Role of Different MicroRNAs in Acute Myeloid Leukemia Fawaz Yahya Hamdi*, Mohammed Nouh Hamdi**, Abdullah Yahya Hamadi***, Naseh Ateahallah Algehainy ****, Ali Mosa Mahzari***** Abstract MicroRNAs (miRNAs) are keys to the pathogenesis of human malignancies and are increasingly recognized as potential biomarkers and therapeutic targets. Hematological malignancies, being the earliest human malignancies linked to aberrant miRNA expression, have consistently underpinned our understanding of the role that miRNAs play in cancer development. Recent studies have significantly improved our understanding of the role that microRNAs (miRNAs) play in regulating normal hematopoiesis. miRNAs are critical for maintaining hematopoietic stem cell function and the development of mature progeny. Thus, perhaps it is not surprising that miRNAs serve as oncogenes and tumor suppressors in hematologic malignancies arising from hematopoietic stem and progenitor cells, such as the myeloid disorders. A number of studies have extensively documented the widespread dysregulation of miRNA expression in human acute myeloid leukemia (AML), inspiring numerous explorations of the functional role of miRNAs in myeloid leukemogenesis.
Keywords: Acute myeloid leukemia (AML), MicroRNAs (miRNAs), miRNAs125, -146, -155, -142, and -29.
Introduction and Review MicroRNAs (miRNAs) are a class of small (~22 nucleotide) noncoding RNAs that are potent regulators of gene expression in animals and plants. In animals, miRNAs bind to target sequences (usually located in the 3′ untranslated region [3′UTR]) in messenger RNAs (mRNAs) and act by negatively regulating gene expression. This binding requires complementarity between the nucleotides 28 of miRNA (so called “seed” region) and the target mRNA. 1 It has been reported that more than 2500 mature human miRNAs have been identified 2 and they are predicted to regulate over 60% of human proteincoding genes 3. This regulatory network can be very complex as one miRNA may potentially regulate
*
several mRNAs, and a given mRNA may possess in its sequence, binding sites for several miRNAs. Since miRNAs control a wide variety of biological processes, including proliferation, apoptosis and differentiation, dysfunctions of the miRNA regulatory network may contribute to tumorigenesis. MicroRNAs can act as both oncogenes and tumor suppressors. MicroRNAs play a crucial role in normal hematopoiesis by controlling the differentiation of hematopoietic stem cells into different types of mature blood cells, while deregulation of miRNA networks has been linked to hematological malignancies. 5 Aberrant miRNA expression profiles have been observed in leukemias and lymphomas, and for several miRNAs there is experimental evidence for their functional involvement in leukemogenesis. 6, 7
Medical Lab Technology, Division of hematology, Toxic Center of Jazan, Ministry of health, Saudi Arabia Medical Lab Technology, Division of hematology, Samtah General Hospital, Jazan, Ministry of Health, Saudi Arabia *** Medical Lab Technology, Division of Immunohematology, Faculty of Applied Medical Sciences, University of Tabuk, Saudi Arabia, Tabuk 71491 **** Medical Lab Technology, Division of Clinical Chemistry, Faculty of Applied Medical Sciences, University of Tabuk, Saudi Arabia, Tabuk 71491 ***** Medical Lab Technology, Division of Biochemistry, Faculty of Applied Medical Sciences, University of Albaha, Saudi Arabia. Correspondence to: Dr. Fawaz Yahya Hamdi, Medical Lab Technology, Division of hematology, Toxic Center of Jazan, Ministry of health, Saudi Arabia. E- mail:
[email protected] **
© ADR Journals 2014. All Rights Reserved.
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Specific miRNA expression signatures can accurately discriminate different leukemia subtypes and are often of great prognostic relevance.8 The importance of miRNAs in carcinogenesis has been inferred by their localization to genomic regions that are frequently deleted or amplified, and to their presence near translocation breakpoints, in various human cancers. 9 The relevance of miRNAs to hematologic malignancies was first established when miR-15 and miR-16 were shown to be the critical genetic elements deleted from chromosome 13q14 in a significant proportion of chronic lymphocytic leukemia (CLL) patients. The gene expression profiling of AML patient blasts revealed a widespread deregulation of miRNAs. These studies also established associations between different miRNA signatures and specific molecular subtypes of disease, suggesting their potential role in AML pathogenesis. 10 The miRNAs for which a functional link between miRNA dysregulation and the development of AML has been established include miRNAs-125, -146, -155, -142, and -29.
Role of miRNAs-29 in AML
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is a small RNA molecule in the shape of a stemloop or hairpin. Each arm of the hairpin can be processed into one member of a closely related family of short non- coding RNAs that are involved in regulating gene expression.11 The processed or "mature" products of the precursor molecule are known as microRNA (miRNA), and have been predicted or confirmed in a wide range of species (see 'MIPF0000009' in miRBase: the microRNA database). Many mammalian genomes encode four closely related miR-29 precursors that are transcribed in two transcriptional units. For example, human miR-29a and miR-29b-1 are processed from an intron of a long non- coding transcript (LOC646329) from chromosome 7. miR-29b-2 (identical in sequence to miR-29b-1) and miR-29c are co- transcribed from chromosome 1. The three main mature miRNAs processed from these precursors are known as hsa-miR-29a, hsa-miR-29b, and hsa-miR-29c. The down- regulation of miR-29b is thought to promote DNA hypermethylation in AML since miR-29b can directly target DNMT3A, DNMT3B, and Sp1 (a transcriptional regulator of DNMT1 as shown in fig. 1.12
The miR-29 microRNA precursor, or pre- miRNA,
Figure 1.MicroRNA-29 family members target multiple genes to mediate their biologic effects
This link between miR-29 expression and methylation status in AML cells prompted the evaluation of miR-29b as a therapeutic target in AML. Investigators have shown that miR-29b oligonucleotide mimics recapitulate the effects of hypomethylating agents, 5-azacytidine and ISSN: 2393 - 8293
decitabine, by demethylating the promoters of tumor suppressors estrogen receptor 1 (ESR1) and p15INK4b, and by promoting the re- expression of these genes in AML cell lines. 12, 13 These results indicate that miR-29b oligonucleotides may be an effective therapeutic strategy in the treatment of AML. It Int. J. Gen. Can. 2014; 1(3&4): 73- 79.
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would also be interesting to evaluate the levels of the DNMTs, TETs, and GDM simultaneously upon miR-29 overexpression in leukemic blasts and normal HSPCs. This would help elucidate whether the effect of miR-29 on DNMTs and TETs are cell context- specific, or whether DNMTs and TETs function to promote the methylation and demethylation of distinct target genes simultaneously.
Role of miRNAs-146 in AML miR-146 is a family of microRNA precursors found in mammals, including humans. The ~22 nucleotide mature miRNA sequence is excised from the precursor hairpin by the enzyme Dicer.14 This sequence then associates with RISC which effects RNA interference. miR-146 is primarily involved in the regulation of inflammation and other processes that function in the innate immune system.15 Loss of functional miR-146 could predispose an individual to suffer from chromosome 5q deletion syndrome.16 miR-146 has also been reported to be highly upregulated in osteoarthritis cartilage, and could be involved in its pathogenesis. 17 miR-146 could be used as a biomarker for sepsis.18 In addition, it was found to be absent from the exosomes of prion infected cells suggesting it could be used as a biomarker for prion infection.19 miR146a could be targeted therapeutically as its depletion has implication in the hyperactive response to infection.20
Role of miRNAs-142 in AML The microRNA-142 is located on human chromosome 17 and miR-142 is expressed in all hematopoietic tissues including the bone marrow, spleen and thymus. It is not expressed in nonhematopoietic tissues. Perhaps it is not surprising that the first role ascribed to miR-142 was in promoting the development of the T-cell and myeloid lineages. 21 miR-142’s importance as a regulator of hematopoiesis was recently underscored by the fact that it is the only miRNA that is recurrently mutated in AML. miR-142 regulates normal hematopoiesis as well as the development of
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lymphoid and myeloid leukemias. By enforcing the expression of miR-142 in Lin- mouse bone marrow cells, 22 it has been shown that miR-142 increases the absolute numbers of T-cells, leads to a minimal decrease in B-cell differentiation (CD19+), and slightly reduces the number of Mac1+ Gr1- myeloid cells. miR-142 also appears to play a role in the development of mature lymphoid malignancies, as the miR-142 locus is 50bp from the breakpoint of t(8;17), a cytogenetic alteration present in a subset of aggressive mature B-cell leukemias. 23 This suggests that MYC, located on chromosome 8, is translocated and regulated by the upstream miR-142 promoter.24 Interestingly, miR-142-3p is significantly downregulated in ALL patients expressing the MLL-AF4 fusion gene. Ectopic expression of miR-142-3p in MLL-AF4+ cell lines suppress cell proliferation, induces apoptosis, and down- regulates multiple genes known to regulate self- renewal including MLL-AF4, HOXA9, HOXA7, and HOXA10. Thus, miR-142-3p likely functions as a tumor suppressor in MLL-AF4+ ALL. 24
Role of miRNAs-155 in AML MicroRNA-155 is a microRNA that, in humans is encoded by the MIR155 host gene or MIR155HG. 25 MiR-155 plays a role in various physiological and pathological processes.26Exogenous molecular control in vivo of miR-155 expression may inhibit malignant growth, viral infections, 27 and enhance the progression of cardiovascular diseases. The MIR155HG was initially identified as a gene that was transcriptionally activated by promoter insertion at a common retroviral integration site in Bcell lymphomas and was formerly called BIC (B-cell Integration Cluster). It was later found that MIR155HG was composed of three exons that span a 13 kb region within human chromosome 21 (Hsa21 band q21.3).28 The MIR155HG is transcribed by RNA polymerase II and the resulting ~1,500 nucleotide RNA is capped and polyadenylated. The 23 nucleotide single- stranded miR-155, which is harbored in exon 3, is subsequently processed from the parent RNA molecule. 29
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Figure 2.Schematic representation of MIR155H
Early phylogenetic analyses demonstrated that the sequence of pre- mir-155 and miR-155-5p was conserved between human, mouse, and chicken. 30 Recent annotated sequencing data found that 22
different organisms including, mammals, amphibians, birds, reptiles, sea squirts, and sea lampreys, express a conserved miR-155-5p.
Figure 3.Summary of miR-125b functions. miR-125b is part of an miRNA locus that encodes other important miRNAs. It is co- transcribed with let-7c and miR-99a. Important targets of miR-125b include several factors important in the maintenance of stem cell properties and cell survival and apoptosis, including lin28, IRF4, BMF, and Bak1.
Currently much less sequence data is available regarding miR-155-3p, therefore, it is not clear how conserved this miRNA is across species. O’Connell et al.31overexpressed miR-155 in RAW 264.7 myeloid cells and showed reductions in the transcripts of several genes (Bach1, Sla, Cutl1, Csf1r, Jarid2, Cebpβ, PU.1, Arntl, Hif1α, and Picalm) known to play critical roles in hematopoiesis; subsequent studies have established that miR-155 directly inhibits src homology 2 domain- containing inositol- 5- phosphatase (SHIP1) as well as CCAAT- enhancer- binding protein- beta (CEBP-β) to mediate leukemogenesis.
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Interestingly, miR-155- mediated hematopoietic malignancies exhibit longer latencies compared to more aggressive miRNA leukemia models, such as those induced by miR-125 overexpression. This raises the possibility that additional mutations are required for full transformation. Intriguingly, miR155 targets mismatch repair genes such as hMLH1, hMSH2, and hMSH6 32 as well as cell- cycle regulators such as WEE1. Thus, miR-155 may increase spontaneous mutation rates in HSPCs, in agreement with observations made in some solid tumor cell lines. 33, 34It would be interesting to investigate this hypothesis in the context of AML and to determine whether such mutations are Int. J. Gen. Can. 2014; 1(3&4): 73- 79.
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required for the full manifestation of disease phenotypes.
in secondary recipients, 41 suggesting that miR-125a cannot maintain long- term HSC self- renewal.
Role of miRNAs-125 in AML
Conclusion
MicroRNA-125 is a member of a group of microRNAs that are ubiquitously expressed in all tissues and as such, has wide ranging function. It has been found to play important roles in some of the most devastating illnesses: cancer, neurological disorders, and cardiovascular disease. MicroRNA125a-5p was found to mediate lipid uptake and to decrease the secretion of some inflammatory factors in oxLDL- stimulated macrophages, suggesting that it may play a protective role against the development of atherosclerosis. In addition to their roles in epigenetic regulation and differentiation, miR29a and miR-29b have been shown to regulate a number of cellular processes. A transcriptomal analysis of human leukemia cell lines transfected with synthetic miR-29b revealed the enrichment of genes that regulate apoptosis, cell cycle progression, and cellular proliferation.
The emergence of novel technologies, especially high- throughput small RNA sequencing methods, will allow more sensitive and efficient microRNA detection in patient samples, and identification of novel microRNAs. There is also the potential to develop microRNA- based therapeutic strategies. It is reasonable to restore lost tumor suppressor microRNA in AML blasts, such as miR-29 or miR181a, using synthetic oligonucleotides that mimic the endogenous mature microRNA or compounds that indirectly up- regulate microRNA expression. It has been demonstrated experimentally that antileukemic effects can be achieved by modulating microRNA expression by pharmacologic agents and/ or increasing low endogenous levels of microRNAs with tumor suppressor function by synthetic microRNA oligonucleotides, or down- regulating high endogenous levels of leukemogenic microRNAs by antisense oligonucleotides (antagomirs). Therefore, it is reasonable to predict the development of novel microRNA- based therapeutic approaches in AML.
Insight into the biological effects of miR-125b comes from both in vitro and in vivo ectopic expression studies. miR-125b overexpression blocks terminal (monocytic and granulocytic) differentiation in HL60 and NB4 AML cell lines 35 and confers interleukin-3 (IL-3) growth independence to the leukemic cell line, 32Dclone3 36 . ABTB1 and CBFB have been identified as miR125b targets that may mediate these anti- apoptotic and pro- proliferative effects. 37 In vivo, primary recipients of HSCs overexpressing miR-125b (35fold) display myeloid-biased differentiation and expansion at the expense of B cells, while secondary recipients develop a lymphoproliferative disease. This increase in lymphocyte output is likely due to the preferential expansion of lymphoid- biased SlamHSCs, as they display intrinsically higher basal apoptotic rates, which makes them more prone to miR-125’s anti- apoptotic effects. Furthermore, a 35-fold overexpression of miR-125b was also associated with an expansion of common lymphoid progenitors. 38Consistent with these studies, enforced expression of miR-125a in BM cells by 1000-fold enhanced long- term reconstitution of all blood lineages following transplantation. This effect persisted in secondary transplants, although it was also associated with increased myeloid cell output.39The majority of these mice also exhibited a myeloproliferative neoplasm (MPN) - like phenotype that occasionally progressed to AML beginning ∼5 months post- transplant. The AML phenotype persisted upon serial transplantation. 40 In a separate study, however, engraftment of HSCs ectopically expressing miR-125a declined over time Int. J. Gen. Can. 2014; 1(3&4): 73- 79.
Acknowledgement The authors highly acknowledge the valuable time given by Dr. AB. Rashid Mir, Assistant Professor, Faculty of Applied Medical Sciences, Supervisor Division of Molecular Genetics, Prince Fahd Bin Sultan Research chair, University of Tabuk, Saudi Arabia, in reviewing this article.
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