AC022306.2. After AC022306.2 was knocked down, the expression of GALK2 was down-regulated. In addition, GALK2 depletion restored the proliferation and aggression capabilities of OC cells after AC022306.2 overexpression. Mechanically, AC022306.2 acted as a competitive endogenous RNA (ceRNA) of miR-369-3p to modulate the expression of GALK2. The up-regulating of miR-369-3p or the down-regulating of GALK2 partially reversed the effect of AC022306.2 overexpressed on cell propagation and aggression in OC. ConclusionsAC022306.2 is a new oncogene in the carcinogenesis and development of OC. AC022306.2 improves the development of OC by regulating the miR-369-3p / GALK2 axis, indicating that AC022306.2 may have the potential to become a new molecular target for the treatment of OC.

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A Yersinia ruckeri TIR Domain-Containing Protein (STIR-2) Mediates Immune Evasion by Targeting the MyD88 Adaptor

International Journal of Molecular Sciences ◽

10.3390/ijms20184409 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4409 ◽

Cited By ~ 3

Author(s):

Tao Liu ◽

Wen-Yan Wei ◽

Kai-Yu Wang ◽

Er-Long Wang ◽

Qian Yang

Keyword(s):

Immune Evasion ◽

Adaptor Protein ◽

Innate Immune ◽

Luciferase Reporter ◽

Yersinia Ruckeri ◽

Reporter System ◽

Tir Domain ◽

Novel Strategy

TIR domain-containing proteins are essential for bacterial pathogens to subvert host defenses. This study describes a fish pathogen, Yersinia ruckeri SC09 strain, with a novel TIR domain-containing protein (STIR-2) that affects Toll-like receptor (TLR) function. STIR-2 was identified in Y. ruckeri by bioinformatics analysis. The toxic effects of this gene on fish were determined by in vivo challenge experiments in knockout mutants and complement mutants of the stir-2 gene. In vitro, STIR-2 downregulated the expression and secretion of IL-6, IL-1β, and TNF-α. Furthermore, the results of NF-κB-dependent luciferase reporter system, co-immunoprecipitation, GST pull-down assays, and yeast two-hybrid assay indicated that STIR-2 inhibited the TLR signaling pathway by interacting with myeloid differentiation factor 88 (MyD88). In addition, STIR-2 promoted the intracellular survival of pathogenic Yersinia ruckeri SC09 strain by binding to the TIR adaptor protein MyD88 and inhibiting the pre-inflammatory signal of immune cells. These results showed that STIR-2 increased virulence in Y. ruckeri and suppressed the innate immune response by inhibiting TLR and MyD88-mediated signaling, serving as a novel strategy for innate immune evasion.

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CircRASSF2 promotes laryngeal squamous cell carcinoma progression by regulating the miR-302b-3p/IGF-1R axis

Clinical Science ◽

10.1042/cs20190110 ◽

2019 ◽

Vol 133 (9) ◽

pp. 1053-1066 ◽

Cited By ~ 21

Author(s):

Linli Tian ◽

Jing Cao ◽

Hui Jiao ◽

Jiarui Zhang ◽

Xiuxia Ren ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Molecular Mechanisms ◽

Laryngeal Squamous Cell Carcinoma ◽

Luciferase Reporter ◽

Circular Rnas ◽

Central Component ◽

Reporter System

Abstract Background: Circular RNAs (circRNAs) are a class of non-coding RNAs (ncRNAs) broadly expressed in cells of various species. However, the molecular mechanisms that link circRNAs with laryngeal squamous cell carcinoma (LSCC) are not well understood. In the present study, we attempted to provide novel basis for targeted therapy for LSCC from the aspect of circRNA–microRNA (miRNA)–mRNA interaction. Methods: We investigated the expression of circRNAs in three paired LSCC tissues and adjacent non-tumor tissues by microarray analysis. Differentially expressed circRNAs were identified between LSCC tissues and non-cancerous matched tissues, including 527 up-regulated circRNAs and 414 down-regulated circRNAs. We focused on hsa_circ_0059354, which is located on chromosome 20 and derived from RASSF2, and thus we named it circRASSF2. Results: circRASSF2 was found to be significantly up-regulated in LSCC tissues and LSCC cell lines compared with paired adjacent non-tumorous tissues and normal cells. Moreover, knockdown of circRASSF2 significantly inhibited cell proliferation and migration in vitro, which was blocked by miR-302b-3p inhibitor. Bioinformatics analysis predicted that there is a circRASSF2/miR-302b-3p/ insulin-like growth factor 1 receptor (IGF-1R) axis in LSCC progression. Dual-luciferase reporter system validated the direct interaction of circRASSF2, miR-302b-3p, and IGF-1R. Western blot verified that inhibition of circRASSF2 decreased IGF-1R expression. Furthermore, silencing circRASSF2 suppressed LSCC growth in vivo. Importantly, we demonstrated that circRASSF2 was up-regulated in serum exosomes from LSCC patients. Altogether, silencing circRASSF2 suppresses progression of LSCC by interacting with miR-302b-3p and decreasing inhibiting IGF-1R expression. Conclusion: In conclusion, these data suggest that circRASSF2 is a central component linking circRNAs to progression of LSCC via an miR-302b-3p/IGF-1R axis.

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Characterization of Hepcidin-Inducing Cytokines in Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.2001.2001 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2001-2001

Author(s):

Ken Maes ◽

Alissa Huston ◽

David Roodman ◽

Seth Rivera ◽

Elizabeta Nemeth ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Promoter Activity ◽

Site Directed Mutagenesis ◽

Luciferase Reporter ◽

Reporter System ◽

Advisory Committees ◽

Hepcidin Expression ◽

Equity Ownership

Abstract Abstract 2001 Poster Board I-1023 Introduction: Hepcidin, the principal iron-regulatory hormone, plays an important role in the development of anemia of inflammation and other iron-restricted anemias. Patients with multiple myeloma (MM) frequently present with anemia not attributable to a known mechanism. We previously found that hepcidin is increased in MM and thus could cause or contribute to the anemia of MM. The BMP and IL-6 pathways are the two known major transcriptional regulators of hepcidin. Methods: To identify cytokines that increase hepcidin in MM patients, we screened patient sera with an in vitro cellular reporter system, consisting of human hepatoma 7 cell line (HuH7) and the hepcidin promoter-firefly luciferase reporter. Using site-directed mutagenesis, the promoter was mutated at the STAT3-binding site (STAT3-BS) and/or two BMP responsive elements (BREs), sequences known to be involved in the regulation of hepcidin expression by IL-6 and BMPs, respectively. Results: As expected, recombinant IL-6 and BMP-4, -6 and -9 activated wild-type hepcidin promoter activity several fold. Of note, IL-6 and BMP-9 interacted synergistically at low doses, at the level of the promoter. Mutations in STAT3-BS abrogated the response to IL-6. Mutations in either BRE site by itself did not abolish the response to BMPs, but concurrent mutagenesis of both sites resulted in a complete loss of hepcidin response. Importantly, STAT3-BS and BREs affected hepcidin promoter response independently from each other. Using the in vitro system, we compared sera from six MM patients with previously measured serum hepcidin levels to sera from healthy controls. Sera of four patients with high hepcidin and one with low hepcidin significantly induced hepcidin promoter activity, while serum of another patient with low hepcidin did not. Mutations in STAT3-BS only abrogated the response to two patient sera, both of which had high hepcidin. Mutations in two BREs abrogated the response in all six sera as did the triple mutation involving STAT3-BS and both BREs. Conclusions: We could separately interrogate the signaling pathways by which IL-6 and BMPs induce hepcidin transcription, allowing us to discriminate between the effects of IL-6-like or BMP-like cytokines in each patient serum. BMP-like cytokines were involved in the upregulation of hepcidin in all tested MM patients, and in some patients IL-6 or related cytokines contributed as well, either independently or through a synergistic interaction. No residual activation of hepcidin promoter by other factors was noted. Antibody neutralization may identify the specific myeloma-associated cytokines that stimulate hepcidin production through the two canonical pathways. Disclosures: Roodman: Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; Acceleron: Consultancy. Nemeth:Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Xenon Pharmaceuticals: Consultancy, Equity Ownership.

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