Ann Hematol (2010) 89:953958 DOI 10.

1007/s00277-010-0980-7

ORIGINAL ARTICLE

Expression of microRNA-451 in normal and thalassemic erythropoiesis

Saovaros Svasti & Shizuka Masaki & Tipparat Penglong & Yasunobu Abe & Pranee Winichagoon & Suthat Fucharoen & Tsukuru Umemura

Received: 22 November 2009 / Accepted: 26 April 2010 / Published online: 12 May 2010 # Springer-Verlag 2010

Abstract MicroRNAs (miRNAs) are negative regulators of gene expression that play an important role in hematopoiesis. Thalassemia, a defective globin synthesis leading to precipitate of excess unbound globins in red blood cell precursors, results in defective erythroid precursors and ineffective erythropoiesis. Expression pattern of miR-451, an erythroid-specific miRNA, was analyzed during differentiation of erythroid progenitors derived from normal and thalassemic peripheral blood CD34-positive cells, after 14 days of culture. A biphasic expression with transient up-regulation of miRNA-451 on day 3 of cultures was observed during thalassemic erythroid differentiation. In contrast, the expression pattern of the miR-451 in erythroid cells obtained from the other extravascular hemolytic

anemia, i.e., hereditary spherocytosis patients showed no transient up-regulation of miR-451 on day 3 of cultures. Our results suggest that early erythroid progenitors in thalassemia have a dysregulated miRNA-451 expression program, and analysis of microRNA is a relevant approach to determine abnormalities of erythropoiesis. Keywords Thalassemia . miRNA . Erythropoiesis

Introduction -Thalassemia occurs from defective -globin synthesis leading to globin chain imbalance and accumulation of free -globins in erythroid cells. This results in chronic anemia secondary to short red cell survival and ineffective erythropoiesis [1]. In comparison to normal subjects, individuals with -thalassemia major showed marked erythroid hyperplasia, and the number of polychromatophilic erythroblasts was higher than the number of orthochromatic erythroblasts [2, 3]. This is because of the existence of strong stimulation to erythroid progenitors, which leads to ineffective erythropoiesis caused by accelerated erythroid expansion [4]. MicroRNAs (miRNAs) are functional RNAs transcribed from non-coding regions of the genome. These miRNAs play important roles in eukaryotic gene regulation, especially in development, differentiation, and cell death [5, 6]. The miRNAs are negative regulators, which suppress the gene functions through translational repression by targeting 3-UTR in messenger RNAs (mRNAs) of protein-coding genes or by inducing instability of mRNAs. The miRNAs are dynamically regulated during hematopoiesis and are controlling normal human hematopoiesis and lineage commitment [7, 8].

No substantial overlapping with previous papers. S. Svasti : T. Penglong : P. Winichagoon : S. Fucharoen Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakornpathom, Thailand S. Svasti Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand S. Masaki : T. Umemura (*) Department of Health Sciences, Faculty of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812 8582, Japan e-mail: ume@shs.kyushu-u.ac.jp Y. Abe Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan

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During erythropoiesis, a complex multistep process encompassing proliferation and differentiation of hematopoietic stem cells to mature erythrocytes, numerous miRNAs are induced or repressed. miR-451 was previously reported to be up-regulated during erythroid maturation with a lineage-specific manner and that miR155 was down-regulated during the early erythropoiesis [9, 10]. In this study, the expression pattern of miRNA451 during erythroid differentiation of CD34 + cells obtained from thalassemia peripheral blood culture was assessed.

Liquid cultures of human erythroid colony-forming cells Erythroid colony-forming cells were prepared as described previously [14]. Briefly, mononuclear cells from peripheral blood were isolated by density gradient centrifugation using Lymphoprep (density 1.0770.001 g/mL, Axis-Shield Poc AS, Norway). After positive selection was performed using anti-CD34 immunomagnetic bead (Miltenyi Biotech, Auburn, CA, USA), the cells were cultured in Iscoves modified Dulbeccos media (GIBCO-Invitrogen, Grand Island, NY, USA) containing 15% heat-inactivated fetal bovine serum (GIBCO-Invitrogen), 15% human AB serum, 2 U/mL erythropoietin (EPREX, Brussels, Belgium), 20 ng/ mL stem cell factor (Promokine, Heidelberg, Germany), and 10 ng/mL IL-3 (Promokine) and incubated in 37C, 5% CO2, and 95% humidity. On days 3, 7, and 10, the media were changed, and the cells were further incubated under the same conditions, without IL-3. Analysis of miRNA expression Each sample containing 5105 cells was sequentially obtained from the liquid cultures on days 3, 5, 7, 9, 12, and 14. After washing with phosphate-buffered saline, the cells were suspended in RNAlater (Applied Biosystems, Foster City, CA, USA) and were stored at 20C. Total RNAs were extracted from cell pellets using the mirVana miRNA Isolation Kit (Applied Biosystems) according to the manufacturer s instructions. The expression of miR-451 and let-7a was measured by qRT-PCR. Briefly, miRNAs were transcribed to cDNAs using a High-Capacity cDNA Archive Kit (Applied Biosystems). Looped RTprimers specific for each microRNA were added according to the manufacturer s instructions (TaqMan MicroRNA Assays Kit, Applied Biosystems). For detecting miRNAs, amplification by real-time PCR was done using TaqMan Micro-

Design and methods Subjects A total of 12 individuals comprised of three normal, seven thalassemia (five -thalassemia/Hb E, one HbH disease, and one HbH-CS disease), and two hereditary spherocytosis patients were examined (Table 1). The classification of patients was done by hematological parameters, clinical data and genotyping. The study was approved by the Institutional Review Boards of Mahidol University (Bangkok, Thailand), and informed consent was obtained from each participant. Complete blood counts and erythrocyte indices from peripheral blood samples were determined using an automated cell counter (ADVIA210, Bayer, Tarrytown, NY, USA). Hemoglobin typing was analyzed by the automatic HPLC system (VARIANT, BioRad, Hercules, CA, USA). DNA analysis to confirm the double heterozygous state for 0-thalassemia and Hb E alleles in all cases was done by reverse dot blot hybridization [11]. For -thalassemic patients, genotyping of -thalassemia 1 and -thalassemia 2, Hb constant spring (Hb CS) mutations were identified by PCR-based methods [12, 13].

Table 1 Characteristics of normal subjects and thalassemic patients

Case no. 1 2 3 4 5 6 7 8 9 10 11 12

Genotype Normal Normal Normal -Thalassemia/Hb E (non-splenectomy) -Thalassemia/Hb E (non-splenectomy) -Thalassemia/Hb E (splenectomy) -Thalassemia/Hb E (splenectomy) -Thalassemia/Hb E (splenectomy) Hb H Hb H-CS Hereditary spherocytosis (splenectomy) Hereditary spherocytosis

Age (year) 26 29 26 25 27 43 37 31 35 60 40 33

Sex F F M F M F F M F F M F

Hb (g/dL) 11.4 13.3 16.9 5.2 6.6 6.2 6.6 5.8 8.6 8.1 9.7 8.1

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RNA Assay probe (Applied Biosystems), Universal PCR Master Mix (Applied Biosystems), and Thermal Cycler Dice (Takara, Shiga, Japan). The relative amount of each miRNA was calculated against the amount on day 7 and Ct calculated using the comparative Ct 2 method. The experiments were carried out in triplicate. Analysis of erythroid-specific mRNA expression Quantitative analysis of -globin, glycophorin-A, and GATA-1 mRNA was performed. The cDNAs were synthesized using the RNA PCR Kit (Takara). The amplification of cDNAs by the qRT-PCR method was done using SYBR Premix Ex Taq (Takara), with -globin: forward primer, 5 GCCCTGGAGAGGATGTTC-3 , reverse primer, 5 AGGGTCACCAGCAGGCAGT-3, glycophorin-A: forward primer, 5-TGGCAATGCACACTTCAACT-3, reverse primer, 5-AGCTCTAGGAGTGGCTGCAT-3, GATA-1: forward primer, 5-TCAATTCAGCAGCCTATTCC-3, reverse primer, 5-TTCGAGTCTGAATACCATCC-3, GATA-2: forward primer, 5-TGTTGTGCAAATTGTCAGACG-3, reverse primer, 5-CATAGGTGCCATGTGTCCAGC-3, -actin: forward primer, 5-AAGAGCTACGAGCTGCCTGA-3, reverse primer, 5-GGCAGTGATCTCCTTCTGCA-3. The amount of the mRNAs expression was normalized with the level of -actin expression. The experiments were carried out in triplicate.

Morphological analysis of in vitro differentiating CD34+ cells from a normal subject and a -thalassemia/Hb E patient is shown in Fig. 1b. On day 3 of cultures, both normal and thalassemic erythroid cell consisted predominantly of progenitor cells. Maturation delay was observed in thalassemic erythroid cells when compared to normal erythroids on days 59. A predominant population of orthochromatic erythroblasts and terminally enucleated RBCs were seen on days 12 and 14 respectively in normal and thalassemia. Expression of miR-451 To evaluate the changes in RNA content during the erythroid differentiation process, let-7a, which is a miRNA ubiquitously expressed in cells of various lineages, was analyzed (Fig. 2a). The expression of let-7a was not different between normal subjects and -thalassemia/Hb E patients. Therefore, expression of miR-451 in normal and thalassemic erythroid cultures was quantitated by RT-qPCR using let-7a as a normalizer. In normal subjects, the expression of miRNA-451 was up-regulated during erythroid differentiation (Fig. 2b), which was consistent with the previous reports [9, 10, 15]. The same expression pattern of miR-451 was observed using U6 as a normalizer, although the expression of U6 was more variable during erythroid differentiation [9]. Surprisingly, a biphasic expression with transient upregulation of miRNA-451 on day 3 was observed during erythroid differentiation in all thalassemic erythroid cells, except one who has very mild clinical symptom (Fig. 2b). The relative level of miR-451 on day 3 compared with the level on day 7 in thalassemia (3.32) was higher than that of normal subjects (0.055) (p <0.04). The level of miR-451 was lowest on day 7, then it was up-regulated with the pattern similar to that of normal subjects during erythroid differentiation. The relative expression of miR-451 on

Results Cellular differentiation in the erythroid cultures Purified CD34+ erythroid progenitors were cultured in the liquid culture system containing SCF and Epo. Cell numbers increased 40120-folds by day 12 of cultures (Fig. 1a).

A
1400

B
percentage of cell (%) percentage of cell (%)

cell number (x105 cells)

1200 1000 800 600 400 200 0 2

/E normal

100 80 60 40 20 0 3 5

normal
100 80 60 40 20 0 Baso E 3

-thalassemia/Hb E

8 day

10

12

14

9 day

12 14 ProE

LargePro

Poly E

9 12 14 day E Ortho E

Fig. 1 Proliferation and morphological changes of cells in the liquid culture system. The viable cells were measured after staining using the Trypan blue exclusion method. a Morphological analysis of cultured cells was done after the staining of cytospin preparations with Giemsa

solution. b LargePro E large proerythroblast, Pro E proerythroblasts, Baso E basophilic erythroblasts, Poly E polychromatophilic erythroblasts, Ortho E orthochromatophilic erythroblasts, E enucleated red blood cell

956 Fig. 2 Expression of miR-451 during erythroid differentiation. a Expression of let-7a was expressed as relative values to the level on day 12. b Expression of miR-451 was normalized by the expression of let-7a. The experiments were carried out in triplicate. Plots for miR-451 shows a relative value to an expression level on day 7 of normal subjects (n =3, black), thalassemia (n =7, red). c The relative expression of miR-451 on day 3 compared to day 7 in -thalassemia/HbE (/E) and hemoglobin H disease (Hb H). d Expression of miR-451 in hereditary spherocytosis (n=2, black) and normal subjects (n=2, gray)

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50 40 30 20 10 0 2 4 6 8 10 days 12 14

B
100 10 1 0.1 0.01
2 4 6

days

10

12

14

C
10
miR-451 day 3/day 7 (fold) miR-451 (fold/day 7)

D
100 10 1 0.1 8 6 4 2 0 -2 -4 normal
/E
normal (n=3) HS (n=2)

0.01 Hb H 2 4 6 8 days 10 12 14

day 3 compared to day 7 in -thalassemia/Hb E and Hb H disease patients was 4.55- and 2.42-folds, respectively, which was much higher than the normal subjects (p <0.03 for each by t test) (Fig. 2c). Moreover, the result showed that relative expression of miR-451 in splenectomized -thalassemia/Hb E patients was higher than that of the non-splenectomized patients, 3.886.73 and 0.183.17, respectively. Interestingly, the ratios are found to be inversely correlated to Hb levels (r value 0.637, p <0.05), but directly correlated to absolute numbers of circulating reticulocytes (r value 0.762, p <0.05, data not shown). To evaluate the influence of accelerated erythropoiesis due to chronic hemolytic anemia on the early up-regulation of miR-451 expression, miR-451 levels in erythroid progenitors from two hereditary spherocytosis patients were examined (Fig. 2d). The results showed that no transient up-regulation of miR-451 on day 3 of cultures was observed in other hemolytic anemia. Expression of erythropoiesis-specific genes As miR-451 was previously reported to be up-regulated during erythroid maturation, the expression of erythroidspecific genes, -globin, and glycophorin-A in normal and -thalassemia/Hb E was compared. The results showed that the expression pattern in thalassemic patients was not different from that of normal (Fig. 3a). The three genes express at very low levels in early erythroid precursors and increased with erythroid maturation. Moreover, the expression of the three genes detected in day 3 cells from thalassemia/Hb E was low and not significant different

from normal, indicating that there was no contamination from more mature erythroid cells in the cells culture. Upregulation of GATA-1 is observed in normal and thalassemia cultures (Fig. 3b). Increment in thalassemia was significantly greater than in normal subjects (4.65 fold and 3.30 fold, respectively, p <0.02). On the other hand, GATA-2 was less down-regulated in thalassemia than in normal (3.42 fold and 7.98 fold, respectively, p <0.0025).

Discussion miR-451 is one of key molecules regulating erythroid maturation [10, 16, 17]. In normal hematopoiesis, miR-451 is lineage specifically up-regulated during normal erythroid differentiation [9, 10, 16]. The content of miR-451 in an erythrocyte is about 10,000 fold higher than that of a granulocyte [9]. In this study, we observed the biphasic up-regulation of miR-451 in cultures of purified erythroid progenitors from thalassemic patients: moderate up-regulation on day 3 of culture and drastic up-regulation on days 12 to 14. In cultures of normal progenitors, there was no up-regulation of miR-451 on day 3. The absence of -globin, glycophorin-A, and GATA1 transcripts excluded the possibility that detection of miR-451 on day 3 was due to the contaminated mature erythroid cells. The expression of miR-451 was higher in patients with less hemoglobin level and increased reticulocytosis. Thus, up-regulation of miR451 on day 3 culture was correlated with abnormal erythropoiesis and disease severity in thalassemia.

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a
1.8 1.6
glycophorin A (fold)

b
60 50 40 30 20 10
-globin (fold)

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0

7 day

12

Fig. 3 Time course expression of erythroid-specific genes. a Glycophorin-A (Gly A), and -globin mRNA in -thalassemia/Hb E (/E) and normal (N) subjects during erythroid cell culture. Expression of glycophorin-A (solid lines: blue normal, red /E), and -globin (broken lines: blue normal, red /E). was normalized by the expression of -actin. b Expressions of GATA-1 and GATA-2 in -thalassemia/Hb

E (/E) (n =2) and normal (N) subjects (n =2) during erythroid cell culture are shown as fold changes: GATA-1, the level on day 3 as 1 (solid lines: black normal, red /E); GATA-2, the level on day 9 as 1 (broken lines: black normal, red /E). The expression levels were normalized by -actin. Each values are means standard deviations

Thalassemia is a complex of hemolytic disorders caused by several different mechanisms. Increase in apoptosis is one of characteristics of thalassemia, resulting more basophilic erythroblasts observed [24]. However, our previous data have shown that differentiated erythroid cells express more miR-451 [9]. Thus, the delay in the maturation of erythroid cells in thalassemia does not explain the biphasic up-regulation of miR-451 in thalassemia cultures. Analysis of the miR-451 expression during erythroid differentiation in cultures using erythroid progenitors purified from two patients with the other extravascular hemolytic anemia, hereditary spherocytosis, showed no increasing in miR-451 expression on day 3. Therefore, erythropoietic stress alone may not explain the mechanism of miR-451 up-regulation in early erythroid progenitor of thalassemia. In sickle cell anemia, circulating red blood cells contain higher levels of miR-451 than in normal [18]. Polycythemia vera, a myeloproliferative disorder which also has the increased erythropoiesis, shows biphasic upregulations of several erythropoiesis-related genes during erythroid differentiation [15]. This study did not examine miRNA expression in erythroid progenitors. Thus, this is the first report to show biphasic regulation of miR-451 in abnormal erythropoiesis. Under hematopoietic stress such as accelerated erythropoiesis, genetic regulation of erythropoiesis changes, for example, enhanced fetal hemoglobin production [19]. In the disease, high persistence of fetal hemoglobin, miR-210, is involved to induce high expression of -globin gene [20]. Abnormal expressions of late erythroid gene in thalassemic erythroid progenitor cells have been observed. Glycophorin-A, a late erythroid lineage-specific protein not normally expressed on CD34+ cells, are expressed in about

20% of -thalassemic BM CD34+ cells compared to 1% of normal BM CD34+ cells [3]. Our observation suggests that the biphasic up-regulation of miR-451 in early erythroid differentiation may be another abnormality specific to thalassemic diseases. Transcription factors GATA-1 and GATA-2 are required for normal hematopoiesis. The two genes have distinct but overlapping patterns of expression in erythropoiesis. GATA-2 is expressed at relatively high levels in early precursors and is gradually replaced by GATA-1 during later stages of maturation [21]. Several groups reported that there is a possibility that the cross talk between miR-144/ 451 and GATA-1/GATA-2 may regulate the expression of miR-451 as well as erythroid-specific genes [16, 17]. Although our observations suggested that there is no direct linkage between expression of GATA-1 and miR-451 on day 3, there is a possibility that more effect of GATA-1 may have some role for up-regulation of miR-451, in which gene has a GATA-1 binding site in the 5 side [16, 17], in the early stage of erythropoiesis in thalassemia. The target gene(s) of miR-451 is still not clear. MDR-1 gene has been shown to be negatively regulated by miR-451 in cancer cells [22]. Other potential miR-451 target mRNAs in erythroid cells have been predicted, which mainly encode for nuclear proteins [16]. Microarray and bioinformatic analysis by Bandreas et al. suggested that macrophage inhibitory factor is a potential target, resulting in suppression of cancer cell proliferation [23]. Tsuchiya et al. reported that miR-451 together with miR-338-3p promotes development of epithelial cell polarity [24]. Thus, miR-451 may function to promote cell differentiation and to suppress cell growth. Although its function in the thalassemic erythroid progenitor cells remains to be determined, analysis of the

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Ann Hematol (2010) 89:953958 human erythropoiesis. Biochem Biophys Res Commun 364:509 514 Zhan M, Miller CP, Papayannopoulou T, Stamatoyannopoulos G, Song CZ (2007) MicroRNA expression dynamics during murine and human erythroid differentiation. Exp Hematol 35:1015 1025 Winichagoon P, Saechan V, Sripanich R, Nopparatana C, Kanokpongsakdi S, Maggio A, Fucharoen S (1999) Prenatal diagnosis of -thalassaemia by reverse dot-blot hybridization. Prenat Diagn 19:428435 Fucharoen S, Fucharoen G, Fukumaki Y (1990) Simple nonradioactive method for detecting haemoglobin Constant Spring gene. Lancet 335:1527 Chong SS, Boehm CD, Cutting GR, Higgs DR (2000) Simplified multiplex-PCR diagnosis of common Southeast Asian deletional determinants of -thalassemia. Clin Chem 46:16921695 Dai CH, Krantz SB, Zsebo KM (1991) Human burst-forming units-erythroid need direct interaction with stem cell factor for further development. Blood 78:24932497 Bruchova H, Yoon D, Agarwal AM, Mendell J, Prchal JT (2007) Regulated expression of microRNAs in normal and polycythemia vera erythropoiesis. Exp Hematol 35:16571667 Dore LC, Amigo JD, Dos Santos CO et al (2008) A GATA-1regulated microRNA locus essential for erythropoiesis. Proc Natl Acad Sci USA 105:33333338 Pase L, Layton JE, Kloosterman WP, Carradice D, Waterhouse PM, Lieschke GJ (2009) miR-451 regulates zebrafish erythroid maturation in vivo via its target GATA2. Blood 113:17941804 Chen SY, Wang Y, Telen MJ, Chi JT (2008) The genomic analysis of erythrocyte microRNA expression in sickle cell diseases. PLoS ONE 3:e2360 Rees DC, Porter JB, Clegg JB, Weatherall DJ (1999) Why are hemoglobin F levels increased in HbE/-thalassemia. Blood 94:31993204 Bianchi N, Zuccato C, Lampronti I, Borgatti M, Gambari R (2009) Expression of miR-210 during erythroid differentiation and induction of -globin gene expression. BMB Rep 42:493 499 Anguita E, Hughes J, Heyworth C, Blobel GA, Wood WG, Higgs DR (2004) Globin gene activation during haemopoiesis is driven by protein complexes nucleated by GATA-1 and GATA-2. EMBO J 23:28412852 Zhu H, Wu H, Liu X, Evans BR, Medina DJ, Liu CG, Yang JM (2008) Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells. Biochem Pharmacol 76:582588 Bandres E, Bitarte N, Arias F et al (2009) microRNA-451 regulates macrophage migration inhibitory factor production and proliferation of gastrointestinal cancer cells. Clin Cancer Res 15:22812290 Tsuchiya S, Oku M, Imanaka Y et al (2009) MicroRNA-338-3p and microRNA-451 contribute to the formation of basolateral polarity in epithelial cells. Nucleic Acids Res 37:38213827

transient up-regulation of miR-451 in the early phase of erythroid differentiation is relevant to understand the abnormality of hematopoietic progenitors in thalassemia.
Acknowledgments This study was supported in part by National Science and Technology Development Agency, Thailand (NSTDA), Mahidol University Research Grant, Japan Society for the Promotion of Science (JSPS), and the National Research Council of Thailand (NRCT). This work was supported in part by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Exploratory Research, 2009, 21659240. Contributions SS contributed to the study design, interpretation of the data, drafting, and editing of the manuscript. SM and TP performed the research and analyzed the data. YA, PW, and SF were responsible for study design and specimen collection. TU contributed to the design, concept of the study, and editing of the manuscript. Final approval of the version to be published was made by all authors.

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15. Conflict of interest None. 16.

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