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Journal ArticleDOI

hsa-miR-503 is downregulated in β thalassemia major.

11 Aug 2012-Acta Haematologica (Karger Publishers)-Vol. 128, Iss: 3, pp 187-189
TL;DR: Whether there is a variance in the expression level of hsa-miR-503 in red blood cells of thalassemic and hematologically normal individuals is investigated to study how the miRNA expression profile is modulated in patients withThalassemia major.
Abstract: 5(G 1 C). All probands were aged between 20 and 30 years. Written informed consent was obtained from them, and the institutional human research committee approved the study. The ethical principles followed by the institute are guided by rules as formulated by the Indian Council of Medical Research and these are in agreement with the Helsinki rules. The CD34+ hematopoietic stem cells were first isolated from peripheral blood samples of these probands via the MACS cell separation technique and they were then grown following a two-step culture system to achieve the unilineage differentiation into mature erythrocytes [6] . Ninety percent of the cells became benzidine positive by day 13 in normal samples. The cells were harvested on the 13th day of culture by selecting for CD235a expression using an immunomagnetic isolation procedure and processed for miRNA and mRNA isolation ( mir VanaTM miRNA Isolation Kit; Ambion). Solution hybridization was performed with an mir Vana miRNA Probe Construction Kit (Ambion) and an mir Vana miRNA Detection Kit (Ambion) following the manufacturer’s instructions. For probe labeling, 32 P CTP (BRIT, India) was used. The intensity of the bands was analyzed using Imagequant software. U6 MicroRNAs (miRNAs) are small, 22to 25-nt-long, endogenous, non-proteincoding RNA molecules which have the potential to downregulate gene expression. The involvement of miRNAs in several biological processes has been demonstrated, and diseased states are characterized by altered expression of miRNAs [1] . We intended to study how the miRNA expression profile is modulated in patients with thalassemia major. Thalassemia is characterized by an enhanced rate of growth of erythropoietic progenitors leading to marrow expansion and resultant iron absorption [2] . Of the various miRNAs expressed during erythropoiesis, hsa-miR-503 has been implicated in regulation of genes involved in cell cycle arrest and apoptosis [3–5] . To investigate whether there is a variance in the expression level of hsa-miR-503 in red blood cells of thalassemic and hematologically normal individuals, four patients with  thalassemia major, four with Ethalassemia major, and two with Ethalassemia intermedia, as well as four normal healthy adult volunteers, were recruited. All  major patients studied here are homozygous for the mutation IVS-I nt 5(G 1 C). The intermedia and severe patients of HbE/ thalassemia are compound heterozygous for HbE and IVS-I nt Received: March 5, 2012 Accepted after revision: May 8, 2012 Published online: August 11, 2012
Citations
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Journal ArticleDOI
TL;DR: Functional studies showed miR-503 suppressed the proliferation of HCC cells by induction of G1 phase arrest through Rb-E2F signaling pathways, and thus may function as a tumor suppressor, and implicate the potential application of mi R-503 in prognosis prediction and miRNA-based HCC therapy.
Abstract: Increasing evidence indicates that deregulation of microRNAs (miRNAs) is involved in tumorigenesis. Downregulation of microRNA-503 has been observed in various types of diseases, including cancer. However, the biological function of miR-503 in hepatocellular carcinoma (HCC) is still largely unknown. In this study we aimed to elucidate the prognostic implications of miR-503 in HCC and its pathophysiologic role. Quantitative reverse transcriptase polymerase chain reaction was used to evaluate miR-503 expression in HCC tissues and cell lines. Western blotting was performed to evaluate the expression of the miR-503 target genes. In vivo and in vitro assays were performed to evaluate the function of miR-503 in HCC. Luciferase reporter assay was employed to validate the miR-503 target genes. miR-503 was frequently downregulated in HCC cell lines and tissues. Low expression levels of miR-503 were associated with enhanced malignant potential such as portal vein tumor thrombi, histologic grade, TNM stage, AFP level and poor prognosis. Multivariate analysis indicated that miR-503 downregulation was significantly associated with worse overall survival of HCC patients. Functional studies showed miR-503 suppressed the proliferation of HCC cells by induction of G1 phase arrest through Rb-E2F signaling pathways, and thus may function as a tumor suppressor. Further investigation characterized two cell cycle-related molecules, cyclin D3 and E2F3, as the direct miR-503 targets. Our data highlight an important role for miR-503 in cell cycle regulation and in the molecular etiology of HCC, and implicate the potential application of miR-503 in prognosis prediction and miRNA-based HCC therapy.

75 citations

Journal ArticleDOI
TL;DR: A large number of approaches based on targeting microRNAs and long non-coding RNAs are expected in the field of molecular diagnosis and therapy, with a facilitated technological transfer in the case of rare genetic diseases, in virtue of the existing regulation concerning these diseases.
Abstract: Since the discovery and classification of non-coding RNAs, their roles have gained great attention. In this respect, microRNAs and long non-coding RNAs have been firmly demonstrated to be linked to regulation of gene expression and onset of human diseases, including rare genetic diseases; therefore they are suitable targets for therapeutic intervention. This issue, in the context of rare genetic diseases, is being considered by an increasing number of research groups and is of key interest to the health community. In the case of rare genetic diseases, the possibility of developing personalized therapy in precision medicine has attracted the attention of researchers and clinicians involved in developing “orphan medicinal products” and proposing these to the European Medicines Agency (EMA) and to the Food and Drug Administration (FDA) Office of Orphan Products Development (OOPD) in the United States. The major focuses of these activities are the evaluation and development of products (drugs, biologics, devices, or medical foods) considered to be promising for diagnosis and/or treatment of rare diseases or conditions, including rare genetic diseases. In an increasing number of rare genetic diseases, analysis of microRNAs and long non-coding RNAs has been proven a promising strategy. These diseases include, but are not limited to, Duchenne muscular dystrophy, cystic fibrosis, Rett syndrome, and β-thalassemia. In conclusion, a large number of approaches based on targeting microRNAs and long non-coding RNAs are expected in the field of molecular diagnosis and therapy, with a facilitated technological transfer in the case of rare genetic diseases, in virtue of the existing regulation concerning these diseases.

48 citations

Journal ArticleDOI
01 Jan 2016-Cell
TL;DR: According to the suppressive effects of certain miRs, it has been possible to increasing γ globin gene expression and fetal hemoglobin (HbF) production and this strategy can be used as a novel therapy in infusing HbF and improving clinical complications of patients with hemoglobi- nopathies.

32 citations


Additional excerpts

  • ...in cells with β-thalassemia mutations (29)....

    [...]

Journal ArticleDOI
Zhang Xueping1
TL;DR: Non-coding linear RNAs come in a variety of forms, including circular RNAs with a continuous closed loop (circRNA), long noncoding RNAs (lncRNA), and microRNAs (miRNA) as mentioned in this paper .
Abstract: Human diseases have been a critical threat from the beginning of human history. Knowing the origin, course of action and treatment of any disease state is essential. A microscopic approach to the molecular field is a more coherent and accurate way to explore the mechanism, progression, and therapy with the introduction and evolution of technology than a macroscopic approach. Non-coding RNAs (ncRNAs) play increasingly important roles in detecting, developing, and treating all abnormalities related to physiology, pathology, genetics, epigenetics, cancer, and developmental diseases. Noncoding RNAs are becoming increasingly crucial as powerful, multipurpose regulators of all biological processes. Parallel to this, a rising amount of scientific information has revealed links between abnormal noncoding RNA expression and human disorders. Numerous non-coding transcripts with unknown functions have been found in addition to advancements in RNA-sequencing methods. Non-coding linear RNAs come in a variety of forms, including circular RNAs with a continuous closed loop (circRNA), long non-coding RNAs (lncRNA), and microRNAs (miRNA). This comprises specific information on their biogenesis, mode of action, physiological function, and significance concerning disease (such as cancer or cardiovascular diseases and others). This study review focuses on non-coding RNA as specific biomarkers and novel therapeutic targets.

11 citations

Journal ArticleDOI
TL;DR: In this paper, a review of the current understanding of interactions between miRNAs, their molecular targets, and their regulatory effects in HbF induction in SCD is presented. But, the authors do not consider the effect of miRNA on the development of SCD.
Abstract: Sickle cell disease (SCD) is a severe hereditary blood disorder caused by a mutation of the beta-globin gene, which results in a substantial reduction in life expectancy. Many studies are focused on various novel therapeutic strategies that include re-activation of the γ-globin gene. Among them, expression therapy caused by the fetal hemoglobin (HbF) at a later age is highly successful. The induction of HbF is one of the dominant genetic modulators of the hematological and clinical characteristics of SCD. In fact, HbF compensates for the abnormal beta chain and has an ameliorant effect on clinical complications. Erythropoiesis is a multi-step process that involves the proliferation and differentiation of a small population of hematopoietic stem cells and is affected by several factors, including signaling pathways, transcription factors, and small non-coding RNAs (miRNAs). miRNAs play a regulatory role through complex networks that control several epigenetic mechanisms as well as the post-transcriptional regulation of multiple genes. In this review, we briefly describe the current understanding of interactions between miRNAs, their molecular targets, and their regulatory effects in HbF induction in SCD.

6 citations

References
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Journal ArticleDOI
01 Dec 2005-RNA
TL;DR: The finding that microRNAs are misexpressed in cancers has reinforced the idea that their regulatory roles are very important and linked a well-known subcellular structure, the cytoplasmic processing bodies (PBs) to the microRNA pathway.
Abstract: MicroRNAs are sequence-specific regulators of post-transcriptional gene expression in many eukaryotes. They are believed to control the expression of thousands of target mRNAs, with each mRNA believed to be targeted by multiple microRNAs. Recent studies have uncovered various mechanisms by which microRNAs down-regulate their target mRNAs and have linked a well-known subcellular structure, the cytoplasmic processing bodies (PBs) to the microRNA pathway. The finding that microRNAs are misexpressed in cancers has reinforced the idea that their regulatory roles are very important.

715 citations

Journal ArticleDOI
01 Nov 2010-Leukemia
TL;DR: This study identifies four PMA-induced microRNAs that when overexpressed cause cell-cycle arrest and partial differentiation and when used in combination induce additional changes not seen by any individual microRNA.
Abstract: Acute myeloid leukemia (AML) involves a block in terminal differentiation of the myeloid lineage and uncontrolled proliferation of a progenitor state. Using phorbol myristate acetate (PMA), it is possible to overcome this block in THP-1 cells (an M5-AML containing the MLL-MLLT3 fusion), resulting in differentiation to an adherent monocytic phenotype. As part of FANTOM4, we used microarrays to identify 23 microRNAs that are regulated by PMA. We identify four PMA-induced microRNAs (mir-155, mir-222, mir-424 and mir-503) that when overexpressed cause cell-cycle arrest and partial differentiation and when used in combination induce additional changes not seen by any individual microRNA. We further characterize these pro-differentiative microRNAs and show that mir-155 and mir-222 induce G2 arrest and apoptosis, respectively. We find mir-424 and mir-503 are derived from a polycistronic precursor mir-424-503 that is under repression by the MLL-MLLT3 leukemogenic fusion. Both of these microRNAs directly target cell-cycle regulators and induce G1 cell-cycle arrest when overexpressed in THP-1. We also find that the pro-differentiative mir-424 and mir-503 downregulate the anti-differentiative mir-9 by targeting a site in its primary transcript. Our study highlights the combinatorial effects of multiple microRNAs within cellular systems.

240 citations

Journal ArticleDOI
TL;DR: This article describes a novel role of Cdc25A down-regulation during differentiation of proliferating myoblasts in response to EMT and describes the mechanism through which this regulation is switched off.
Abstract: This article describes a novel role of Cdc25A down-regulation during differentiation of proliferating myoblasts.

197 citations

Journal ArticleDOI
04 Jun 2008-PLOS ONE
TL;DR: It is found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs, and poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation.
Abstract: Background Since mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS). Methods and Findings Using a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation. Conclusions In summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases.

176 citations

PatentDOI
TL;DR: In this paper, a two-step culture method for mass production in vitro of erythroid cells from either CD34 + or light-density (10 6 cells/mL) cells purified from the blood of normal donors and thalassemic patients was described.
Abstract: We describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34 + (10 5 cells/mL) or light-density (10 6 cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10 −6 M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated about 1-2×10 7 erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45 low /glycophorin (GPA) neg /CD71 low cells at day 7, 50-60% of which became CD45 neg /GPA+/CD71 high by days 15-20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (>90% benzidine pos and CD45 neg /GPA + /CD71 medium ) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.

134 citations