Biosynthesis And Mechanism Of Micrornas

Biosynthesis and mechanism of microRNAs
In the early 1990s, Rosallnd Lee and colleagues found that lin-4, a gene was involved in the regulation of developmental timing of postembryonic in C. elegans, does not encode a protein but instead produce two small lin-4 transcripts that contain sequences complementary to a sequence in the 3'untranslated region(UTR) of lin-14 mRNA[1]. Since then, the function and mechanism of the tiny RNAs that is called microRNAs (miRNAs) now, which is highly conserved from animals to plants [2] , have been revealing gradually. miRNAs are small non-coding RNA molecules in the length of 19-25 nt that regulate gene expression by targeting mRNAs to inhibit translation of protein[3]. miRNA genes are transcribed by RNA polymerase II (pol II) to produce a primary-miRNA (pri-miRNA) that has long nucleotide sequences usually[4, 5]. Pri-miRNA is cropped into pre-miRNA which has a hairpin shape by nuclear RNase' Drosha[6]. The pre-miRNA , as the important intermediate of the maturation of miRNA, is transported to the out of the nucleus by exportin-5(Exp 5) (a member of the Ran-dependent nuclear transport receptor family)[7].In the cytoplasm, pre-miRNA is subsequently cleaved by Dicer to generate a miRNA[8].
MDS and its classification

MDS comprise a group of disorders of hematopoietic stem cell leading to ineffective hematopoiesis and propensity to develop acute myeloid leukemia(AML).The morphologic features of this disease include a hypercellular bone marrow with 'megaloblastoid changes,' atypical megakaryocytes, erythroid hyperplasia, defective maturation in the myeloid lineage, and increased blasts or ringed sideroblasts[13]. In 1982, French'American'British (FAB) Cooperative Group proposed a classification of myelodysplastic syndromes (MDS) based on morphological features in blood and bone marrow, including refractory anemia (RA), refractory anemia with ringed siderolasts (RARS), refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMML) [14]. In 2001, World Health Organization (WHO) proposed a new classification of MDS, based on the FAB classification, CMML and RAEB-T were removed and RAEB was split into refractory anemia with excess blasts-1(RAEB-1) and refractory anemia with excess lasts-2(RAEB-2). 5q- syndrome was separated out, and RA or RARS with 2 or 3 excess blasts were considered as a new group called refractory cytopenia with multilineage dysplasia (RCMD). In addition, myelodysplastic syndrome unclassified (MDS-U) is also a separated group [15]. In 2008, World Health Organization (WHO) published a new classification of MDS. A new subtype called refractory cytopenia with unilineage dysplasia (RCUD), consisting of RA, refractory neutropenia(RN), refractory thrombocytopenia(RT),was involving[16].
miRNAs have been implicated in tissue morphogenesis, cellular processes like apotosis, major signaling pathways [9].Hematopoiesis is an elegant developmental model to observe normal changes in miRNA expression and to identify the effects of miRNA on differentiation and tumorigenesis[10].In recent years, there are more and more researches focus on the relationship between microRNAs and hematological malignancies, including myelodysplastic syndromes (MDS) with certainty.
miRNAs expression profiles of MDS
miRNA expression profile is various in different diseases, there are unique miRNAs expression profiles in different subtypes MDS. Recent studies provide novel findings of unique miRNAs expression profile of MDS that form a basis for further investigations of diagnostic biomarkers of this disease.
1. MDS are usually diagnosed based on findings in BM and peripheral blood (PB), the latter is an important specimen in diagnosis due to its accessibility. Therefore, PONS et al. [] assessed the expression of these hematopoiesis-related miRNAs in bone marrow (BM) and peripheral blood (PB) of 25 patients with MDS. Twelve miRNAs were overexpressed in BM of MDS, six of which were also overexpressed in PB: miR-17-3p, miR-17-5p, miR-18a, miR-15a, and miR-21, and miR-142-3p. miR-15a in BM and miR-16 in PB were differentially expressed between low-risk and high-risk group. Expression of miR-181a and miR-222 was progressively increased from controls to early-stage MDS to advanced MDS to post-MDS AML, which provide a evidence that these miRNAs may contribute to leukemic transformation of MDS.
(Hematopoiesis-related microRNA expression in myelodysplastic Syndromes)

1. In another study [], miRNAs were collected from unsorted BM cells of 24 MDS patients and three control samples. By using low-density real-time PCR, this study revealed that MDS entities with distinct chromosomal aberrations have a different miRNA expression profile to that of the corresponding MDS entities without cytogenetic aberrations .For example, RA/RCMD with +8 expressed six downregulated miRNAs as compared with RA/RCMD with a normal karyotype. MDS patients with del(5q) also showed a distinct miRNA profile relative to RA/RCMD with a normal karyotype . Interestingly, several miRNAs not encoded on chromosome 5 were significantly up-regulated in MDS-del(5q).This special miRNA expression pattern was not found in '7 and +8. Thus, in contrast to isolated '7 or +8, the del(5q) aberration appears to influence increased expression of these miRNAs which are not encoded on the affected chromosomal region.(Aberrant microRNA expression pattern in myelodysplastic bone marrow cells)
2. Votavova, H et al [19] focused on differential expression of miRNAs in CD34+ cells of 5q- Syndrome. Twenty-one differently expressed miRNAs in 5q- patients compared to controls was identified by using microarrays. Of these miRNAs, miR-34a was markedly over-expressed, whereas miR-128b showed most significant decrease. Out of four miRNAs at del (5q), miR-143 and miR-145 expressions showed slight up-regulation and transcript levels of miR-378 and miR-146a were reduced. So the researchers thought that gene expression of the miRNAs in the deleted region is not significantly affected by the loss of one allele. (Differential Expression of MicroRNAs in CD34+ Cells of 5q- Syndrome) Interestingly, another study showed the opposite results that miR-145 was significantly lower in CD34+ cells of 5q- syndrome.[18]
3. To determine different miRNA expression between MDS subtypes, Merkerova D.M et al [94] analyzed miRNA expression in CD34+ cells of five types: 5q-syndrome, low-risk MDS, RAEB-1, RAEB-2 and AML evolved from MDS. They identified 45 differentially expressed miRNA between these five subtypes. Of note, a significant difference was observed between RAEB-1 and RAEB-2 expression patterns. The miRNA profile of RAEB-1 patients was more similar to controls and early MDS subtypes, whereas RAEB-2 profiles rather resembled those of secondary AML, suggesting transformation of the disease as early as between RAEB-1 and RAEB-2. For instance, expression of miR-422a, which correlated with disease progression, was lower in normal samples and in patients with early MDS than in patients with advanced MDS and post-MDS AML, with elevation of their expression level between RAEB-1 and RAEB-2. (Distinctive microRNA expression profiles in CD34+ bone marrow cells from patients with myelodysplastic syndrome)
4. By using miRNA microarray, expressions of 13 miRNAs were identified decreased in bone marrow mononuclear cells (BM-MNCs) of 10 MDS patients (7 RCMD, 1 RAEB-1 and 1 RA). RT-PCR further validated expression of 8 miRNAs was significantly decreased in paraffin embedded tissue samples of 24 MDS patients (19 RCMD, 2 RAEB-1 and 3 RAEB-2). c-Myb was predicted to be regulated by three different miRNAs (miR-103, miR-150, and miR-342). IHC studies on these paraffin-embedded samples demonstrated that c-Myb is overexpressed in MDS bone marrow biopsies. In addition, IHC studies also demonstrated that the Hedgehog pathway tumor suppressor Sufu, which is a potential target of miR-378, was overexpressed in MDS bone marrow biopsies compared to normal controls. Of note, the researchers used paraffin-embedded tissues as a valid source of miRNAs for the study of MDS, which may provide a basis of the use of this readily available tissue for future diagnostic purposes in MDS (Diagnostic microRNAs in myelodysplastic syndrome).
5. Mesenchymal stromal cells (MSC) are a small, nonhematopoietic, BM microenvironment cell population. Regarded as the osteoblastic progenitors, they are also a key components in the hematopoietic microenvironment['''''2]. Santamar??a et al [] firstly detected miRNAs expression in MSC from 21 low-risk MDS with the use of qPCR array. They found 159 down-regulated miRNAs in MDS samples compared to healthy controls. They further analyzed some miRNAs involved in hematopoiesis regulation by using real-time PCR, a decreased expression of miR-155, miR-181a and miR-222 in MSC from MDS patients was observed. In addition, the expression of DICER1 is down-regulated in MSC from MDS. DICER1 is a RNase III enzyme that play an important role in miRNA biogenesis. Together, all these findings suggested that the key machinery regulating microRNA biogenesis in MSC from MDS patients is impaired.(Impaired expression of DICER, DROSHA, SBDS and some microRNAs in mesenchymal stromal cells from myelodysplastic syndrome patients)
Given the differences in patient populations, samples, and discovery platforms, it is not surprising that there are differences among all these studies. Ultimately, it is hoped that miRNA expression profiles of MDS will provide new markers for the diagnosis of MDS.
The role of miRNAs in pathogenesis and progression of MDS
Array comparative genomic hybridization and traditional metaphase cytogenetics studies has identified numerous karyotypic and cryptic gains and losses in MDS. In addition, MDS has been associated with a global hypermethylation state. However, the mechanisms leading to MDS pathogenesis and disease progression remain to be fully elucidated. Recent studies try to revealing the relationship between miRNAs and different pathways, as well as other epigenetic regulation in MDS, which may provide a guide to better understanding of pathogenesis of MDS.
1'5q-syndrome '
5q-syndrome is a unique subtype of MDS, which is associated with an isolated deletion of chromosome arm 5q and characterized with
macrocytic anemia, variable neutropenia, and abnormal megakaryocytes associated with normal or slightly elevated platelets counts[17].There are several studies tried to explore the effect of miRNAs on 5q- syndrome phenotype. Early in 2002, a common deleted region (CDR) for the 5q-syndrome has been mapped that spans approximately 1.5 megabases, encompassing 40 protein-coding genes and some miRNAs genes['b''2].
a. Starczynowski, DT et al [18] analysis 13 miRNAs presented within CDR in bone marrow of six patients with 5q- syndrome. qRT-PCR revealed three miRNAs 'miR-143, miR-145and miR-146a'whose expression was significantly lower in MDS samples with del(5q) compared to MDS samples and controls with a normal karyotype. To determine whether miR-145 and miR-146a contribute to features of 5q' syndrome, they knocked down both two miRNAs in mouse HSPCs, and the loss of these two miRNAs in mouse results in hematopoietic abnormalities similar with 5q' syndrome, including hypolobated megakaryocytes and peripheral thrombocytosis. Moreover, two key genes of the innate immune response pathway, Toll-interluekin-1 receptor domaincontaining adaptor protein (TIRAP) and tumor necrosis factor receptor-associated factor-6 (TRAF6), are targeted by miR-145 and miR-146a, respectively. TIRAP interacts with TRAF6 and subsequently results in activation of nuclear factor-k B (NF- k B). Mouse that was transplanted with TRAF6-over-expressing marrow was showed to be associated with either bone marrow failure or AML. These findings proved that these two microRNAs have a pathogenic role in 5q' syndrome.
b. Ebert BL['''''3]have previously found that decreased expression of RPS14,causes the erythroid phenotype of the 5q-syndrome. RPS14 is a gene located in CDR and encoding a member of the 40S ribosomal subunit. But RPS14 deficiency does not explain the full clinical phenotype of the 5q- syndrome. Recently, a study [] found that patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1,which is a putative target of miR-145. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. (miR-145'RSP14'''''''''''5q-'''''''''')'
2) '''MDS'''''miRNA''''''

a.SHIP-1 is a lipid phosphatase to regulate the levels of phosphoinositides in phosphatidylinositol (PI) 30kinase signaling implicated as a critical pathway of cell survival control in epithelial and hematological malignancies[']. Lee DW et al []showed that tansient transfection of wild-style SHIP-1 increased the proportion of apoptotic THP-1 cells and decreased the number of colonies from primary AML cells compared with mutant SHIP-1 or vector alone, which proved SHIP-1 can act as a tumor suppressor in human myeloid disease. Further, they detected that SHIP-1 was consistently decreased in high-risk MDS, not in low-risk MDS, suggesting a disease-specific tumor suppressor activity. Levels of miR-210 and miR-155 transcripts, which target SHIP-1, were increased in CD34+ MDS cells compared with their normal counterparts. Direct binding of miR-210 to the 3'untranslated region of SHIP-1 was confirmed by luciferase reporter assay. Transfection of a myeloid cell line with miR-210 resulted in loss of SHIP-1 protein expression. Thus, the miR/SHIP/Akt pathway could serve as a clinical biomarker for disease progression, and miR-155 and miR-210 might serve as novel therapeutic targets in MDS.
b. Expression levels of miR10a and miR10b were significantly higher in CD34+ marrow cells from 28 patients with MDS than in CD34+ cells from healthy donors. miR10a/b levels in selected CD34+ MDS marrow cells showed a statistically highly significant association with TWIST-1 mRNA levels. Moreover, miR10a/b levels in the leukemia-derived cell line (KG1a) with TWIST-1 knockdown were also decreased. In two leukemia-derived cell lines'KG1a and PL-21, miRs10a/b were stably knocked down (KD); KD cells were co-cultured with HS5 stroma and exposed to TNF??. Apoptosis was significantly enhanced in KG1a KD and PL-21 KD cells. Thus, Expression of miRs10a/b is controlled by TWIST-1 and appears to control sensitivity to TNF'??induced (and stroma-dependent) apoptosis in clonal myeloid cells. These findings suggest a role for miR10 family members in the pathogenesis of MDS.
c. Bhagat TD et al. [] analyzed the 3'UTR of SMAD7 gene, product of which is a negative regulator of TGF-??receptor-I kinase, and determined that it contains a highly conserved putative binding site for miR-21. They observed significantly elevated levels of miR-21 in MDS marrow samples when compared with age matched controls. Dual luciferase activity was measured and showed decreased SMAD7 luciferase activity caused by expression of miR-21 compared with cells transfected with control mimics. This was cancelled by mutation in its binding site on the UTR, demonstrating a direct effect of miR-21 on the 3'UTR of SMAD7.Subsequently, they observed that inhibition of miR-21 led to an increase in SMAD7 protein expression in leukemic cell lines and in primary MDS patient samples. In contrast, overexpression of miR-21 led to decreased SMAD7 protein levels in leukemic cells and led to quantitative and qualitative reduction in erythroid colony formation from primary CD34+ cells [miR-21 mediates hematopoietic suppression in MDS by activating TGF-''signaling].

d. To explore the role of miR-22 in hematopoietic stem cell function and malignancy, scientists generated transgenic mice conditionally expressing miR-22 in the hematopoietic compartment. These mice displayed reduced levels of global 5-hydroxymethylcytosine (5-hmC) and increased hematopoietic stem cell self-renewal accompanied by defective differentiation. Conversely, miR-22 inhibition blocked proliferation in both mouse and human leukemic cells. Over time, miR-22 transgenic mice developed MDS and hematological malignancies. These results strongly suggest that miR-22 can enhance HSPC function and contribute to leukemia development in vivo. They also identified TET2 as a key target of miR-22 by using luciferase reporter assay, as well as real-time qPCR and western blot to reveal a marked reduction in levels of Tet2 mRNA and protein, respectively, in miR-22 transgenic mice. In addition, ectopic expression of TET2 suppressed the miR-22-induced phenotypes. Downregulation of TET2 protein is also correlated with miR-22 overexpression in MDS patients.
3) Mechanism of miR-125 family in myeloid transformation of MDS
Chromosomal translocations are frequent in MDS and AML, but the t (2; 11) (p21; q23) chromosomal translocation is a rare event. In addition, the t (2; 11) (p21; q23) translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34 + cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines [Myeloid cell differentiation arrest by miR-125b-1 in myelodysplasic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation]. In a vivo experiment, miR-125b 32Dclone3 cells produced an aggregate tumor burden greater than 1cm in diameter within 65 to 75 days in all subcutaneously injected nude mice but no tumors were observed following injection with control 32Dclone3 cells [MicroRNA-125b transforms myeloid cell lines by repressing multiple mRNA]. In another vivo experiment[MicroRNA miR-125b causes leukemia] ,all mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative neoplasm. Moreover, miR-125b is likely to transform cell lines by repressing multiple mRNA, which playing a key role in myeloid differentiation and apoptosis, such as CBFB, multiple genes involved in the p53 pathway. Therefore, overexpression of miR-125b may represent a new mechanism of myeloid cell transformation [MicroRNA-125b transforms myeloid cell lines by repressing multiple mRNA].
ASXL1 mutations (ASXL1-MTs) are common in patients with hematologic malignancies associated with myelodysplasia, including MDS, and chronic myelomonocytic leukemia ['''''5'8]. Recently, Inoue D et al. [] identified ASXL1-MT mice displayed features of human-associated MDS, including multilineage myelodysplasia, pancytopenia, and occasional progression to overt leukemia. Expression of miR-125a was increased in the murine myeloid cell line (32Dcl3 cells) expressing ASXL1-MT when compared with those expressing ASXL1-WT or the empty vector. They identified a recognition site of miR-125a in the 3'UTR of mouse Clec5a and 2 recognition sites in the 3'UTR of human Clec5a. When miR-125a was expressed in 32Dcl3 cells, Clec5a expression was reduced both at mRNA levels and protein expression levels compared with 32Dcl3 cells transduced with empty vector. Moreover, these cells became more resistant to G-CSF'induced differentiation. These results clearly demonstrate that miR-125a targets Clec5a expression, leading to the inhibition of differentiation, which may identify a new critical pathway for miR-125a-mediated myeloid transformation. Indeed, more in vivo studies are needed to confirm the effect of miR-125a in transformation of MDS to AML.
4) Role of hypermethylation of miRNA promoters in pathogenesis of MDS
MDS has been associated with a global hypermethylation state. ''MDS'''''''''''''''There are several researches try to revealing hypermethylation state of some MDS- associated miRNA.
By expressing EVI1 in murine BM cells, a mouse model of MDS was generated, which was characterized by dysplastic erythropoiesis and megakaryopoiesis, progressive pancytopenia, severe anemia, and BM failure['''''2]. It was reported that miR-124 expression inversely correlated with the degree of promoter methylation[''2''''58]. Methylation and silencing of miR-124 by EVI1 contributes to deregulation of cell division and self-renewal in murine BM cells and in the hematopoietic cell line 32Dcl3, which led to BM failure in the mice ultimately [Methylation and silencing of miRNA-124 by EVI1 and self-renewal exhaustion of hematopoietic stem cells in murine myelodysplastic syndrome]. Howerver, the mechanism of methylation of miR-124 by EVI1 in pathogenesis of MDS is still unclear.

miR-34b promoter hypermethylation was investigated directly in 112 AML patients, 28 MDS patients and 17 patients with juvenile myelomonocytic leukemia (JMML). MS-PCR and western blot analysis revealed that 66% AML patients had miR-34b promoter hypermethylation and high CREB protein levels, respectively, whereas preleukemic MDS and JMML were not associated with miR-34b hypermethylation or CREB overexpression. Primary BM samples from patients with MDS and the corresponding, transformed AML revealed that cells acquired miR-34b promoter hypermethylation during the evolution to AML. The inhibition of miR-34b levels in healthy BM and fetal liver cells promotes cell proliferation and clonogenic potential, leading to aberrant myelopoiesis. miR-34b overexpression suppressed tumor growth in vivo. In sum, hypermethylation of miR-34b induces overexpression of CREB, and a potential downstream mechanism was involved in the pathogenesis of AML transformation.
In another study, several intragenic miRNAs and their host genes showed similar expression patterns during myeloid maturation, suggesting their co-regulation. In addition, WWP2 (host gene for miR-140), EVL (host gene for miR-342) and ZNF207 (host gene for miR-632) showed significantly decreased in MDS, and these three miRNAs are also underexpressed in MDS. The methylation status of selected CpG loci in the promoters of miRNAs/host genes was further examined and increased promoter methylation of miR-140/WWP2, miR-378/PPARGC1B and miR-632/ZNF207 was found. Therefore, hypermethylation of several MDS-associated miRNA promoters might contribute to the down-regulation of MDS-associated miRNAs and their associated host genes. Moreover, treatment with DNA methyltransferase inhibitors (DNMTIs) appears to result in normalization of the methylation at these sites.
These studies identify a role of hypermethylation of miRNA promoters in the down-regulation of MDS-associated miRNAs, unifying research on miRNAs in MDS and epigenetic regulation in MDS into a common pathway.
MiRNAS associated with prognosis and treatment in MDS
The International Prognostic Scoring System (IPSS) relies on the the number of cytopenias, cytogenetic profile, and the percentage blasts in the bone marrow to group patients with MDS into one of 4 prognostic categories: low risk(LR), intermediate 1 risk(IN-1), intermediate 2 risk(IN-2), and high risk(HR). This prognostic-scoring system provides a useful method for predicting survival and guiding therapeutic management in MDS patients [46]. In recent years many efforts have been made to improve the efficiency of this prognostic-scoring system in MDS. There are different miRNAs described as reliable prognostic biomarkers for risk-stratification and management of MDS.
In the bone marrow mononuclear cells of forty-four MDS patients, four members of miR-181 family( miR-181a, miR-181b, miR-181c, miR-181d) were up-regulated in patients with higher risk (INT-2/High) MDS compared to those of patients with lower risk (INT-1/Low) MDS. In patients with lower risk MDS, median survival time was longer in patients with low expression of miR-181 family than in patients with high expression of these miRNAs[47]. Recently, a few studies also showed that increased miR-181b-5p/a expression levels were found to linked with a poor overall survival(OS) outcome in patients with AML [63, 64]. Interestingly, increased expression of miR-181 is significantly associated with favorable outcome in cytogenetically abnormal AML (CA-AML) patients [48, 49, 62]. The miR-181 family plays a key role in the negative regulation of differentiation of all hematopoietic lineages [50].
The elevation of miR-155 expression has been validated correlated with poor prognosis in hematological malignancies including MDS [47, 56, 57, 58, 59]. Recently, a study showed loss of SHIP-1 protein expression in high-risk myelodysplastic syndromes was associated with miR-210 and miR-155, so that miR-210/SHIP-1/Akt pathways could serve as clinical biomarkers for disease progression [61]. Additional, An aggressive malignant lymphoproliferation, with organomegaly and lymphadenopathy and similar to Burkitt lymphoma, was observed in miR-155 transgenic mice [55].
Besides aforementioned two miRNAs, there are a few other miRNAs predicts prognosis of MDS. Zuo, Z et al. [65] showed that circulating let-7a predicts OS and progression-free survival (PFS) in MDS patients, and patients with low circulating let-7a level have better OS than patients with high circulating let-7a level. However, the conclusion of another study runs counter to aforementioned conclusion. This discrepancy could be attributed to different samples they used. The former measured let-7a expression levels in CD34+ cells, whereas the latter measured let-7a expression levels in plasma [66]. Recently, a study showed patients with let-7a-3 over-expression had significantly shorter OS than those without let-7a-3 over-expression among 51 AML patients obtained CR. That is to say let-7a-3 over-expression is associated with poor clinical outcome in AML [70].Certainly, more studies are needed to confirm the prognostic value of let-7a in MDS. Previous studies have proved that let-7a is associated with pathogenesis of hematological

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