scholarly journals POS0419 ABERRANT SPLICEOSOME AND ALTERED EXPRESSION OF IFN-RESPONSE RELATED GENES ARE HALLMARKS OF MONOCYTES FROM LUPUS PATIENTS WITH RENAL DISEASE

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 438.2-439
Author(s):  
A. M. Patiño-Trives ◽  
C. Perez-Sanchez ◽  
A. Ibañez-Costa ◽  
M. Luque-Tévar ◽  
M. D. C. Abalos-Aguilera ◽  
...  
Keyword(s):  
Renal Disease ◽  
Renal Involvement ◽  
Aberrant Expression ◽  
Altered Expression ◽  
Inflammatory Status ◽  
Inflammatory Profile ◽  
Active Release ◽  
Splicing Machinery

Background:To date, novel mechanisms such as the involvement of splicing machinery components in lupus nephropathy and its interplay with the transcriptome in innate immune cells have not been evaluated.Objectives:1- To identify altered transcriptomic signatures associated with the immune response of monocytes from SLE patients and its association with clinical features. 2- To evaluate the role of the spliceosome linked to the transcriptomic profile of SLE monocytes. 3- To analyze mechanistically the impact of anti-dsDNA antibodies (Ab) and the modulation of the spliceosome in the SLE monocytes activity.Methods:Sixty SLE patients and forty healthy donors (HD) were included in the study. Infiltration rate of myeloid cells and its association with clinical features were analyzed in kidney biopsies by Immunohistochemistry. In parallel, circulating monocytes were purified from peripheral blood by immune-magnetic selection. The expression of a set of 770 genes related to autoimmune/inflammatory diseases was evaluated using NanoString Technologies. The levels of the main 45 components of the splicing machinery were further analyzed in these samples using a microfluidic qPCR array (Fluidigm). An extensive clinical/serological evaluation was also performed, comprising disease activity, renal involvement parameters, autoAb profile, and the systemic inflammatory status (27-plex Assay). Finally, in vitro studies involving anti-dsDNA-IgG Ab treatment and over/down-expression of splicing machinery components were carried out to analyze their effects in the monocyte activity.Results:Infiltration of CD68 expressing cells was confirmed in kidney biopsies and associated with parameters of kidney failure (C3/C4, chronic index), highlighting the key role of the myeloid compartment in lupus nephropathy. Gene expression profiling recognized 156 genes differentially expressed in SLE monocytes compared with HDs, including 87 genes up-regulated and 69 down-regulated. Functional analysis showed that most dysregulated genes were associated with the IFN response (i.e. IFIT1, IFI44, IFI44L, RSAD2). In parallel, the altered expression of 27 spliceosome components was demonstrated in SLE monocytes compared with HD, including 3 up-regulated and 24 down-regulated. Correlation studies demonstrated that the aberrant expression of splicing machinery components was linked to the altered interferon signature and the plasma inflammatory profile. This aberrant profile at molecular level was associated with the disease activity status, anti-dsDNA positivity and C3/C4 levels. Interestingly, SLE patients with renal disease displayed a simultaneous alteration of both, the IFN and the spliceosome signatures in monocytes, along with an enlarged pro-inflammatory profile in plasma. Logistic regression models that integrated the concomitant alteration of some splicing machinery components and IFNs genes identified lupus nephritis patients with high accuracy. Mechanistic studies showed that in vitro treatment of monocytes from HDs with anti-dsDNA promoted a concomitant deregulation of the IFN signature and the expression of several spliceosome components (i.e. PTB, RBM17, RNU6ATAC). Finally, the over/down-expression of selected spliceosome components (PTB and RBM17) in monocytes from SLE patients reduced the active release of inflammatory cytokines and their adhesion capacity.Conclusion:1) Monocytes from SLE patients with renal involvement exhibit a remarkable alteration of genes associated with the IFN response, further linked with the aberrant expression of several splicing machinery components. 2) Anti-dsDNA promoted the dysregulation in monocytes of both the IFN and spliceosome signatures, along with an active release of pro-inflammatory mediators. 3) The modulation of key splicing components in monocytes from SLE patients reduce their pro-inflammatory status and migration capacity. Ongoing studies may provide novel biomarkers and therapeutic tools to treat lupus nephropathy.Acknowledgements:Funded by ISCIII, PI18/00837 and RIER RD16/0012/0015 co-funded with FEDERDisclosure of Interests:None declared

THU0234 INFLUENCE OF THE SPLICING MACHINERY DYSREGULATION ON THE PATHOGENESIS OF LUPUS AND THE KIDNEY INVOLVEMENT

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 344.1-344
Author(s):  
C. Lopez-Pedrera ◽  
M. Luque-Tévar ◽  
A. Ibañez-Costa ◽  
A. M. Patiño-Trives ◽  
M. A. Aguirre ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lupus Nephritis ◽  
Renal Involvement ◽  
Research Support ◽  
Altered Expression ◽  
Kidney Involvement ◽  
Inflammatory Profile ◽  
Potential Biomarkers ◽  
Splicing Machinery

Background:Objectives:The aim of this study was to evaluate whether alterations in the splicing machinery of immune cells from Systemic Lupus Erythematosus (SLE) patients could influence the development and activity of the disease and the kidney involvement.Methods:The study was conducted in 41 SLE patients and 34 healthy donors (HD). Monocytes, lymphocytes and neutrophils were purified by immunomagnetic selection. Then, selected elements of the splicing machinery and a set of genes related to inflammation, renal and cardiovascular disease (including interleukins, adipocytokines, chemokines, and oxidative stress markers, among others) were evaluated using a microfluidic qPCR array (Fluidigm). Besides, the inflammatory profile in plasma, including the analysis of 27 proteins, was evaluated by using the Bioplex assay. In parallel, an extensive clinical/serological evaluation was performed; comprising disease activity and cardiovascular and renal involvement along with autoantibodies, complement factors, and acute phase reactants. Correlation and association studies and logistic models among those clinical and analytical parameters were developed. Mechanistic in vitro studies were carried out by incubation of HD-leukocytes with anti-dsDNA-IgG purified from SLE patients and changes promoted in both, splicing machinery and leukocyte inflammatory profile, were assessed.Results:A significantly altered expression of spliceosome components was found in all the leukocyte subsets: 27, 12 and 11 components were differentially expressed in monocytes, lymphocytes and neutrophils, respectively, in SLE patient’s vs HD. In parallel, a number of genes codifying for proteins involved in inflammation, fatty acid metabolism, oxidative stress and migration were found altered and correlations with spliceosome components were further identified. Besides, those statistical analyses demonstrated multiple links among altered spliceosome components and the clinical profile of these patients, such as the activity of the disease (SLEDAI), the occurrence of obstetric complications and the presence of arterial hypertension. Logistic regression and ROC curve analyses identified a signature composed in each leukocyte subset by 3 altered spliceosome components that could differentiate between SLE and HDs. Remarkably, the levels of a high number of those altered components were associated to the presence of lupus nephritis (LN). Moreover, ROC curve analyses allowed to identify several cell-specific spliceosome components as potential biomarkers of renal disease. In patients with LN we could also identify a distinctive inflammatory profile in plasma in relation to patients without renal involvement, which further correlated with the altered expression of a number of spliceosome components in each leukocyte subset. Lastly, the in vitro treatment of HD leukocytes with anti-dsDNA promoted the alteration of several spliceosome components found also altered in vivo in SLE lymphocytes, monocytes and neutrophils.Conclusion:1)The splicing machinery is greatly altered in leukocytes from SLE patients, regulated, at least partially, by anti-dsDNA antibodies and closely related to the activity of the disease, including obstetrical complications, hypertension and lupus nephritis. 2)Specific components of the spliceosome in SLE leukocytes subsets might be used as potential biomarkers to typify the disease, particularly kidney involvement.Acknowledgments :Funded by ISCIII (PI18/00837 and RIER RD16/0012/0015) co-funded with FEDER.Disclosure of Interests: :Chary Lopez-Pedrera Grant/research support from: ROCHE and Pfizer., María Luque-Tévar: None declared, Alejandro Ibañez-Costa: None declared, Alejandra M. Patiño-Trives: None declared, Maria A Aguirre: None declared, Pérez Sánchez Laura: None declared, Maria del Carmen Abalos-Aguilera: None declared, Iván Arias de la Rosa: None declared, Pedro Seguí Azpilcueta: None declared, Mario Espinosa: None declared, Nuria Barbarroja Puerto Grant/research support from: ROCHE and Pfizer., Speakers bureau: ROCHE and Celgene., Eduardo Collantes-Estévez Grant/research support from: ROCHE and Pfizer., Speakers bureau: ROCHE, Lilly, Bristol and Celgene., Justo Pastor Castaño Fuentes: None declared, Raúl Miguel Luque Huertas: None declared, Carlos Perez-Sanchez: None declared

scholarly journals Splicing machinery is impaired in rheumatoid arthritis, associated with disease activity and modulated by anti-TNF therapy

2021 ◽  
pp. annrheumdis-2021-220308
Author(s):  
Alejandro Ibáñez-Costa ◽  
Carlos Perez-Sanchez ◽  
Alejandra María Patiño-Trives ◽  
Maria Luque-Tevar ◽  
Pilar Font ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Disease Activity ◽  
Synovial Fibroblasts ◽  
Data Set ◽  
Altered Expression ◽  
Healthy Donors ◽  
Inflammatory Profile ◽  
Splicing Machinery

ObjectivesTo characterise splicing machinery (SM) alterations in leucocytes of patients with rheumatoid arthritis (RA), and to assess its influence on their clinical profile and therapeutic response.MethodsLeucocyte subtypes from 129 patients with RA and 29 healthy donors (HD) were purified, and 45 selected SM elements (SME) were evaluated by quantitative PCR-array based on microfluidic technology (Fluidigm). Modulation by anti-tumour necrosis factor (TNF) therapy and underlying regulatory mechanisms were assessed.ResultsAn altered expression of several SME was found in RA leucocytes. Eight elements (SNRNP70, SNRNP200, U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1 and SRSF10) were equally altered in all leucocytes subtypes. Logistic regressions revealed that this signature might: discriminate RA and HD, and anti-citrullinated protein antibodies (ACPAs) positivity; classify high-disease activity (disease activity score-28 (DAS28) >5.1); recognise radiological involvement; and identify patients showing atheroma plaques. Furthermore, this signature was altered in RA synovial fluid and ankle joints of K/BxN-arthritic mice. An available RNA-seq data set enabled to validate data and identified distinctive splicing events and splicing variants among patients with RA expressing high and low SME levels. 3 and 6 months anti-TNF therapy reversed their expression in parallel to the reduction of the inflammatory profile. In vitro, ACPAs modulated SME, at least partially, by Fc Receptor (FcR)-dependent mechanisms. Key inflammatory cytokines further altered SME. Lastly, induced SNRNP70-overexpression and KHDRBS1-overexpression reversed inflammation in lymphocytes, NETosis in neutrophils and adhesion in RA monocytes and influenced activity of RA synovial fibroblasts.ConclusionsOverall, we have characterised for the first time a signature comprising eight dysregulated SME in RA leucocytes from both peripheral blood and synovial fluid, linked to disease pathophysiology, modulated by ACPAs and reversed by anti-TNF therapy.

scholarly journals miR-29a sensitizes the response of glioma cells to temozolomide by modulating the P53/MDM2 feedback loop

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Qiudan Chen ◽  
Weifeng Wang ◽  
Shuying Chen ◽  
Xiaotong Chen ◽  
Yong Lin
Keyword(s):  
Molecular Mechanism ◽  
Feedback Loop ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Transcriptional Level ◽  
Aberrant Expression ◽  
Altered Expression ◽  
P53 Signaling

AbstractRecently, pivotal functions of miRNAs in regulating common tumorigenic processes and manipulating signaling pathways in brain tumors have been recognized; notably, miR‐29a is closely associated with p53 signaling, contributing to the development of glioma. However, the molecular mechanism of the interaction between miR-29a and p53 signaling is still to be revealed. Herein, a total of 30 glioma tissues and 10 non-cancerous tissues were used to investigate the expression of miR‐29a. CCK-8 assay and Transwell assay were applied to identify the effects of miR-29a altered expression on the malignant biological behaviors of glioma cells in vitro, including proliferation, apoptosis, migration and invasion. A dual-luciferase reporter assay was used to further validate the regulatory effect of p53 or miR-29a on miR-29a or MDM2, respectively, at the transcriptional level. The results showed that miR-29a expression negatively correlated with tumor grade of human gliomas; at the same time it inhibited cell proliferation, migration, and invasion and promoted apoptosis of glioma cells in vitro. Mechanistically, miR-29a expression was induced by p53, leading to aberrant expression of MDM2 targeted by miR-29a, and finally imbalanced the activity of the p53-miR-29a-MDM2 feedback loop. Moreover, miR-29a regulating p53/MDM2 signaling sensitized the response of glioma cells to temozolomide treatment. Altogether, the study demonstrated a potential molecular mechanism in the tumorigenesis of glioma, while offering a possible target for treating human glioma in the future.

scholarly journals KLF7/VPS35 axis contributes to hepatocellular carcinoma progression through CCDC85C-activated β-catenin pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yarong Guo ◽  
Bao Chai ◽  
Junmei Jia ◽  
Mudan Yang ◽  
Yanjun Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Luciferase Reporter ◽  
Aberrant Expression ◽  
Proliferation And Invasion ◽  
Hcc Cells

Abstract Objective Dysregulation of KLF7 participates in the development of various cancers, but it is unclear whether there is a link between HCC and aberrant expression of KLF7. The aim of this study was to investigate the role of KLF7 in proliferation and migration of hepatocellular carcinoma (HCC) cells. Methods CCK8, colony growth, transwell, cell cycle analysis and apoptosis detection were performed to explore the effect of KLF7, VPS35 and Ccdc85c on cell function in vitro. Xenografted tumor growth was used to assess in vivo role of KLF7. Chip-qPCR and luciferase reporter assays were applied to check whether KLF7 regulated VPS35 at transcriptional manner. Co-IP assay was performed to detect the interaction between VPS35 and Ccdc85c. Immunohistochemical staining and qRT-PCR analysis were performed in human HCC sampels to study the clinical significance of KLF7, VPS35 and β-catenin. Results Firstly, KLF7 was highly expressed in human HCC samples and correlated with patients’ differentiation and metastasis status. KLF7 overexpression contributed to cell proliferation and invasion of HCC cells in vitro and in vivo. KLF7 transcriptional activation of VPS35 was necessary for HCC tumor growth and metastasis. Further, co-IP studies revealed that VPS35 could interact with Ccdc85c in HCC cells. Rescue assay confirmed that overexpression of VPS35 and knockdown of Ccdc85c abolished the VPS35-medicated promotion effect on cell proliferation and invasion. Finally, KLF7/VPS35 axis regulated Ccdc85c, which involved in activation of β-catenin signaling pathway, confirmed using β-catenin inhibitor, GK974. Functional studies suggested that downregulation of Ccdc85c partly reversed the capacity of cell proliferation and invasion in HCC cells, which was regulated by VPS35 upregulation. Lastly, there was a positive correlation among KLF7, VPS35 and active-β-catenin in human HCC patients. Conclusion We demonstrated that KLF7/VPS35 axis promoted HCC cell progression by activating Ccdc85c-medicated β-catenin pathway. Targeting this signal axis might be a potential treatment strategy for HCC.

scholarly journals Identification of a microRNA signature in renal fibrosis: role of miR-21

2011 ◽  
Vol 301 (4) ◽  
pp. F793-F801 ◽  
Author(s):  
Abolfazl Zarjou ◽  
Shanzhong Yang ◽  
Edward Abraham ◽  
Anupam Agarwal ◽  
Gang Liu
Keyword(s):  
Epithelial Cells ◽  
Renal Fibrosis ◽  
Transforming Growth Factor ◽  
Response To Treatment ◽  
Tubular Epithelial Cells ◽  
Altered Expression ◽  
Tnf Α ◽  
Tgf Β1

Renal fibrosis is a final stage of many forms of kidney disease and leads to impairment of kidney function. The molecular pathogenesis of renal fibrosis is currently not well-understood. microRNAs (miRNAs) are important players in initiation and progression of many pathologic processes including diabetes, cancer, and cardiovascular disease. However, the role of miRNAs in kidney injury and repair is not well-characterized. In the present study, we found a unique miRNA signature associated with unilateral ureteral obstruction (UUO)-induced renal fibrosis. We found altered expression in UUO kidneys of miRNAs that have been shown to be responsive to stimulation by transforming growth factor (TGF)-β1 or TNF-α. Among these miRNAs, miR-21 demonstrated the greatest increase in UUO kidneys. The enhanced expression of miR-21 was located mainly in distal tubular epithelial cells. miR-21 expression was upregulated in response to treatment with TGF-β1 or TNF-α in human renal tubular epithelial cells in vitro. Furthermore, we found that blocking miR-21 in vivo attenuated UUO-induced renal fibrosis, presumably through diminishing the expression of profibrotic proteins and reducing infiltration of inflammatory macrophages in UUO kidneys. Our data suggest that targeting specific miRNAs could be a novel therapeutic approach to treat renal fibrosis.

scholarly journals HNRNPA2B1 promotes multiple myeloma progression by increasing AKT3 expression via m6A-dependent stabilization of ILF3 mRNA

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Fengjie Jiang ◽  
Xiaozhu Tang ◽  
Chao Tang ◽  
Zhen Hua ◽  
Mengying Ke ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cellular Proliferation ◽  
Aberrant Expression ◽  
The Stability ◽  
Induced Apoptosis ◽  
Proliferation In Vitro

AbstractN6-methyladenosine (m6A) modification is the most prevalent modification in eukaryotic RNAs while accumulating studies suggest that m6A aberrant expression plays an important role in cancer. HNRNPA2B1 is a m6A reader which binds to nascent RNA and thus affects a perplexing array of RNA metabolism exquisitely. Despite unveiled facets that HNRNPA2B1 is deregulated in several tumors and facilitates tumor growth, a clear role of HNRNPA2B1 in multiple myeloma (MM) remains elusive. Herein, we analyzed the function and the regulatory mechanism of HNRNPA2B1 in MM. We found that HNRNPA2B1 was elevated in MM patients and negatively correlated with favorable prognosis. The depletion of HNRNPA2B1 in MM cells inhibited cell proliferation and induced apoptosis. On the contrary, the overexpression of HNRNPA2B1 promoted cell proliferation in vitro and in vivo. Mechanistic studies revealed that HNRNPA2B1 recognized the m6A sites of ILF3 and enhanced the stability of ILF3 mRNA transcripts, while AKT3 downregulation by siRNA abrogated the cellular proliferation induced by HNRNPA2B1 overexpression. Additionally, the expression of HNRNPA2B1, ILF3 and AKT3 was positively associated with each other in MM tissues tested by immunohistochemistry. In summary, our study highlights that HNRNPA2B1 potentially acts as a therapeutic target of MM through regulating AKT3 expression mediated by ILF3-dependent pattern.

The role of 5-HT7 receptors on isoproterenol-induced myocardial infarction in rats with high-fat diet exacerbated coronary endothelial dysfunction

2020 ◽  
Vol 39 (8) ◽  
pp. 1005-1018 ◽  
Author(s):  
I Cinar ◽  
Z Halici ◽  
B Dincer ◽  
B Sirin ◽  
E Cadirci
Keyword(s):  
Myocardial Infarction ◽  
Endothelial Dysfunction ◽  
High Fat Diet ◽  
Density Lipoprotein ◽  
Heart Tissue ◽  
High Fat ◽  
Inflammatory Status

The presence of 5-HT7r’s in both human and rat cardiovascular and immune tissues and their contribution to inflammatory conditions prompted us to hypothesize that these receptors contribute in acute myocardial infarction (MI) with underlying chronic endothelial dysfunction. We investigated the role of 5-HT7 receptors on heart tissue that damaged by isoproterenol (ISO)-induced MI in rats with high-fat diet (HFD). In vitro and in vivo effects of 5-HT7r agonist (LP44) and antagonist (SB269970) have been investigated on the H9C2 cell line and rats, respectively. For in vivo analyses, rats were fed with HFD for 8 weeks and after this period ISO-induced MI model has been applied to rat. To investigate the role of 5-HT7r’s, two different doses of LP44 and SB269970 were evaluated and compared with standard hypolipidemic agent, atorvastatin. In vitro studies showed that LP44 has protective and proliferative effects on rat cardiomyocytes. Also in in vivo studies stimulating 5-HT7r’s by LP44 improved blood lipid profile (decreased total cholesterol, low-density lipoprotein-C, and triglyceride, increased high-density lipoprotein), decreased cardiac damage markers (creatine kinase and troponin-I), and corrected inflammatory status (tumor necrosis factor-α, interleukin-6). Our results showed significant improvement in LP44 administered rats in terms of histopathologic analyses. In damaged tissues, 5-HT7 mRNA expression increased and agonist administration decreased this elevation significantly. We determined for the first time that 5-HT7r’s are overexpressed in ISO-induced MI of rats with underlying HFD-induced endothelial dysfunction. Restoration of this overexpression by LP44, a 5-HT7r agonist, ameliorated heart tissue in physiopathologic, enzymatic, and molecular level, showing the cardiac role of these receptors and suggesting them as future potential therapeutic targets.

scholarly journals MiR-1188 at the imprinted Dlk1-Dio3 domain acts as a tumor suppressor in hepatoma cells

2015 ◽  
Vol 26 (8) ◽  
pp. 1416-1427 ◽  
Author(s):  
Wei Cui ◽  
Zhijun Huang ◽  
Hongjuan He ◽  
Ning Gu ◽  
Geng Qin ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Normal Liver ◽  
Hepatoma Cells ◽  
Genomic Region ◽  
Migration And Invasion ◽  
Aberrant Expression ◽  
Profiling Analysis

The aberrant expression of microRNAs (miRNAs) has frequently been reported in cancer studies; miRNAs play roles in development, progression, metastasis, and prognosis. Recent studies indicate that the miRNAs within the Dlk1-Dio3 genomic region are involved in the development of liver cancer, but the role of miR-1188 in hepatocellular carcinoma (HCC) and the pathway by which it exerts its function remain largely unknown. Here we demonstrate that miR-1188 is significantly down-regulated in mouse hepatoma cells compared with normal liver tissues. Enhanced miR-1188 suppresses cell proliferation, migration, and invasion in vitro and inhibits the tumor growth of HCC cells in vivo. Moreover, overexpressed miR-1188 promotes apoptosis, enhances caspase-3 activity, and also up-regulates the expression of Bax and p53. MiR-1188 directly targets and negatively regulates Bcl-2 and Sp1. Silencing of Bcl-2 and Sp1 exactly copies the proapoptotic and anti-invasive effects of miR-1188, respectively. The expression of apoptosis- and invasion-related genes, such as Vegfa, Fgfr1, and Rprd1b, decreases after enhancement of miR-1188, as determined by gene expression profiling analysis. Taken together, our results highlight an important role for miR-1188 as a tumor suppressor in hepatoma cells and imply its potential role in cancer therapy.

The Role of Microrna-196a-1 and Microrna-196b in Acute T-Lymphoblastic Leukemia and Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1607-1607
Author(s):  
Ebru Coskun ◽  
Eva Kristin von der Heide ◽  
Cornelia Schlee ◽  
Nicola Goekbuget ◽  
Dieter Hoelzer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lymphoblastic Leukemia ◽  
Mirna Precursor ◽  
Acute Leukemias ◽  
Aberrant Expression ◽  
Gm Csf ◽  
Fold Reduction ◽  
Precursor Molecules

Abstract Abstract 1607 Poster Board I-633 INTRODUCTION Overexpression of the gene ERG (v-ets erythroblastosis virus E26 oncogene homolog) is an adverse prognostic factor in adult patients with acute T-lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). However, the underlying biology remains unknown. The aim of this study was to investigate the regulation of ERG expression by microRNAs (miRNAs) and to explore their potential role in acute leukemia and normal hematopoiesis. METHODS: A bioinformatic database search was carried out using the Targetscan, Pictar, and Human microRNA target tools to predict ERG regulating miRNAs. Verification of ERG as potential target of predicted miRNAs was performed by AMAXA transfection of miRNA precursor molecules in the myeloid leukemic cell line KG1a. After 24 hours (hrs) and 48 hrs total RNA was extracted using the Trizol reagent. Overexpression of the miRNAs was confirmed by TaqMan MicroRNA assays and ERG expression was determined by real-time RT-PCR. Moreover, specific binding of miRNAs to the 3'UTR of ERG was verified by luciferase reporter assays co-transfecting the ERG 3'UTR cloned into the psiCHECK-2 luciferase vector with miRNA precursor molecules. To investigate the expression of miRNAs during hematopoietic maturation, CD34 positive bone marrow cells from healthy individuals were in vitro cultured using the cytokines SCF and IL-3 (maintenance culture) with the addition of EPO or G-/GM-CSF. Cells were harvested after 3, 6, 9, 13, 16, and 20 days and miRNA expression levels were measured. The expression of miR-196a-1 and miR-196b was also studied in acute leukemias including bone marrow samples of adult patients with newly diagnosed T-ALL (n=105) and AML (n=34). RESULTS: By the database search, a total of 13 miRNAs were predicted to potentially regulate ERG and were further studied. Of these, only the miRNAs miR-196a-1 and miR-196b induced a significant reduction of ERG expression levels. After 24 hrs ERG was significantly down-regulated by 36% (after miR-196a-1 transfection) and by 42% (after miR-196b transfection) as well as after 48 hrs by 43% (after miR-196a-1 transfection) and by 47% (after miR-196b transfection) compared to the controls. The luciferase assays revealed a 30% and 40% luciferase activity reduction in miR-196a-1 and miR-196b transfected cells, respectively, compared to the miRNA-missense transfected cells. This confirmed the direct binding of these miRNAs to the ERG 3'UTR. During hematopoietic differentiation of normal CD34 positive progenitors, expression of miR-196a-1 was constant over time using the different cytokine conditions. In contrast, the expression of miR-196b decreased substantially during the in vitro differentiation (maintenance culture: 20-fold reduction; EPO: 18-fold reduction; G-/GM-CSF: 13-fold reduction - from day 0 to day 9). In acute leukemia, we found that miR-196a-1 was significantly higher expressed in AML compared to bone marrow samples of healthy donors (P=0.02). In T-ALL, miR-196a-1 was significantly up-regulated in patients with aberrant expression of myeloid markers (P=0.04), and miR-196b expression correlated with CD34 expression (P=0.003). In contrast to the reported adverse prognostic impact of ERG, expression of these miRNAs had no prognostic significance in T-ALL. CONCLUSION: This study identifies miR196a-1 and miR-196b as ERG regulators. We show that miR-196b is specifically down-regulated during hematopoietic differentiation, thus indicating a specific role of this miRNA in hematopoiesis. Moreover, the aberrant expression of miR-196a-1 and miR-196b in T-ALL and AML points to a potential role of these miRNAs in acute leukemias. Disclosures No relevant conflicts of interest to declare.

Polycomb Group Member Rybp Is a Functional Tumor Suppressor Repressed By Mir-9 in MLL-Rearranged AML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 871-871
Author(s):  
Colles Price ◽  
Ping Chen ◽  
Shenglai Li ◽  
Zejuan Li ◽  
Yuanyuan Li ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cancer Biology ◽  
Chromosomal Rearrangements ◽  
Polycomb Group ◽  
Aberrant Expression ◽  
Molecular Abnormalities ◽  
Group Member

Abstract MicroRNAs (miRNAs), are small non-coding RNA molecules known to be important regulators of cancer biology. Notably, we and others have shown that miRNAs play important roles in Acute Myeloid Leukemia (AML), a heterogeneous malignancies with multiple chromosomal and molecular abnormalities. Patients with chromosomal rearrangements involving mixed lineage leukemia (MLL), the mammalian homology of trithorax gene, are associated with poor survival. Previously, we have found that MLL-rearranged AML drives aberrant expression of several miRNAs, most notably microRNA-9 (miR-9). Expression of miR-9 with MLL-AF9, a common MLL-translocation, was sufficient to promote transformation normal hematopoietic progenitor cells in vitro and leukemogenesis in vivo. We previously found that miR-9 reduces expression of several genes but we did not know which genes were critical tumor suppressors. We found that the polycomb group member RING1- and YY1-Bindin Protein (RYBP) was consistently inhibited upon miR-9 expression. To assess the regulation of RYBP we used publically available data from the Cancer Genome Atlas (TCGA) and looked at genome-wide Illumina 450K methylation data. We did not find a strong correlation with methylation and RYBP expression, suggesting that expression of RYBP is likely not regulated by the DNA methylation machinery in patients. Upon looking at copy number alterations we found that a small population of AML patients contained either homozygous or heterozygous loss of RYBP, suggesting a potential role of RYBP in leukemia pathogenesis. To assess the role of RYBP we did a series of in vitro experiments. We found that expression of RYBP was sufficient to attenuate colony-forming growth driven by MLL- AF9. Furthermore, RYBP expression was able to reduce proliferation, increase apoptosis, and significantly reduce immature cell population. To determine the role of RYBP expression in vivo, we transplanted lethally irradiated mice with progenitors retrovirally transduced with MLL-AF9 compared to MLL-AF9 and RYBP. We found that expression of RYBP was sufficient to reduce leukemia burden in vivo as well as induce differentiation as shown by flow cytometry and histological analysis. Thus, this demonstrates that RYBP is a functional tumor suppressor in MLL-rearranged AML. In conclusion, we have demonstrated that chromosomal rearrangements involving MLL, the mammalian homology of trithorax, downregulates a member of the polycomb complex through upregulation of miR-9. Disclosures No relevant conflicts of interest to declare.

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