Analysis of Let-7a and Mir-17-5p Micro-RNAs Expression In Patients with Adult De Novo Myelodysplastic Syndromes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4965-4965
Author(s):  
D. Vassilatou ◽  
Vassiliki Pappa ◽  
F. Kontsioti ◽  
S. Papageorgiou ◽  
C. Kontos ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Small Sample ◽  
Micro Rna ◽  
Clinicopathological Parameters ◽  
Control Group ◽  
Ras Proteins ◽  
Expression Levels ◽  
Micro Rnas ◽  
Cd34 Cells

Abstract Abstract 4965 Introduction. MicroRNAs are small non-coding RNAs that act at the post-transcriptional level, regulating protein expression by repressing translation or destabilizing mRNA target. Since their discovery, microRNAs have been associated with almost every normal cell function, including proliferation, differentiation and apoptosis. Several studies suggest that they have an important role in normal hematopoiesis and hematological malignancies. Let-7a negatively regulates the expression of Ras proteins, which are overexpressed in patients with Myelodysplastic syndromes (MDS). In addition mir-17-5p contributes to the down-regulation of E2F1, which is overexpressed in MDS. Purpose. The purpose of the current study was to evaluate the expression of let-7a and mir-17-5p in CD34 cells from the bone marrow of patients with MDS. Material and methods. We evaluated 29 patients with MDS (25 men, 4 women) with median age 73 years. FAB classification was as follows: 9 RA, 15 RAEB, 2 RAEB-t and 3 AML. According to IPSS our study included 9 patients with low, 8 with Int-1, 7 with Int-2 and 5 with high risk disease. We isolated CD34+ cells from bone marrow of patients using magnetic beads. Extraction of microRNA and total RNA was performed and cDNA of let-7a and mir-17-5p was amplified using real time PCR, to study the relative expression of these microRNAS according to the expression of RNU48 (a snoRNA used as control). The control group included donors of CD34+ cells for stem cell transplantation. Results. The median miR17-5p expression level in patients was lower compared to controls [23vs 47.4]. The median let-7a expression levels were higher in MDS patients than in healthy donors [32.85 vs 8]. However these differences were not significant as shown by the Mann-Whitney U test. Moreover, ROC analysis demonstrated that neither miR17-5p nor let-7a expression had significant discriminatory value to efficiently distinguish MDS patients from non-MDS. In addition Spearman analysis did not reveal any correlations between these two microRNA expression levels and other continuous variables examined in the current study (age, hemoglobin concentration, numbers of neutrophils, platelets, and BM blasts). Finally, we found no associations between either miR17-5p or let-7a expression and clinicopathological parameters of MDS patients using chi-square (χ2) or Fisher's exact test where appropriate. Conclusion. The micro RNA let-7 was overexressed although not significantly in CD34 cells from MDS patients demonstrating that this is not a mechanism contributing to the increased expression RAS proteins in MDS. The micro RNA-mir17-5 is underexpressed in CD34 cells and needs further investigation for the establishment of a role in the overepxression of E2F in MDS. The lack of significant differences in the expression levels of both micro RNAs could be related to the admixture of normal and dysplastic CD34 cells in the bone marrow of MDS or to the small sample size. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Effect of Micro RNA-34a and carboplatin combination on the inducing apoptosis of Breast Cancer Cell line

2021 ◽  
Author(s):  
Sajjad Eslamkhah ◽  
Nazila Alizadeh ◽  
Sahar Safaei ◽  
Mohammad Amini ◽  
ahad Mokhtarzadeh ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Viability ◽  
Cancer Cells ◽  
Cell Line ◽  
Caspase 3 ◽  
Cancer Cell Line ◽  
Micro Rna ◽  
Control Group ◽  
Expression Levels ◽  
Micro Rnas

Abstract Aim: Breast cancer (BC) has been classified among the main causes of death owing to females' cancer. Carboplatin is a platinum-based chemotherapeutic drug that is an important treatment option for BC. But high and frequent doses of carboplatin usually reducing the reaction of cancer cells to medication. There is an immediate need to establish methods for increasing the carboplatin susceptibility to BC cells. For instance, micro RNAs (miRNAs) such as MiR34a demonstrate significant potential. Considering that, this research was planned to explore the better clinical effect and underlying mechanism of miR-34a as a possible tumor inhibitor and drug resistance regulator in compound with carboplatin chemotherapy drug in the cell lines of BC in humans. Methods: MCF-7 cell line was transfected with miR-34a to perform functional analyses. Subsequently, the MTT assay was applied to assess cell viability. Cell viability and cell death associated gene expression amounts including Bax, Bcl-2, caspase-3, MDR1, P53, and mir34-a, were examined through real-time quantitative PCR. Results: Findings showed that miR-34a upregulation significantly decreased MCF7 cell viability in comparison with control group. Furthermore, separate treatment of cells with miR-34a mimics and carboplatin could significantly increase Bax, Caspase-3, P53, and decrease in Bcl-2 mRNA expression levels evaluated to the non-treated group. Moreover, by reduction in expression levels of the MDR1 gene, BC cells' reaction to carboplatin has increased via miR-34a. Conclusion: In line with the findings, it could be inferred that miR-34a may improve the responsiveness of breast cancer cells to carboplatin chemotherapy with downregulation of MDR1.

Bone Marrow Mesenchymal Stem Cell (BM-MSC) Release Microvesicles/Exosomes That Incorporate Into Hematopoietic Cells From MDS Patients and May Modify Their Behaviour

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 863-863 ◽  
Author(s):  
Sandra Muntión ◽  
Post Doc Fellowship ◽  
Teresa Ramos ◽  
Bruno Paiva ◽  
Beatriz Roson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
De Novo ◽  
Annexin V ◽  
Micro Rna ◽  
Bioactive Molecules ◽  
Advisory Committees ◽  
Micro Rnas ◽  
Cd34 Cells

Abstract A new mechanism of intercellular communication has been proposed consisting in the secretion of exosomes/ microvesicles (MVs). Such mechanism has been shown to modify the functional properties of recipient cells by the transfer of proteins, mRNA, or micro-RNAs. The hypothesis of the present work was that MSC from MDS patients could differentially modify the HPC properties throughout the shedding of MVs when compared with those from controls due to their different content. Material and methods: MVs were isolated from MSC from bone marrow (BM) samples 18 patients diagnosed with ‘de novo’ and untreated low risk MDS and from MSC from 12 healthy BM. BM-MSC at third passage were cultured in DMEM deprived of FCS, and supernatants were collected after 6 or 24 hours. MVs purification was performed in the majority of the experiments (16 MDS/ 9 Controls) using the ExoQuick-TC exosome precipitation solution (ExoQuick; System Biosiences). To confirm the isolation of MVs by exosome precipitation solution, in some cases (2 MDS/3 Controls) the MVs were obtained by ultracentrifugation; MVs identification was done by transmission electron microscopy (TEM) as well as by flow cytometry (FC). To evaluate if the micro-RNA content into MSC-MVs from patients and controls was different, expression analysis of miRNAs was done using Megaplex™ RT Primers pool (Applied Biosystems) and 384-well microfluidic cards (TaqMan® MicroRNA Array A) were loaded with retro-transcription product and PCR runs were performed on a 7900HT Fast Real-time PCR system (eight MVs from MDS and 4 from HD).To demonstrate the incorporation of MVs from MSC into human hematopoietic progenitors (HPC: CD34+ cells obtained by immunomagnetic selection) HPC were co-cultured with MVs from MSC. Incorporation of Vybrant Dil-labelled MVs into HPC was evaluated at 1, 3, 6, and 24 h. by FC. To detect the incorporation of MVs by confocal microscopy (CM) an intracellular primary Ab for CD90 (Santa Cruz, Biotechnology) was used as MVs marker and anti-CD45 to detect HPC. A Zeiss LSM 510 CM connected to a digital camera (Leica DC 100) were used to obtain confocal images. Apoptotic rate of CD34+ cells that had the MVs-MSC from MDS and controls were evaluated by FC by using APC H7 Annexin V DY634 (Immunostep) and 7AAD (BD Biosciences). Results: More than 95% of MVs isolated by ExoKit system from supernatants of cultured MSC from 6 HD and 6 MDS patients showed scatter intensities lower than of 6µm beads. We observed, in all cases, the same FC pattern. Also, MVs/exosomes isolated by ultracentrifugation (3 MDS/ 5 HD) showed the same FC pattern. MVs from MDS and controls isolated by ultracentrifugation were identified by TEM (fig1). When co-cultures of CD34+ HPC and MVs were studied in both HD and MDS, MVs were incorporated into HPC in all cases (fig2). When the content of miRNAS in the MVs from MDS and HD were compared significant differences were observed between both groups. Twenty-one out of 384 evaluated miRNAs were over-expressed in the MVs from patients compared with the controls. To confirm these results, the expression of miR10a and miR-132 was analyzed by RT-PCR. In both cases their expression was significantly increased in MVs from patients. Recently, it has been suggested that the cargo of these structures are bioactive molecules, therefore we explored the possibility that MVs could modify the behavior of the target cell. For this purpose we searched in which pathways the overexpressed miRNAs could be involved and apoptosis was among them. Since it is considered a very important process in MDS pathophysiology we compared apoptosis by FC, after co-culturing CD34+ cells with MVs from MSC of MDS and HD. Interestingly, preliminary results show that the MVs from MDS protected better from apoptosis CD34+ cells than MVs obtained from controls. In summary, in the present study we show that BM-MSC produce MVs/exosomes with different microRNAs content according to their origin, MDS or HD. These structures can be incorporated into HPC and can modify their properties. Funding: Instituto de Salud Carlos III. PI12/01775. Junta de Castilla y León.GRS 873/A/13. Portuguese FST Grant. SFRH/BD/86451/2012 Disclosures: Diez-Campelo: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. San Miguel:Jansen, Celgene, Onyx, Novartis, Millenium: Membership on an entity’s Board of Directors or advisory committees. del Cañizo:Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

scholarly journals Prostate-Specific antigen value and micro RNAs as potential diagnostic biomarkers for prostate cancer

2021 ◽  
Vol 8 (2) ◽  
pp. 107-113
Author(s):  
Aydemir Asdemir ◽  
Sevgi Durna Dastan ◽  
Esat Korgali ◽  
Tugba Yildiz Asdemir ◽  
Huseyin Saygin ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Specific Antigen ◽  
Diagnostic Value ◽  
Micro Rna ◽  
Control Group ◽  
Diagnostic Biomarkers ◽  
Expression Levels ◽  
Non Invasive ◽  
Cancer Group ◽  
Micro Rnas

Objective: It is necessary to provide PSA alternatives or methods that can be used in conjunction with PSA to regress complications rising from negative biopsies and to increase diagnostic value. Patients and Methods: The study is consisting of 59 men as the sample group. Blood samples from the individuals are grouped as prostate cancer and BPH (benign prostatic hyperplasia) groups. 27 prostate cancer patients whom some of them also operated are assembled in the patients group and the other 32 individuals are grouped as BPH group. Micro RNA expression levels evaluated by RT-PCR. Results: Prostate cancer group when compared with the control group, it is observed that expression levels of miRNA-221 and miRNA-432 increased while expression levels of miRNA-17-5p, miRNA-30c, miRNA-107, miRNA-141, miRNA-145, miRNA-181a-2, miRNA-331-3p, miRNA-574-3p decreased and expression levels of miRNA-21 and miRNA-375 are quite similar between the groups. Conclusion: The prospect of strong and sensitive serum miRNA expression levels in prostate cancer cases which are easily detectable by non-invasive methods as biomarkers is a promising field of study. Nevertheless, it is currently necessary to work in conjunction with both tissue and serum to enhance both sensitivity and specificity of miRNAs as biomarkers. As such, expression levels of the same miRNAs in tissue and serum provide different expression values which in turn make it difficult to indicate a common biomarker.

scholarly journals POS0191 THE VALUE OF MIR-20B, MIR-22, MIR-26A, MIR-125B AND MIR-221 IN RHEUMATOID ARTHRITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 309.1-310
Author(s):  
M. Ciesla ◽  
B. Kolarz ◽  
M. Majdan ◽  
M. Dryglewska
Keyword(s):  
Rheumatoid Arthritis ◽  
Rheumatoid Factor ◽  
Micro Rna ◽  
Clinical Variable ◽  
Tender Joint Count ◽  
Control Group ◽  
Altered Expression ◽  
Expression Levels ◽  
Das 28 ◽  
Micro Rnas

Background:Micro-RNAs (miRNAs) are an endogenous small, single-stranded, non-coding RNAs with a 18-25 nucleotide long and have been reported as a potential extracellular biomarkers of various diseases. They mainly decrease the gene expression by inhibiting the translation or cause mRNA destabilization.Objectives:The aim of the study was to identify miRNAs whose plasma expression level is associated with RA prevalence and/or activity.Methods:A total of 74 unrelated individuals, 50 with RA and 24 in a control group were enrolled to the study. Real-time PCR was used to evaluate the plasma expression levels of 5 miRNAs: miR-20b, miR-22 miR-26a, miR-125b, miR-221.Results:We found the differences in four out of five evaluated miRs between RA and HC group – miR-26a, p<0.0001; miR-125b, p <0.0001; miR-20b p<0.0001; miR-22, p=0.005. Graphical presentation of the results is shown in Figure 1. The logistic regression results showed that miR-22 (p=0.0003) and miR-26a (p=0.049) are the most important molecules distinguishing RA patients and healthy controls in the study. MiR-22 was positively correlated with ESR (rs=0.41), CRP (rs=0.49) and DAS28 (rs=0.33) and miR-26a was positively correlated with ESR (rs=0.49), CRP (rs=0.41), number of swelling joints (rs=0.43), number of painful joints (rs=0.74) and DAS28 (rs=0.63). Moreover, miR-22 expression was different between rheumatoid factor (RF) positive and RF negative patients (p=0.04).Conclusion:The results showed that miR-22 (p=0.0003) and miR-26a (p=0.049) may be the most useful in evaluated panel of miRs distinguishing RA patients and HCs. In this study we demonstrated for the first time that plasma concentration of miR-22 may be considered as a potential molecular marker of RA exacerbation.References:[1]Ouboussad, L., Hunt, L., Hensor, E.M.A. et al. Profiling microRNAs in individuals at risk of progression to rheumatoid arthritis. Arthritis Res Ther 2017 19, 288.[2]Churov AV, Oleinik EK, Knip M. MicroRNAs in rheumatoid arthritis: altered expression and diagnostic potential. Autoimmun Rev. 2015 14(11):1029-37.Figure 1.Diagrams from A to D show the expression levels of miR-22, miR-26a, miR-125b and miR-20b, respectively. The median values of expression have been connected by a line.Table 1.Spearman’s rank correlation between micro-RNA-22 and micro-RNA-26 and the clinical variables.miRs \ clinical variableAgeDisease durationESRCRPSJCTJCDAS-28ACPARFmiR-220.09-0.270.410.490.260.210.330.240.16miR-26a0.20.140.490.410.430.740.630.260.16The significant correlations were bolded and indicated by red. Abbreviations: ACPA, anti-citrullinated protein antibodies; CRP, C-reactive protein; SJC, swollen joint count; TJC, tender joint count; DAS-28, disease activity score 28; ESR, erythrocyte sedimentation rate; RF, rheumatoid factor.Disclosure of Interests:None declared

scholarly journals EVALUATION OF THE PLASMA MICRO RNA EXPRESSION LEVELS IN SECONDARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS

2013 ◽  
Vol 5 (1) ◽  
pp. e2013066 ◽  
Author(s):  
Ali Bay ◽  
Enes Coskun ◽  
Serdar Oztuzcu ◽  
Sercan Ergun ◽  
Fatih Yilmaz ◽  
...  
Keyword(s):  
Hemophagocytic Lymphohistiocytosis ◽  
Laboratory Data ◽  
Study Groups ◽  
Clinical Findings ◽  
Micro Rna ◽  
Expression Level ◽  
Control Group ◽  
Healthy Children ◽  
Expression Levels ◽  
Micro Rnas

Background: Hemophagocytic lymphohistiocytosis (HLH) is a life threatening hyper inflammatory disease. Micro RNAs (miRNA) are about 22 nucleotide-long, small RNAs encoded with genes, and they have regulatory functions in immune response. Objective: To determine the miRNA expression levels of 11 secondary HLH patients, we evaluated the associations of miRNA levels with pathogenesis, clinical presentation, and prognosis of the disease. Patients and Methods: Patients who were diagnosed with secondary HLH from January 2011 to December 2012 were included in this study. We profiled the expressions of 379 miRNAs in plasma of both HLH patients and healthy controls. Patients were evaluated regarding with age, clinical findings, miRNA expresions, laboratory data, treatment, and prognosis, by using descriptive statistics. Results: A total of 11 secondary HLH patients and 11 healthy children were included in this study. miR-205-5p was expressed in all case and controls and expression level of miR-205-5p was found 6.21 fold higher than control group (p=0.01). We detected the second highest expression percent in miR-194-5p with 81% of cases and controls. Expression level of miR-194-5p was found to have 163 fold higher than controls (p= 0.009). miR-30c-5p showed 77% expression percent in cases and controls together. The expression level of this miRNA was detected 9 fold decreased in HLH patients compared to healthy children (p= 0.031). Conclusion: We showed that miR-205-5p, miR-194-5p and miR-30c-5p could be useful plasma biomarkers for HLH. Further research is needed in larger and homogenous study groups, especially for these miRNAs as biomarkers for HLH.

Abstract 263: Role of Micro RNA-21 in Atherosclerosis

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Rihab E Hamed-Berair ◽  
Srinivas D Sithu ◽  
Nalinie Wickramasinghe ◽  
Jasmit Shah ◽  
Abhinav Agawral ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Oxidized Ldl ◽  
Ldl Receptor ◽  
Micro Rna ◽  
Limited Information ◽  
Micro Rnas ◽  
Late Apoptosis ◽  
Apoptosis And Inflammation

Micro RNAs (miR) are short non-coding RNAs that regulate several genes under pathophysiological conditions. Accumulating evidence suggest the involvement of miR in atherogenesis. However, limited information is available about atherogenic miR and the underling mechanisms by which miR affect atherogenesis. Our data shows that 12 weeks of western diet (WD) in LDL receptor-knockout (LDLR-KO) mice upregulated 99 and downregulated 50 miR in the aorta. Among the 41 differentially expressed miR associated with macrophage inflammation and apoptosis, expression of micro RNA-21 (miR-21) was increased by 1.4-fold (P<0.05). WD also increased the expression of miR-21 by 1.5-fold in bone marrow derived macrophages (BMDM). In vitro , LDL, oxidized LDL, acetylated LDL and LPS induced miR-21 by 2-3-fold (P<0.05) and down regulated its target protein PDCD4 in BMDM. Basally, miR-21 deficient BMDM showed increased secretion of IL-6, IL-9 and CXCL-2,-3,-4, and -10 (P<0.05)); and increased early and late apoptosis (2-3-fold, P<0.05). We also observed 40% decrease in the survival of F4/80+ cells during differentiation of bone marrow derived cells isolated from miR-21-KO mice. Stimulation of miR-21-KO BMDM with LPS significantly increased the activation of NF-κB and enhanced the secretion of several pro-inflammatory cytokines including TNFα, IL-6, IL-12 and CXCL-2 (2-10 fold; P<0.05); interferon gamma+LPS polarized the macrophages to pro-inflammatory M1 phenotype (increased expression of CD11c and CD86). Staurosporin and oxidized lipids derived aldehyde 4-hydroxynonenal significantly increased both early and late apoptosis of miR-21-KO BMDM (2-4-fold, P<0.05). This was accompanied by increased cleavage of caspase -3, -7 and -9. Transplantation of bone marrow cells from miR-21-KO into LDLR-KO mice, followed by 12 weeks of WD increased the lesion formation (1.7-fold, P<0.05), apoptosis (3-fold, P<0.05) and necrosis (1.6-fold, P<0.05) in the aortic valve of the chimeric mice. Collectively, these data suggest that miR-21 prevents atherosclerosis, at least in parts, by preventing macrophage apoptosis and inflammation.

scholarly journals Prognostic significance of Wilms tumor 1 mRNA expression levels in peripheral blood and bone marrow in patients with myelodysplastic syndromes

2016 ◽  
Vol 17 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Sumiko Kobayashi ◽  
Yasunori Ueda ◽  
Yasuhito Nannya ◽  
Hirohiko Shibayama ◽  
Hideto Tamura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mrna Expression ◽  
Myelodysplastic Syndromes ◽  
Peripheral Blood ◽  
Wilms Tumor ◽  
Prognostic Significance ◽  
Expression Levels ◽  
Wilms Tumor 1 ◽  
Mrna Expression Levels

Definition of RAEB-I and RAEB-Ii According to the Who Classification of Myelodysplastic Syndromes (MDS): Problems Emerging from a Large Multicenter Registry.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1432-1432
Author(s):  
Alessandro Levis ◽  
L. Godio ◽  
M. Girotto ◽  
M. Bonferroni ◽  
T. Callegari ◽  
...  
Keyword(s):  
Internal Medicine ◽  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Bone Marrow Cells ◽  
Clinical Information ◽  
Bone Marrow Trephine Biopsy ◽  
A Value ◽  
Cd34 Cells ◽  
Definition Of

Abstract BACKGROUND. TheWHO classification of myelodysplastic syndromes (MDS) is based on the evaluation of bone marrow morphology. The two categories of REAB-I and RAEB-II are apparently easy to differentiate on the basis of bone marrow blast percent. However there are no so far data about the differences among cytology, histology and immunophenotypic evaluation of blasts in order to discrimante non-RAEB from RAEB-I and RAEB-II categories. PATIENTS AND METHODS. The Piemonte MDS Registry was born in 1999 thanks to the cooperation of both Haematology and Internal Medicine departments of our region, with the following aims: a) to follow homogeneous guidelines in diagnosis and treatment of MDS; b) to collect epidemiological and clinical information on a large group of patients; c) to cryopreserve bone marrow cells for molecular biology studies. When obtaining an informed consent, data of patients were prospectively centrally recorded through our web site. A retrospective analysis on differences in diagnosing RAEB, comparing conventional cytology on bone marrow smears (CBM), histochemical evaluation of CD34+ cells on bone marrow trephine biopsy (HBM), and cytofluorimetric count of CD34+ and CD117+ cells (IBM) has been done. RESULTS. From June 1999 to December 2003, 633 MDS patients were registered from 37 different institutions: 364 (57%) from haematology and/or academic institutions and 269 (43%) from internal medicine departments of community hospitals. Mean age was 72 (range 23–69). The actual diagnostic distribution of cases according to the WHO criteria based on only morphology evaluation of bone marrow smears was: non-RAEB 429 (68%), RAEB-I 134 (21%), and RAEB-II 70 (11%). Information about the quantification of blasts with both CBM and HBM techniques was avilable in 243 cases. An IBM evaluation was also available in 89 out of this 243 cases. A disagreement between CBM and HBM was evident in 65/243 cases (27%), with HBM over-evaluating and under-evaluating WHO class on the basis of blasts count in 54/243 (22%) and 11/243 cases respectively. When comparing CBM and IBM the disagreement was even higher in 29/89 cases (33%), with IBM over-evaluating blast percent in 9 (10%) and under-evaluating it in 20 cases (23%). The disagreement betwen HBM and IBM was maximum with a value of 39%. The role of CBM in predicting a different prognosis of non-RAEB, RAEB-I and RAEB-II was confirmed. However, when comparing the prognostic value of the three different methods of computing bone marrow blasts, IBM was the best in order to define the good prognostic non-RAEB group. CONCLUSIONS. The distinction among non-RAEB, RAEB-I and RAEB-II is far from beeing highly accurate and reproducible. Important differences are present among CBM, HBM and IBM. While CBM remain the conventional standard system, IBM could offer a tool better and more reproducible than CBM in order to define MDS categories on the basis of blast percentage. A large multicenter study could be useful in order to clarify this point.

Safe and Efficient Expansion of Human HSC with Sendai Virus Vector Expressing HoxB4 in Fetal Sheep.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 693-693
Author(s):  
Shigeo Masuda ◽  
Tomoyuki Abe ◽  
Makoto Inoue ◽  
Mamoru Hasegawa ◽  
Satoshi Hayashi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Insertional Mutagenesis ◽  
Sendai Virus ◽  
Retroviral Vectors ◽  
Transduction Efficiency ◽  
Control Group ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Colony Pcr ◽  
Cd34 Cells

Abstract Abstract 693 Background: Homeobox B4 (HoxB4) has been shown to be a potent stem cell self-renewal gene, especially in hematopoietic stem cells (HSC). Accumulating evidence from murine studies indicates that the overexpression of HoxB4 enhances in vivo and ex vivo expansion of HSC. Although no leukemia has been observed after transplantation of HoxB4-transduced cells in murine models, the study using large animals such as dogs and non-human primates with retroviral vectors expressing HoxB4 showed the frequent development of leukemia. Regarding retroviral vectors expressing HoxB4, there is another concern, that is, insertional leukemogenesis, which has been elucidated in the hematopoietic stem cell gene therapy for X-SCID. To avoid the insertional mutagenesis, other vectors may be considered, including Epstein-Barr nuclear antigen (EBNA)-1 based episomal vectors or the transposon; however, problems are left, i.e. low transduction efficiency with EBNA vectors and unclear safety with transposon vectors. To avoid both the persistent HoxB4 expression and insertional mutagenesis leading to leukemogenesis, we have developed a new type of Sendai virus (SeV) vector; it lacks the polymerase gene, namely P-defective SeV (SeV/δP) vector. SeV is an enveloped virus with a non-segmented, negative-stranded RNA genome. SeV-based vectors are non-integrating, cytoplasmic vectors. They replicate exclusively in the cytoplasm of transduced cells, and do not go through a DNA phase; therefore, there is no concern about the unwanted integration of foreign sequences into chromosomal DNA of the host. We have previously shown that the transduction efficiency of human CD34+ cells with the SeV vector was very high; around 70% (100 multiplicity of infections). On the other hand, SeV/δP vectors are incapable of self-replication, thus enabling transient gene expression without spoiling their ability to efficiently transduce CD34+ cells. Here, using the SeV/δP vector expressing HoxB4 (SeV/δP/HoxB4 vector), we examined the effectiveness and safety of human HSC expansion after in utero transplantation to fetal sheep. Methods: After enrichment of CD34+ cells from cryopreserved human umbilical cord blood, these cells were repeatedly exposed to SeV/δP/HoxB4 vector every 24 h for 4 days. The transduced cells (3.2–11.7 × 105) were transplanted into the abdominal cavity of fetal sheep at 45–50 gestational days (full term, 147 days) that have premature immune system (HoxB4 group, n = 4; control group, n = 4). The engraftment of hematopoietic cells derived from human HSC in the lambs after birth was quantitatively evaluated by colony PCR of the bone marrow. The development of leukemia was assessed by regular sampling of peripheral blood and bone marrow. Results: The human–sheep chimeric ratio in the bone marrow of HoxB4 group was calculated 4.8-times higher than that of control group after birth, as assessed by colony PCR. The SeV genome was no longer detectable in the bone marrow and peripheral blood of lambs as assessed by RNA-PCR, confirming the SeV vectors were cleared. No leukemia developed in any of the sheep in either group at present (at 12 months after transplantation). Conclusion: The SeV/δP vector would be suitable for transient expression of HoxB4 in human CD34+ cells, enabling 4.8-times expansion of human HSC as assessed by their repopulating ability in sheep. The expansion of human HSC with the SeV vector was comparable to that with HoxB4-retroviral vectors. In addition, the SeV/δP vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors. Disclosures: No relevant conflicts of interest to declare.

Let-7a, Mir-17 and Mir-20a Expression Levels in CD34+ Bone Marrow Cells of Patients with Myelodysplastic Syndromes (MDS) Are Associated with Established Prognostic Factors, Supporting Their Implication in the Pathogenesis of the Disease,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3792-3792 ◽  
Author(s):  
Christos K Kontos ◽  
Vassiliki Pappa ◽  
Diamantina Vasilatou ◽  
Maria-Angeliki S Pavlou ◽  
Frida Kontsioti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Prognostic Factors ◽  
High Risk ◽  
Mirna Expression ◽  
Who Classification ◽  
Low Risk ◽  
Expression Levels ◽  
Cd34 Cells ◽  
Bone Marrow Blasts

Abstract Abstract 3792 Introduction: MicroRNAs are single, small non-coding RNA molecules of approximately 21–26 nucleotides, which regulate the expression of numerous genes. miRNAs may act either at the post-transcriptional or the post-translational level to repress gene expression; still, upregulation of gene expression has been noticed in some cases as a direct effect of miRNA function. The importance of miRNAs in carcinogenesis is emphasized by the association of cancers with alterations in miRNA expression. Many miRNAs, including let-7a and those of the miR-17-92 cluster (miR-17, miR-20a, etc.), have been shown or are predicted to affect the activities of targeted mRNAs encoding proteins that have oncogenic or anti-oncogenic functions. let-7a downregulates KRAS, while miR-17 and miR-20a downregulate E2F1. Both these proteins are overexpressed in myelodysplastic syndromes (MDS) and have been shown to be involved in the pathobiology of the disease. Purpose: In the current study, we examined the prognostic value of let-7a, miR-17 and miR-20a levels in MDS and their potential as novel molecular biomarkers. Furthermore, we investigated the protein expression levels of validated targets of these three miRNAs in bone marrow CD34+ cells of MDS patients. Material and Methods: We evaluated 43 patients with MDS (34 men, 9 women) with a median age of 73 years (range 45–87). According to WHO classification, 12 patients (27.9%) were diagnosed with RA, 6 (13.9%) RCMD, 8 (18.6%) with RAEB-I, 7 (16.3%) with RAEB-II, 8 (18.6%) with AML, and 2 (4.7%) with CMML. According to IPSS, 13 patients (32.5%) had low risk, 14 (35.0%) intermediate I risk, 6 (15.0%) intermediate II, and 7 (17.5%) high risk disease. WPSS classification was: 8 (23.5%) very low risk, 5 (14.7%) low risk, 8 (23.5%) intermediate, 9 (26.5%) high risk, and 4 (11.8%) very high risk. We isolated CD34+ cells from bone marrow mononuclear cells from MDS patients, as well as from peripheral blood of donors of CD34+ cells for stem cell transplantation, using magnetic beads. Extraction of small RNA-containing total RNA from CD34+ cells was performed and cDNA of let-7a, miR-17 and miR-20a was synthesized using specific primers. miRNA expression levels were determined using quantitative real-time PCR, the TaqMan® chemistry and the relative quantification (2−ΔΔCT) method. The snoRNA RNU48 was used as reference gene. Furthermore, total protein was extracted from CD34+ cells using a lysis buffer and subsequently quantified using the Bradford assay. Western blot analysis was carried out for MYC, E2F1, Cyclin D1 (CCND1), BCL2 and KRAS, while Actin was used as reference protein. Results: In MDS patients, let-7a expression levels were 0.053–506.1 copies/RNU48 copies, while miR-17 and miR-20a expression levels were 0.005–2694.5 and 0.003–3116.7 copies/103RNU48 copies, respectively. No significant differences were found between patients and controls regarding let-7a, miR-17 and miR-20a expression. let-7a underexpression was associated with high (>10%) bone marrow blasts percentage (P =0.036), presence of WHO classification subtypes with poor prognosis (RAEB-I, RAEB-II and AML) (P =0.020), and high IPSS (P =0.037). Furthermore, miR-17 underexpression was related to high (>10%) bone marrow blasts percentage (P =0.008), intermediate and/or high risk karyotype (P =0.018) and high IPSS (P =0.016). Moreover, miR-20a underexpression was associated with high IPSS (P =0.037) and WPSS (P =0.013). Interestingly, protein expression levels of all targets analyzed in the current study were shown to be lower in samples overexpressing let-7a, miR-17 and/or miR-20a, in comparison with the corresponding protein levels noticed in specimens showing lower expression of these three miRNAs. Conclusion: To the best of our knowledge, this is the first study showing that expression levels of let-7a, miR-17 and miR-20a are associated with established prognostic factors in MDS, including IPSS and WPSS. Furthermore, these three miRNAs seem to be implicated in the pathogenesis of the disease, most probably by finely tuning the expression of target proteins that are involved in highly important molecular pathways, therefore affecting key cellular functions, such as cell cycle control, apoptosis, cell proliferation, and regulation of gene expression. Undoubtedly, further studies are needed to confirm the present findings and clarify their association with the pathogenesis of different MDS subgroups. Disclosures: No relevant conflicts of interest to declare.

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