Role of miRNAs and lncRNAs in hematopoietic stem cell differentiation

Non-coding RNAs (ncRNAs) have diverse roles in the differentiation of hematopoietic cells. Among these transcripts, long ncRNAs (lncRNAs) and microRNAs (miRNAs) have especial contribution in this regard particularly by affecting levels of transcription factors that define differentiation of each linage. miR-222, miR-10a, miR-126, miR-106, miR-10b, miR-17, miR-20, miR-146, miR-155, miR-223, miR-221, miR-92, miR-150, miR-126 and miR-142 are among miRNAs that partake in the differentiation of hematopoietic stem cells. Meanwhile, this process is controlled by a number of lncRNAs such as PU.1-AS, AlncRNA-EC7, EGO, HOTAIRM1, Fas-AS1, LincRNA-EPS and lncRNA-CSR. Manipulation of expression of these transcripts has functional significance in the treatment of cancers and in cell therapy. In this paper, we have provided a brief summary of the role of miRNAs and lncRNAs in the regulation of hematopoietic stem cells.


Introduction
Non-coding RNAs (ncRNAs) have diverse roles in the biologic processes. Compared with the mRNA-coding transcripts, ncRNA transcripts more abundant in the human genome [1]. Two groups of ncRNAs have attracted attention of researchers due to their regulatory roles on the expression of genes. These groups of transcripts are long ncRNAs (lncRNAs) and microRNAs (miRNAs) [1]. In addition to acting as enhancers of transcription, lncRNAs can function as signals, decoys, scaffold transcripts and guide transcripts to directly regulate gene expression or recruit other regulatory molecules to alter gene expression [2]. The regulatory role of miRNAs on gene expression is exerted via their incorporation into the RNA-induced silencing complex (RISC). Subsequently, they can decrease expression of their targets. Based on the extent of similarity between the miRNA and target sequences, they degrade mRNA or inhibit its translation [3]. Both lncRNAs and miRNAs can regulate differentiation of hematopoietic cells [4,5]. Fig. 1 represents a summary of ncRNAs with critical roles in the differentiation of hematopoietic cells.
In the present review, we have provided a brief record of the role of miRNAs and lncRNAs in the regulation of HSCs.

miRNAs role in differentiation of HSCs
After assessment of miRNA signature in normal human bone marrow, Georgantas et al. have described expression of more than 30 miRNAs in CD34 + hematopoietic stem-progenitor cells (HSPCs). Subsequently, they integrated miRNA signature with mRNA profile of these cells and predicted miRNA-mRNA interaction data. Among the identified miRNAs has been miR-155, a miRNA that can regulate myelopoiesis and erythropoiesis. miRNA-155 has been shown to decrease both myeloid and erythroid colony construction from HSPCs [9]. Another pioneer study in this field has shown the role of various miRNA families in controlling HSC self-renewal and differentiation with HSCs being described by a certain miRNA profile in each differentiation phase. For instance, expressions of miR-125a, miR-125b, miR-155, miR-99a, miR-126, miR-196b, miR-130a, miR-542-5p, miR-181c, miR-193b and let7e have been significantly increased in long term-HSCs (LT-HSCS) [10]. Over-expression of miR-125b-5p, miR-126-3p and miR-155 in bone marrow cells has led to a competitive engraft enhancement in the bone marrow in all downstream lineages, while miR-196b, miR-181c, let7e and miR-542-5p have conferred an opposite effect. These observations have suggested the functional effect of these miRNAs in the regulation of HSC homeostasis instead of a certain role in differentiation to some specific phenotypes [10].
Using a high throughput combinatorial technique, Petriv et al. have assessed miRNA signature in 27 different cell populations and categorized these cells based on miRNA profile into six chief groups namely stem cell populations and multipotent progenitor cells, lymphoid cells, and four diverse principal classes of myeloid cells. They have reported alterations in the expressions of numerous miRNAs at distinctive nodes. Notably, miR-125b, miR-196a/b, miR-130a, let-7d, miR-148b and miR-351 have been the utmost differentially expressed miRNAs between stem cell populations and progenitor cells compared with the more mature cells [11]. Chen et al. have reported specific expression of three miRNAs in the hematopoietic cells. They have also demonstrated dynamic regulation of their expression throughout early hematopoiesis and lineage definition. Among these small transcripts, miR-181 has been mostly expressed in the B-lymphoid cells, and its expression in HSPCs has resulted in the preferential expansion of B-lineage cells [12]. miR-23a cluster is also involved in the regulation of lymphopoiesis since deficiency of this cluster in mice has resulted in the enhancement of B lymphopoiesis at the cost of myelopoiesis. However, HSPCs have not been altered. Concomitant deletion of mirn23a and mirn23b in adult bone marrow has also twisted HSPC differentiation to B cells at the cost of myeloid cells. Notably, double-knockout of these miRNAs has reduced bone marrow cellularity and diminished HSC and HSPC populations, demonstrating the exacerbation of the phenotype detected in mirn23a deficient mice [13]. On the other hand, miR-29a has a prominent role in controlling differentiation of myeloid lineage. This miRNA is over-expressed in early progenitors contributing in preservation of the undifferentiated status, whereas its expression has been decreased in the course of differentiation [14]. Therefore, forced over-expression of miR-29a in mice HSCs has conferred self-renewal aptitudes of myeloid precursors, enhancing myelopoiesis [14]. Another study has demonstrated the impact of miR-125b over-expression in bone marrow in induction of a myeloproliferative condition that might lead to myeloid leukemia [10]. Felli et al. have shown the role of miR-221 and miR-222 in reduction of proliferation of CD34 + progenitors and enhancement of differentiation of erythropoietc cells. These effects have been complemented by a significant reduction of kit protein. Besides, miR-221 and miR-222 treated CD34 + cells had lower engraftment capability and impaired stem cell activity upon transplantation in NOD-SCID animals. Taken together, under-expression of miR-221 and miR-222 increases kit protein synthesis, therefore resulting in the development of early erythroblastic cells [15]. Garzon et al. A high throughput expression profiling of CD34+-derived megakaryocytes has shown under-expression of miR-10a, miR-126, miR-106, miR-10b, miR-17 and miR-20. miR-130a has been shown to alter expression of MAFB, a transcription factor which stimulates expression of platelet-related protein GPIIB. Besides, miR-10a reduces expression of HOXA1. Evaluation of miRNA signature in the megakaryoblastic leukemic cells and in vitro differentiated megakaryocytes has demonstrated over-expression of miR-101, miR-126, miR-99a, miR-135, and miR-20 in the former cells [16]. Fazi et al. have uncovered the role of miR-223, NFI-A and C/EBPα in the regulation of differentiation of human granulocytes. They have also demonstrated a competition between NFI-A and C/EBPα for binding with promoter of miR-223. While NFI-A retains miR-223 expression low, C/EBPα enhances miR-223 expression after induction of cell differentiation by retinoic acid. Therefore, miR-223 participates in the process of granulopoiesis. It also down-regulates NFI-A expression to further mediate gene reprogramming in the granulocyte lineage [17]. miR-150 is among miRNAs with specific expression in the hematopoietic cells. This miRNA has been shown to be predominantly expressed in the lymph nodes and spleen, being over-expressed in the course of development of mature T and B cells with a sharp up-regulation in the immature B cell phase. Forced up-regulation of miR-150 in HPSCs has impaired the development of mature B cells, with no significant effects Fig. 1. Non-coding RNAs (ncRNA) include abundant small regulatory RNAs namely microRNAs (miRNAs), in addition to lots of polyadenylated and nonpolyadenylated long ncRNAs (lncRNAs). Currently, ncRNAs are proven as regulators of hematopoiesis and leukemogenesis [6]. Figure represents the most important ncRNA contributing in the differentiation of hematopoietic stem cells into functional blood cells [7,8].
on the development of mature CD8 + and CD4 + T cells, granulocytes or macrophages upon transplantation. Besides, early expression of miR-150 has obstructed the conversion of pro-B cells to the pre-B cell lineage. Taken together, miR-150 possibly inhibits expression of transcripts that have critical roles in development of pre-and pro-B cells [18]. miR-150 has been predicted to target c-Myb, a transcription factor governing numerous stages of lymphocyte expansion. miR-150 precisely regulates c-Myb expression to fundamentally influence lymphocyte development [19]. miR-125a is another miRNA whose role in enhancing the number of HSCs has been displayed in vivo. This process is completed via a specific inhibition of apoptosis in immature progenitors of this lineage, probably through regulation of expression of a number of pro-apoptotic gene targets among them is Bak1 which is directly targeted by miR-125a [20]. Table 1 sums up the results of investigations that appraised the role of miRNAs in HSC differentiation.

LncRNAs role in differentiation of HSCs
The function of lncRNAs in the differentiation of HSPCs has been investigated in numerous studies. LncRNAs can regulate expression of transcription factors which regulate hematopoiesis. Luo et al. have assessed lncRNA profile of HSCs by high throughput sequencing and recognized more than 300 unannotated lncRNAs. Comparison of expression of these lncRNAs in differentiated lineages has led to identification of 159 HSC-enriched lncRNAs (LncHSCs). Silencing of two LncHSCs has conferred specific impact on HSC self-renewal and lineage commitment possibly through modulation of principal hematopoietic transcription factor, namely E2A [41]. Expression of the transcription factor PU.1 has been controlled by an antisense lncRNA which is transcribed from the same locus namely PU.1-AS. This lncRNA has been shown to suppress PU.1 expression through regulating its translation [42]. Notably, others have described that high level of PU.1 is required for the development of macrophage compared with neutrophils [43]. Therefore, fine-tuning of PU.1 expression by its antisense transcript might define the lineage development. Paralkar et al. have identified more than 1000 polyadenylated lncRNAs expressed in erythroblastic cells, megakaryocytes, and megakaryocyte-erythroid precursor cells of mouse, and about 600 lncRNAs in human erythroblasts. The majority of these lncRNAs have been shown to be controlled by chief transcription factors including GATA1 and TAL1 [44]. Wagner et al. have reported over-expression of EGO in human bone marrow and in mature eosinophilic cells. This lncRNA has been shown to be transcribed from an intronic region of the ITPR1 gene. Stimulation of CD34 + hematopoietic progenitors with IL-5 has enhanced expression of EGO. EGO knock down has reduced expression of MBP and EDN in developing CD34 + hematopoietic progenitors [45]. HOTAIRM1 is another antisense transcript originating from the same CpG island that is around the initiation site of HOXA1 gene. HOTAIRM1 is the most noticeable intergenic RNA which is over-expressed in the course of induced granulocytic differentiation of hematopoietic cells. This lncRNA contributes in the myelopoiesis cia regulation of HOXA cluster [46]. Expression of Fas-AS1 has also been induced in the course of erythropoiesis via the activity of important erythroid transcription factors GATA-1 and KLF1. This lncRNA is inhibited by NF-κB. Besides, up-regulation of Fas-AS1 in HSPCs-originated erythroblasts has decreased surface levels of Fas and induced defense against Fas-mediated apoptosis [47]. LincRNA-EPS has a role in the erythroid differentiation as its suppression has blocked erythroid differentiation and enhanced apoptosis. This lncRNA has been shown to suppress expression of the pro-apoptotic gene Pycard [48]. Linc-MAF-4 is a chromatin-related lncRNA with specific expression in T helper 1 cells. Its expression has been inversely correlated with expression of the T helper 2-associated transcription factor MAF. Linc-MAF-4 silencing has twisted T cell differentiation to the T helper 2 route [49]. H19 is another lncRNA with critical role in the emergence of HSCs. Absence of H19 in the early developmental stages has suppressed endothelial-to-hematopoietic transition. Besides, H19 deficiency in pre-HSCs has resulted in promoter hypermethylation and simultaneous down-regulation of numerous important hematopoietic transcription factors, such as Runx1 and Spi1. The detected defects in the hematopoietic system following H19 deficiency has been attributed to the enhanced function of S-adenosylhomocysteine hydrolase, a controller of DNA methylation [50]. An animal study has indicated the role of Xist RNA in the suppression of hematologic cancer as deletion of this lcRNA in the blood of mice has resulted in initiation of an extremely aggressive myeloproliferative condition being described by a number of characteristics including myelofibrosis and leukemia. Deficiency of this   [81,89] lncRNA-THRIL TNF-and hnRNPL-related immunoregulatory lncRNA Macrophage/DC Regulates homeostasis and activation of inflammatory reaction; necessary for expression of inflammatory cytokines [90] lncRNA-PACER p50-associated COX-2 extragenic RNA Macrophage/DC Has an important role in decoy molecule in the NF-kB signaling pathway [91] lncRNA-NKILA NF-kB-interacting lncRNA Macrophage/DC [92,93] (continued on next page) S. Ghafouri-Fard et al. lncRNA in HSCs has resulted in abnormal maturation and age-dependent defects [51]. Dlk1-Gtl2 is another ncRNA with an important impact in inhibition of LT-HSCs. This locus contains a miRNA mega-cluster locus that inhibits the whole PI3K-mTOR pathway, suppressing mitochondrial synthetic processes and metabolic function and protecting LT-HSCs from reactive oxygen species (ROS) [52]. Table 2 reviews the investigations that assessed the role of lncRNAs in HSC differentiation.

Discussion
NcRNAs have critical regulatory functions in cell proliferation, programmed cell death, organ development, and differentiation. Both miRNAs and lncRNAs are important elements of the molecular pathways that regulate hematopoiesis. A number of these transcripts influence the expression of transcription factors that regulate differentiation of certain lines of hematopoietic cells. Few antisense transcripts have been identified that modulate expression of transcription factors in cis. Identification of other overlapping complementary transcripts with regulatory roles on the expression of transcription factors would facilitate clarification of molecular mechanisms of HSPCs differentiation. The majority of lncRNAs in the hematopoietic cells which have been identified through high throughput methods are unannotated, highlighting the prospect for novel discovery via investigating specialized cell kinds [44]. Several of lncRNAs which are extensively expressed during erythropoiesis have been shown to be controlled by critical erythroid transcription factors such as GATA1, TAL1, or KLF1 [53], revealing the mutual interactions between transcription factors and lncRNAs.
Notably, a vast body of literature about the contribution of ncRNAs in the differentiation of hematopoietic cells has come from the animal studies. Although these studies have provided invaluable clues about this subject, verification of their results in the human cells is a necessary step for implementations of these results in the clinical settings. Few comparative studies have demonstrated lack of conservation of hematopoietic cell-associated lncRNAs between mammalian species [44], signifying the importance of assessment of expression of these transcripts in each species.
Notably, exosomes originated from HSPCs have been shown to encompass ncRNAs, therefore transferring these transcripts to the recipient cells to modulate their function [23]. Exosome-mediated transfer of ncRNAs represents an important way of modulation of bone marrow microenvironment.
High throughput sequencing methods have shown significant differences in the miRNA profile between hematopoietic and nonhematopoietic cells. In addition, miRNA signature is slightly different within the hematopoietic group. Notably, completely differentiated effector cells and precursors at parallel stages of differentiation share miRNA pattern to a high extent. Therefore, miRNAs have critical functions during hematopoietic cell differentiation and in the process of maintenance of characteristics of different cells [100]. Some miRNAs have been shown to be specifically expressed in mature hematopoietic cells, but not their progenitors [19], thus regulating certain stages of development of hematopoietic cells. It is possible that miRNAs regulate the expression of only limited numbers of crucial target proteins in specific cellular settings [19]. Besides, miRNAs have a cell-stage-specific regulatory role in HSCs through which they control the stem cell bulk [20].
Manipulation of expression of these transcripts has functional significance in the treatment of cancers and in cell therapy. In vitro studies have shown the effects of silencing or over-expression of a number of ncRNAs in changing the differentiation process of hematopoietic cells, suggesting these methods as putative enrichment strategies before bone marrow transplantation.

Declaration of competing interest
The authors declare they have no conflict of interest. Regulates monocyte/macrophage differentiation [99] Th: lymphocyte T helper; Treg: lymphocyte T regulatory; NFAT1; nuclear factor of activated T-cells 1, MFAP4; microfibril associated protein 4, IFNs; interferon, STAT3: signal transducer and activator of transcription 3, DC: Dendritic cells, NFATs: nuclear factor of activated T cells.