8 results on '"Anna Cordeiro"'
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2. Zebrafish Models Provide Novel Insights into the Disease Biology of Parn-Mutant Dyskeratosis Congenita and DNAJC21-Mutant Shwachman-Diamond Syndrome
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Yigal Dror, Anna Cordeiro, Jason N. Berman, Sarada Ketharnathan, and Sergey V. Prykhozhij
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Genetics ,Shwachman–Diamond syndrome ,Immunology ,Mutant ,Cell Biology ,Hematology ,Disease ,Biology ,medicine.disease ,biology.organism_classification ,Biochemistry ,medicine ,Zebrafish ,Dyskeratosis congenita - Abstract
Inherited bone marrow failure syndromes (IBMFS) are a group of rare inherited genetic disorders defined by impaired hematopoiesis and multi- or unilineage cytopenias. Dyskeratosis Congenita (DC) is characterized by pancytopenia, abnormal skin pigmentation and leukoplakia and is caused by mutations in genes involved in telomere biogenesis and/or RNA processing such as TERT, TERC, DKC1 and PARN. Shwachman-Diamond syndrome (SDS) is characterized by neutropenia and exocrine pancreatic insufficiency and occurs as a result of mutations in genes required for ribosome subunit maturation such as SBDS, DNAJC21 and EFL1. How the disruption of such ubiquitous cellular processes leads to distinct cytopenic phenotypes is not fully understood. Further, IBMFS patients have a high risk of developing myelodysplastic syndrome and/or acute myeloid leukemia with a cumulative incidence of 36% by 30 years of age for SDS patients and 13% for DC patients who do not undergo a stem cell transplant. As IBMFS are rare and large numbers of primary human samples are not readily available for mechanistic studies, we employed zebrafish (Danio rerio) given their highly conserved hematopoietic program and ease of genetic manipulation. Using CRISPR-Cas9 genomic editing, we generated deletion mutations predicted to introduce premature stop codons in the zebrafish orthologs, parn and dnajc21, two more recently identified causes of DC and SDS, respectively. Homozygous zygotic parn mutants had normal morphology and were viable to adulthood. However, fertile homozygous adult females were not recoverable in subsequent generations, implicating parn as an essential factor in oocyte specification. In contrast, homozygous dnajc21 mutants showed normal development to adulthood. Using whole mount in situ hybridization (WISH), we found increased expression of hematopoietic precursor markers (erythroid - gata1 and myeloid - lcp1) and concurrently decreased expression of mature hematopoietic markers (erythroid - hbbe3, myeloid - mpx and thrombocyte - CD41) in parn mutants at 24- and 48-hours post-fertilization (hpf). Further, o-dianisidine staining showed reduced hemoglobinized erythrocytes at 48 hpf. These data indicate that multilineage embryonic hematopoiesis is compromised in parn mutants, recapitulating the pancytopenia observed in patients with DC. WISH for mpx in dnajc21 mutants revealed reduced expression at 24 and 48 hpf, recapitulating the neutropenia seen in SDS. Activation of the TP53 tumor suppressor pathway has been suggested to mediate marrow failure and leukemic progression in some types of IBMFS. Using quantitative PCR to measure the expression of tp53 and its downstream effector p21 at 48 hpf, we observed no significant changes in parn mutants but significantly upregulated tp53 expression in dnajc21 mutants. To further study the role of tp53 in dnajc21-mutant SDS, we crossed dnajc21 mutants with a zebrafish line carrying a tp53 R217H point mutation that confers anti-apoptotic phenotypes. Heterozygous tp53 loss in a dnajc21-/- or +/- background resulted in reduced overall larval growth and abnormal yolk sac development, suggesting defective lipid metabolism. Using the lipophilic dye, Oil Red O, we observed reduced lipid distribution in the vasculature and caudal hematopoietic tissue region (equivalent to mammalian fetal liver) of dnajc21 mutants at 48 hpf. In summary, these findings support a role for PARN in hematopoietic lineage specification through mechanisms that are predominantly TP53-independent. By contrast, DNAJC21 is required for neutrophil specification and normal lipid metabolism and may function in a TP53-dependent manner. These zebrafish models provide new insights into the unique biology underlying these IBMFS and can serve as an in vivo platform for identifying therapeutic compounds that restore normal hematopoiesis and prevent leukemic transformation. Disclosures Dror: Alexion Canada: Other: Received funding for a Marrow Failure and Myelodysplasia conference that I organized April 2021; RepaetDiagnostic Laboratory: Other: Received funding for a Marrow Failure and Myelodysplasia conference that I organized April 2021. Berman: Oxford Immune Algorithmics: Membership on an entity's Board of Directors or advisory committees.
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- 2021
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3. Characterizing Dyskeratosis Congenita Caused By Parn Mutations in the Zebrafish
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Jason N. Berman, Yigal Dror, Santhosh Dhanraj, Anna Cordeiro, and Adam P Deveau
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Mutation ,Telomerase ,Point mutation ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease ,medicine.disease_cause ,biology.organism_classification ,Biochemistry ,Dyskeratosis ,Cell biology ,Telomerase RNA component ,medicine ,Cell aging ,Zebrafish ,Dyskeratosis congenita - Abstract
Inherited bone marrow failure syndromes (IBMFs) are a group of rare genetic disorders characterized by deficient hematopoiesis and extra-hematologic traits. Most known entities are related to a specific gene or group of genes, but others still remain unclassified. In many cases, the involved proteins are required for critical processes involved in cell survival, such as ribosome biogenesis, maintenance of telomere length, and DNA repair. Importantly, patients with IBMFs have higher risks of developing a variety of cancers from leukemia to solid tumours of the head and neck. In 2015, we published four patients from three different families with mutations in the Poly(A)-specific ribonuclease (PARN) gene. This gene encodes a ribonuclease which is involved in degradation of the poly(A) tails, which regulate mRNA turnover, and thus gene expression. Three of the patients presented with several degrees of mental illness and/or developmental delay. The fourth patient, harbored both a monoallelic deletion and a point mutation at the catalytic domain of the protein, and presented with bone marrow failure and hypomyelination, similar to a severe form of dyskeratosis congenital (DC) known as Hoyeraal-Hreidarsson syndrome. In the last two decades, zebrafish (Danio rerio) has emerged as an excellent animal model for human disease, and is especially relevant in hematology, since many of the transcription factors and cell types are highly conserved. Zebrafish have a single PARN ortholog,with 64% sequence identity to the human gene. Using CRISPR-Cas9 genome editing and a combination of six sgRNAs, we generated a 1.2 kb deletion in the zebrafish parn ortholog extending from exon 5 to 13, causing a premature stop codon. Homozygous fish were generated by incrossing to replicate the complete loss-of-function observed in the patient with the DC-like phenotype. Using whole-mount in situ hybridization (WISH) at 48 hours post-fertilization (hpf), we observed a decrease in the number of several mature myeloid cell lineages including neutrophils (labeled with mpx; p PARN is described as a protein involved in RNA processing, but has also been associated with telomere maintenance. This latter process is crucial for cell senescence and genome integrity, and is a known cause of several IBMFSs. The telomerase ribonucleoprotein complex is highly active in hematopoietic stem and progenitor cells (HSPCs) and plays a role in cell differentiation. This complex is composed of a reverse transcriptase (TERT), RNA template (TERC), and the dyskerin protein complex (DKC1), mutations of which represent a common cause of DC. qPCR analysis in zebrafish parn mutants revealed a 2.98-fold reduction in tert expression compared with the wild type fish. Combined, these findings suggest that PARN plays an important role in HSC differentiation into myeloid and erythroid lineages, resulting in a bone marrow failure phenotype. Our model provides a unique in vivo platform to study the role of PARN in hematopoiesis and for identifying compounds that restore normal blood cell ratios, which may have the potential to prevent future leukemic transformation. Disclosures No relevant conflicts of interest to declare.
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- 2019
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4. A Zebrafish JAK1-A634D Gain-of-Function Model Provides New Insights into the Pathogenesis of Familial Hypereosinophilia
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Kate L. Del Bel, Stuart E. Turvey, Sergey V. Prykhozhij, Jason N. Berman, Adam P Deveau, and Anna Cordeiro-Santanach
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Ruxolitinib ,Myeloid ,biology ,Transgene ,Immunology ,Cell Biology ,Hematology ,Eosinophil ,biology.organism_classification ,Biochemistry ,Blood cell ,Andrology ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Stem cell ,Zebrafish ,medicine.drug - Abstract
Introduction: JAK/STAT signaling is of major importance for hematopoiesis and immune function. A heterozygous JAK1 c.1901C>A de novo point mutation was identified in three members of a family (a mother and two male sons). These patients presented with a unique clinical phenotype of severe atopic dermatitis, markedly elevated peripheral blood eosinophil counts with eosinophilic infiltration of the liver and gastrointestinal tract, hepatosplenomegaly, and failure to thrive. Treatment with ruxolitinib, a JAK1/2 inhibitor, resulted in reduced eosinophilia and improved growth (Del Bel et al. JACI 2017). However, the role of this JAK1 mutation in hematopoiesis remains unclear. Methods: ubi-JAK1-WT or A634D-P2A-sfGFP-pA zebrafish transgenic lines were generated incorporating the human genes. Both of these constructs were injected into embryos, but the survival rate for the JAK1-A634D embryos was very low. Six hour post-fertilization (hpf) embryos expressing the mutant transgene were treated with 1 µM ruxolitinib for 24h, which dramatically increased survival. Whole-mount in situ hybridization (WISH) was used to assess changes in hematopoietic stem cells/blood cell populations due to wild-type or JAK1-A634D expression at 24, 28, 36 and 48 hpf using a battery of blood lineage specific probes. O-dianisidine staining was employed to evaluate hemoglobin levels. ImageJ cell counter pluggin was used for cell quantification. Gata1 and o-dianisidine staining intensity was analyzed using Ilastik and CellProfiler. Six groups of 30-50 embryos were analyzed by RNA sequencing (RNAseq): control, JAK1-WT and JAK1-A634D at 28 and 36 hpf. RNA was extracted using Trizol. RNAseq data was analyzed with edgeR and enrichment analysis was performed using DAVID 6.8. Results: JAK1-A634D was toxic to zebrafish embryos but they survived to sexual maturity post-ruxolitinib therapy. F1 embryos from JAK1-A634D founders with high expression of the transgene exhibited abnormal development (70% of embryos) which could be reduced to 12% of embryos following 24h exposure to 1 µM ruxolitinib. This rescue suggests that JAK1-A634D is highly active in zebrafish and its activity needs to be under tight regulation for normal development. Both JAK1-WT and A634D mutant fish demonstrated increased HSPCs (c-myb/runx1 expression) at 36 hpf (pWT>controls; p=0.0308 and p=0.0045, respectively). pu.1/spi1 early myeloid expression showed a similar expression pattern at 28 hpf, A634D>WT>controls; p Conclusions: JAK1-A634D plays an important role in zebrafish development and specifically impacts the myeloid lineage, particularly the granulocyte branch, from which eosinophils arise. Ruxolitinib can revert the arrested development phenotype, demonstrating the specificity of this targeted therapy. Our model provides the first in vivo evidence of the effects of the JAK1-A634D mutation on hematopoiesis and will provide further insight in understanding the cause of familial hypereosinophilia. Disclosures No relevant conflicts of interest to declare.
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- 2018
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5. Exploring the Expression Profile of Long Non-Coding RNA (lncRNA) in Different Acute Myeloid Leukemia (AML) Subtypes: t(8;16)(p11;p13)/MYST3-Crebbp AML Harbors a Distinctive Lncrna Signature
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Alfons Navarro, Ruth M. Risueño, Marina Díaz-Beyá, Joan J. Castellano, Mariano Monzo, Jordi Esteve, Marta Pratcorona, Anna Cordeiro, Meritxell Nomdedeu, María Rozman, and Miguel Ángel Torrente
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Genetics ,NPM1 ,Immunology ,GATA2 ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,Biochemistry ,MYST3 ,Long non-coding RNA ,Gene expression profiling ,CEBPA ,microRNA - Abstract
Introduction: Long non-coding RNAs (lncRNAs) have recently emerged as important actors in the regulation of multiple cellular processes including cancer. Acute myeloid leukemia (AML) is a heterogeneous disease; most of the main cytogenetic AML subgroups harbor a specific gene expression profile. AML with translocation t(8;16)(p11;p13) (t(8;16) AML) is a subtype with specific clinical and biological characteristics including a distinctive gene (Camós et al, Cancer Research 2006) and microRNA (Díaz-Beyá et al, Leukemia 2013) expression profile. In this translocation, MYST3 on chromosome 8p11 fuses with CREBBP on chromosome 16p13.3. The MYST3-CREBBP fusion protein is able to interact with multiple transcription factors (TF) producing a disturbed transcriptional program. However, the lncRNA expression pattern of different cytogenetic AML subtypes, including t(8;16) AML, have not been described yet. Aims: To examine the expression profile of lncRNAs within different AML subtypes, and to characterize the expression pattern of lncRNAs in t(8;16) AML in comparison to other AML subtypes. Patients and Methods: 46 AML patients, 4 normal bone marrow (NBM) and 3 CD34+ NBM samples were included in the study. Samples included different AML subtypes: intermediate-risk cytogenetic AML (IR-AML, n=18), t(15;17) (APL, n=4), t(8;21) AML (n=4), inv(16) AML(n=2), t(6;9) AML (n=7), AML with monosomal karyotype (n=4), t(3;3) AML (n=1), t(9;11) AML (n=1) and t(8;16) AML (n=5). Within IR-AML patients with a different mutational profile: FLT3-ITD (n=7), NPM1 (n=5), CEBPA (n=7) and DNMT3A (n=6) were included. The lncRNA expression was studied using Affymetrix® Human Gene 2.1 ST platform which includes 9698 lncRNAs transcripts. The filtering and normalization of the array data was performed using oligo package from Bioconductor. Statistical analyses were performed with TiGR MultiExperiment Viewer, BRB tools and R. The Transcription factor Affinity Prediction Web Tool was used to determine the putative transcription factors binding to the differentially expressed lncRNAs promoters. Results: The hierarchical cluster analysis showed that all 4 NBM as well as all 3 CD34+ NBM clustered together according to their lncRNA expression. Interestingly, all 5 t(8;16) AML samples clustered together, as well as the 3 APL, the 7 t(6;9) AML and 5 out of 7 cases with CEBPA mutations. The specific lncRNA signature of APL was composed of 79 differentially expressed lncRNA and t(6;9) AML lncRNA signature comprised of 15 differentially expressed lncRNAs. When we focused on t(8;16) AML lncRNA profile, we identified an specific 113-lncRNA signature in the supervised analysis (Figure). Interestingly, when we analyzed which (TF) had motifs overrepresented in the promoters regions of the t(8;16) AML lncRNA signature, we identified GATA2 as the TF with significantly overrepresented motifs for GATA2 (p Conclusions: LncRNAs expression profile seems specific of several AML subtypes, including t(8;16) AML. Some of the lncRNAs of this distinctive signature in t(8;16) AML are located in the HOXA genomic region, and correlate with several of the characteristic microRNAs previously described in this entity. Interestingly, we have predicted in silico GATA2, which interacts with CREBBP, as the most significant TF that could potentially regulate this lncRNAs signature. Nonetheless, further investigation is warranted to determine the mechanisms leading to this lncRNA signature and to identify the specific targets of these lncRNAs. Río Hortega CM13/00205, FIS PI13/00999 Disclosures No relevant conflicts of interest to declare.
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- 2015
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6. The LincRNA HOTAIRM1, Located in the HOXA genomic Region, impacts Prognosis in Acute Myeloid Leukemia and Is Associated with a Distinctive microRNA Signature
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Jordi Esteve, Olga Salamero, Josep-Maria Ribera, Jorge Sierra, Marta Pratcorona, Meritxell Nomdedeu, Lourdes Escoda, Mar Tormo, Rafael F. Duarte, Josep M Marti-Tutusaus, Anna Cordeiro, Salut Brunet, Josep F. Nomdedeu, Alfons Navarro, David Gallardo, Ruth M. Risueño, Carmen Pedro, Mariano Monzo, and Marina Díaz-Beyá
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Oncology ,Regulation of gene expression ,NPM1 ,medicine.medical_specialty ,Myeloid ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,Bioinformatics ,medicine.disease ,Biochemistry ,Leukemia ,medicine.anatomical_structure ,Internal medicine ,Gene expression ,CEBPA ,microRNA ,medicine - Abstract
Background: Non-coding RNAs (ncRNAs) have recently emerged as key regulators of diverse cellular processes, including leukemia. ncRNAs are classified according to their size as short (eg, microRNAs) or long ncRNAs. lincRNAs are long ncRNAs located in intergenic regions and have multiple regulatory functions, including gene expression regulation. Interestingly, active crosstalk between microRNAs and lincRNAs has been observed. lincRNAs are known to be deregulated in some cancers but their importance in acute myeloid leukemia (AML) is so far unknown. HOX genes play an important role in hematopoiesis and are deregulated in AML. lincRNAs are especially abundant in the clusters of HOX genes. HOTAIRM1, a myeloid lineage-specific lincRNA, is located at the 3’end of the HOXA cluster and seems to play a regulatory role in myelopoiesis. However, to date the potential prognostic role of HOTAIRM1 expression in AML has not been examined. Aims: To investigate first whether the expression of the lincRNA HOTAIRM1 is associated with the clinical, cytogenetic and molecular characteristics and microRNA expression in AML patients. Secondly, since intermediate risk (IR) AML patients have a highly diverse prognosis, we analyzed the potential prognostic value of HOTAIRM1 expression in IR-AML patients. Methods: To explore the expression level of HOTAIRM1 among different AML subtypes, we analyzed samples from 244 AML patients including CBF-rearranged AML (n=5), APL (n=4), MLL-rearranged AML (n=3), EVI1-rearranged AML (n=3), t(6;9) AML (n=9), AML with monosomal karyotype (n=3), and a large cohort of IR-AML (described below). For the analysis of prognostic value of HOTAIRM1, we analyzed specifically the outcome of 217 IR-AML patients (median age, 52; 51% males) sequentially included in CETLAM trials during the period 1995-2009. Molecular genotyping of this group identified NPM1 mutation (NPM1mut), FLT3-ITD, and biallelic CEBPA mutation (CEBPA mut) in 99 (45%), 79 (36%) and 17 (11%), respectively. The expression of HOTAIRM1 was analyzed using TaqMan® Gene Expression Assays (Applied Biosystems). microRNA and mRNA expression data were obtained in previous studies (Díaz-Beyá, Leukemia 2013). Statistical analyses were performed with BRB Array Tools, SPSS v20 and R v3.0. MaxStat package from R software was used to determine the optimal cutoff point of HOTAIRM1 expression. Results: Among all 244 patients, HOTAIRM1 expression was significantly different among the 7 included genetic subgroups (ANOVA p=0.0024), with the lowest levels observed in APL-AML patients and the highest in the t(6;9)AML patients. Within the IR-AML group, HOTAIRM1 overexpression was observed in NPM1mut patients (p The prognostic study showed that high HOTAIRM1 expression was associated with shorter 5-year overall survival (OS) (27+11% vs.47+8%; p=0.009) shorter 5-year disease-free survival (LFS) (22+12% vs. 53+9%; p Supervised analysis by means of t-test based on multiple permutations revealed a distinctive 33-microRNA signature which correlated with HOTAIRM1 expression including miR-196b (p Conclusion: The expression level of the lincRNA HOTAIRM1 varied among different molecularly-defined AML. Interestingly, HOTAIRM1 expression level showed independent prognostic value within the IR-AML group. Moreover, HOTAIRM1 expression strongly correlates with its neighboring HOXA4 gene and harbors a distinctive microRNA signature. Our findings can pave the way for further studies of HOX-related lincRNAs and microRNAs regulatory networks and their influence on clinical outcome. Acknowledgments: ISCIII RH13/00205, SEHH, FIS13/00999 Disclosures No relevant conflicts of interest to declare.
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- 2014
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7. Piwirna-651 Expression Influences Treatment Response and Impacts Survival in Classical Hodgkin Lymphoma Patients through Regulation of ABCC5
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Carmen Martinez, Antonio Martínez, Dolors Fuster, Blanca Gonzalez-Farre, Anna Gaya, Marina Díaz-Beyá, Anna Cordeiro, Jordi Esteve, Mariano Monzo, and Alfons Navarro
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medicine.diagnostic_test ,Immunology ,RNA ,Piwi-interacting RNA ,Cell Biology ,Hematology ,In situ hybridization ,Argonaute ,Biology ,Biochemistry ,Molecular biology ,Western blot ,Complementary DNA ,DNA methylation ,medicine ,Gene - Abstract
Introduction: PiwiRNAs (piRNAs) are small non-coding RNAs processed by PIWI proteins, a subfamily of Argonaute proteins. The main functions of piRNAs include regulation of chromatin and genome organization, DNA methylation, transposon repression and regulation of protein synthesis. Until recently, PIWI/piRNA functions were believed to be limited to germinal stem cells and early embryonic development, where they play a crucial role in the regulation of genomic stability. However, piRNA expression has now been identified in some tumors, although the specific prognostic impact of piRNAs remains to be elucidated. We have examined the potential reactivation of the PIWI/piRNA pathway in classical Hodgkin lymphoma (cHL) and its impact on prognosis in cHL patients. Methods: Formalin fixed and paraffin embedded tumor samples from 96 cHL patients were studied. All patients were diagnosed and treated in a single institution. HIV+ was an exclusion criterion. Median age was 33 years (range, 15–89); 47.9% were male; 30.2% were EBV+. The most frequent histological subtype was nodular sclerosis (65.6%). First-line treatment consisted of ABVD-type therapies in 90% of patients. 12 reactive lymph nodes (RLNs) were used as controls. The PIWI proteins PIWIL1 and PIWIL2 were studied by immunohistochemistry. The expression levels of three piRNAs (piR-651, piR-20365, piR-20582) were analyzed by Real-time PCR. cDNA was synthesized using miScript II RT Kit and Real-time PCR was performed using miScript SYBR Green PCR Kit (Qiagen). PiRNA expression was correlated with treatment response, disease-free survival (DFS) and overall survival (OS). Paired serum samples from seven prospectively collected cHL patients at diagnosis and at complete response and from 10 healthy controls were used to study the expression of piRNAs in serum. In addition, 5 cHL cell lines were used for the functional analysis. RNA hybrid, Renilla/Luciferase assay and Western Blot were used to identify and validate the piRNA targets. Statistical analyses were performed using R v2.13. Results: PIWIL1 and PIWIL2 proteins were expressed in the cytoplasm of the Hodgkin/Reed Sternberg (HRS) cells, indicating that the piRNA pathway is active in cHL. This was corroborated by the finding that the 3 piRNAs were also expressed in all tumor samples and in the 5 cHL cell lines. piR-651 (p In situ hybridization with LNA probes (Exiqon) showed that piR-651 was highly expressed in the cytoplasm of HRS cells. We studied putative targets genes of piR-651 using RNA hybrid. We identified several candidate genes related to drug metabolism, including ABCC5, an efflux pump of multiple drugs including doxorubicin. We cloned the 3’UTR region of ABCC5 in the psicheck2 vector and performed a Renilla/Luciferase assay. We observed a significant increase of 28% (p=0.047) in the Renilla luciferase levels in the cells transfected with 200nM of piR-651 inhibitor compared to oligo control. To further validate this, we transfected 3 HL cell lines with piR-651 inhibitor or oligo control and analyzed the protein levels of ABCC5 by Western Blot, observing a mean increase of 18% in the cells transfected with piR-651 inhibitor. These results showed that piR-651 inhibits the translation of ABCC5. Conclusions:The PIWI/piRNA pathway is reactivated in cHL and the expression of piRNAs affects tumorogenesis and cHL patient outcome. piR-651 influences treatment response and survival through regulation of ABCC5. Our findings provide the groundwork for further investigation of the novel role of piRNAs in HL. Acknowledgments : AECC-Catalunya (2014), APIF-UB (AC). Disclosures No relevant conflicts of interest to declare.
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- 2014
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8. Prognostic Significance Of a 4-Microrna Signature Targeting JAK2 In Classical Hodgkin Lymphoma
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Alfons Navarro, Anna Gaya, Anna Cordeiro, Blanca Gonzalez-Farre, Marina Díaz-Beyá, Dolors Fuster, Carmen Martinez, Jordi Esteve, Antonio Martinez, and Mariano Monzó
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Oncology ,medicine.medical_specialty ,Expression vector ,medicine.diagnostic_test ,Immunology ,Cell Biology ,Hematology ,Transfection ,Biology ,Biochemistry ,Western blot ,B symptoms ,Internal medicine ,microRNA ,TaqMan ,medicine ,Lymph ,medicine.symptom ,Gene - Abstract
Introduction The behavior of classical Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the Hodgkin/Reed Sternberg (HRS) cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. One of the genetic lesions most frequently found in HRS cells involves members of the JAK/STAT signaling pathway. However, few studies have identified microRNAs (miRNAs) that target JAK2 in cHL. In a previous study, our group identified miR-135a as a JAK2 regulator and showed the prognostic impact of this miRNA in a small set (n=89) of cHL patients (Navarro A. et al. Blood 2009). In the present study, we have examined novel miRNAs targeting JAK2 and evaluated the prognostic impact of their expression levels in 170 lymph nodes of cHL patients. Methods TargetScan and miRbase were used to identify JAK2-related miRNAs. The conserved miRNAs with higher scores were selected and further validated by Renilla-Luciferase assay and Western Blot. We performed a Renilla-Luciferase assay with a modified expression vector psiChecK-2 containing the complete 3’UTR region of JAK2 cloned in the 3’UTR region of Renilla luciferase gene. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in the HDMYZ cHL cell line together with 1μM of modified psicheck2 vector using Lipofectamine 2000, and luminescence was read at 24 hours. Further validation of the miRNA target was performed by Western Blot in three cHL cell lines (L428, L-1236 and HDMYZ). The expression levels of the miRNAs identified as targeting JAK2 were then assessed in lymph nodes from 170 cHL patients (median age, 33 years; male, 51%; EBV, 43.8%; HIV, 12%) diagnosed and treated in a single institution. Moreover, 15 reactive lymph nodes (RLN) were used as normal controls. RNA was purified from formalin-fixed paraffin-embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation kit. The miRNA expression was analyzed using TaqMan microRNA assays. Statistical analyses were performed using R v2.13 and PASW Statistics 18. Results The bioinformatic analysis identified seven microRNAs as possible regulators of JAK2 (miR-34a, miR-101, miR-135a, miR-204, miR-216a, miR-216b and miR-375). The Renilla-Luciferase assay confirmed that in addition to miR-135a, miR-101 (61%; p=0.01), miR-204 (46%; p=0.003) and miR-216a (64.8%; p=0.02) also targeted JAK2. These findings were confirmed by Western Blot analysis of JAK2 levels after transfection with pre-miRNAs (median % of protein reduction in the 3 cell lines of 21.1%, 46.7% and 24%, respectively). The analysis of these miRNAs in RLN showed that miR-101, miR-135a and miR-204 were significantly downregulated in lymph nodes of cHL patients (p Conclusions MiR-101, miR-135a, miR-204 and miR-216 regulate JAK2 and are independent prognostic factors in cHL. Acknowledgments SDCSD of School of Medicine of UB. AC is an APIF-UB fellow. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
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