Overexpression of mir-708 and Its Targets in the Childhood Common Precursor B-Cell ALL

Transcription

1 Overexpression of mir-708 and Its Targets in the Childhood Common Precursor B-Cell ALL Pediatr Blood Cancer 2013;60: Xue Li, MMed, 1 Dong Li, PhD, 2 Yong Zhuang, MMed, 1 Qing Shi, BSc, 2 Wei Wei, MMed, 1 and Xiuli Ju, MD, PhD 1 * Background. The critical function of micrornas in the pathogenesis and prognosis of hematopoietic cancer has become increasingly apparent. However, only a few mirnas have been reported to be altered in acute lymphocytic leukemia (ALL). Procedures. To uncover aberrantly expressed mirnas in pediatric B-cell ALL, our study employed genome-wide mirna microarray analysis and stem-loop real-time quantitative polymerase chain reaction (qrt-pcr) to examine common precursor B-cell ALL samples. The target genes of mirna-708 were then identified and verified by bioinformatics, dual-luciferase reporter assay, qrt-pcr, and Western blot. Results. Significant upregulation of mir-708, mir- 210, and mir-181b, and downregulation of mir-345 and mir-27a were observed in common precursor B-cell ALL (common-all) samples (P < 0.05). In addition, elevated expression of mir-708 and mir-181b were found in high-risk common-all compared to standard and intermediate ones. mir-708 inhibited luciferase reporter activity by binding to the 3 -untranslated regions (3 - UTRs) of CNTFR, NNAT, and GNG12 mrna in HEK-293 cell line and suppressed the protein levels of CNTFR, NNAT, and GNG12 in Jurkat cells. In addition, mrna levels of CNTFR and NNAT, but not of GNG12, were found to be downregulated in high risk common-all samples. Mutational analysis revealed that mir-708 binds to the bp sequence region of the 3 -UTR of CNTFR mrna. Conclusion. The expression level of mir-708 reflects differences among the clinical types of common-all, and CNTFR, NNAT, and GNG12 were identified as targets of mir-708. Pediatr Blood Cancer 2013;60: # 2013 Wiley Periodicals, Inc. Key words: mirna; pediatric acute lymphoblastic leukemia; regulatory region; target gene INTRODUCTION Acute lymphocytic leukemia is the most common leukemia subtype observed in children, and approximately three-quarters of patients with precursor B-cell ALL exhibit the common precursor B-cell immunophenotype. With effective therapeutic schedules, most patients can achieve remission from ALL and nearly 80% of these patients are eventually cured [1]. However, high-risk ALL is not highly responsive to chemotherapy, because of which leukemic cells, although undetectable, are not completely eradicated after treatment [2,3]. As such, biochemical markers with altered expressions that may be utilized for risk stratification should be identified to give more chances to cure clinical high-risk ALL. MicroRNAs (mirnas, mir) are a class of small noncoding RNAs that modulate gene expression via base-pairing to sites in the 3 -untranslated region (3 UTR) based on partial mirna/mrna complementarity. Normal levels of mirnas are accurately regulated in cells to guarantee proper cellular function and differentiation [4]. Dysregulation of mirna can be observed in several pathological processes, including oncogenesis. Identification of target genes of mirnas involved in cancer has revealed that mirnas have pivotal roles in cancer initiation, progression, and metastasis [5]. Oncogenes or tumor suppressors regulate the expression of specific mirnas [6]. Targeting oncogenic micro- RNAs is thus a promising strategy for cancer therapy. The involvement of mirnas in hematological malignancies has been recently confirmed. The first report linking mirna and cancer showed the downregulation of mir-15 and mir-16 in chronic lymphocytic leukemia [7]. Tumors can become addicted to oncogenic micrornas, as shown in an in vivo model of microrna-21-induced B-cell lymphoma [8]. mirnas can also exert cooperative effects on genes, pathways, and processes involved in tumor suppression. A small set of mirnas, including mir-19b, mir-20a, mir-26a, mir-92, and mir-223, produce overlapping and cooperative effects on tumor suppressor genes implicated in the pathogenesis of T-cell ALL [9]. Although many mirnas have been confirmed to be dysregulated in childhood C 2013 Wiley Periodicals, Inc. DOI /pbc Published online 23 August 2013 in Wiley Online Library (wileyonlinelibrary.com). ALL, further research about ALL-related mirnas and their targets may help us better understand the pathogenesis of this disease [10,11]. In the present study, we employed microarray analysis to determine the aberrant expression of mirna in pediatric common precursor B-cell ALL samples. Then, we selected 11 markedly dysregulated mirnas for validation by stem-loop RT-PCR. Finally, we identified the downstream targets of the highly upregulated mir-708. Our report demonstrates the important functions of specific mirnas in pediatric common precursor B-cell ALL and highlights candidate mirna/mrna interactions that may drive the formation or progression of common precursor B-cell ALL. METHODS Samples Bone marrow and peripheral blood were obtained from children newly diagnosed with common precursor B-cell ALL (CD10 positive and no surface or cytoplasmic immunoglobulin) at Qilu Hospital, Jinan, China. Monocytes were isolated from these 1 Department of Pediatrics, Cryomedicine Laboratory, Qilu Hospital, Shandong University, Shandong, PR China; 2 Cryomedicine Laboratory, Qilu Hospital, Shandong University, Shandong, PR China Grant sponsor: Shandong Province Natural Science Foundation; Grant numbers: ZR2011HM007; 2010G ; Grant sponsor: Major State Basic Research Development Program; Grant number: 2012CB966504& ; Grant sponsor: Innovation Fund Project of Shandong University; Grant number: 2012ZD023 Conflict of interest: Nothing to declare. Correspondence to: Xiuli Ju, Department of Pediatrics, Cryomedicine Laboratory, Shandong University, Qilu Hospital, Jinan, Shandong , China. shellysdcn07@gmail.com Received 8 January 2013; Accepted 7 April 2013

2 mir-708 and Its Targets in Childhood B-ALL 2061 samples in lymphocyte separation medium and stored at 80 C until use. The patients were diagnosed using morphological, immunological, cytogenetic, and molecular standards, and treated according to the Chinese Children s Leukemia Group 2008 (CCLG-2008) protocol [12]. Risk stratification of ALL patients was also done following the CCLG-2008 protocol. Patient characteristics are presented in detail in Table I. As controls, bone marrow was obtained from consenting agematched individuals (n ¼ 5) undergoing orthopedic surgery without tumor or antecedent primary hematologic abnormalities. ALL samples were collected after approval of the institutional review board and provision of informed consent by parents or legal guardians. Cell Cultures Human embryonic kidney cell line (HEK-293 cells) was cultured in High-glucose DMEM medium (Gibco, GD) with 10% fetal bovine serum (Gibco) at 37 C in a 5% CO 2 atmosphere. Jurkat (human T-ALL cell line) cells were cultured in RPMI-1640 medium (Gibco) with 10% FBS (Gibco) at 37 C in a 5% CO 2 atmosphere. RNA Isolation and mirna Microarray Profiling Total RNA, including mirna, was extracted from 34 common precursor B-cell ALL and five normal bone marrow monocyte cell samples using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer s protocol. To identify the specific expression of mirnas in common precursor B-cell ALL compared with healthy controls, microarray preparation, and hybridization were performed in three common precursor B-cell ALL samples and three normal samples by CapitalBio Corporation (Beijing, China) following the instructions of the manufacturer. Up to 1 mg of total RNA purified from each of the sample preparations was labeled using a Biotin RNA labeling kit for mirna arrays (Genisphere, PA). Hybridization was performed using a Hybridization, Wash, and Stain Kit (Affymetrix, CA) containing 7,815 probe sets according to the manufacturer s protocol. Processed slides were scanned using a GeneChip Scanner 3000 (Affymetrix). Raw data from mirna chips were normalized and analyzed using Affymetrix mirna QC tools. Backgroundsubtracted intensities were thresholded to 10 and log-transformed. Flagged spots corresponding to absent or low-quality signals were removed from the analysis before global median normalization. TABLE I. Characteristics of Pediatric Patients with ALL Type of samples Characteristics Median (range) No. (%) Initial diagnosed Age at diagnosis, y 5.0 ( (N ¼ 34 Sex Male 18 (52.9) Female 16 (47.1) WBC count, 10 9 /L 16.4 ( ) Less than (67.6) 50 or higher 11(32.4) NCI risk group SR 22 (64.7) HR 12(35.3) Cytogenetics t(9,22) or BCR-ABL 1(2.9) t(4,11) or MLL-AF4 2(5.9) E2A/PBX1 0(0) N/A 31(91.2) Immunophenotype B 34(100) T 0(0) CNS-ALL YES 1(2.9) NO 33(97.1) Prednisone response Good response 29(85.3) Poor response 5(14.7) Day 15 BM M1 32(94.1) M2 2(5.9) M3 0(0) Day 33 BM MRD <0.01% 31(91.2) 0.01% 3(8.8) Risk group SR 18 (52.9) IR 10 (29.4) HR 6 (17.7) MRD, minimal residual disease; BM, bone marrow; WBC, white blood cells; CNS-ALL, central nervous system acute lymphocytic leukemia.

3 2062 Xue et al. TABLE II. The Expression of mirna in the 34 Pediatric ALL and Five Control Normal Cases (x s) Group Cases mir-708 mir-181b mir-210 mir-345 mir-27a Experiment Control Z-value P-value < < mirnas with expression ratios greater than 0.4 compared to the controls were considered abnormal. Quantitative RT-PCR Analysis To verify the expression of specific mirnas in common precursor B-cell ALL, stem-loop RT-PCR was performed as described previously [13] on 34 common precursor B-cell ALL samples (18 standard-risk ALL, 10 intermediate-risk ALL, and 6 high-risk ALL) and five normal bone marrow samples. First-strand cdna was synthesized using 1 mg of total RNA in a 20 ml reverse transcriptase reaction mixture using the Revertaid H Minus First Strand cdna Synthesis kit (Tiangen, Beijing, China) with mirnaspecific primers. Quantitative RT-PCR was performed on an ABI Prism 7500 sequence detection system using SYBR Green PCR Master Mix (Tiangen) in a 20 ml reaction mixture. Expression of the U6 small nuclear RNA was used as an internal control to normalize all results. The total RNA of the normal bone marrow sample or was used as a normal control. Data were analyzed by Sequence Detection Software 1.4 (Applied Biosystems, Carlsbad, CA). The relative quantity of the transcript was calculated using the 2 DCT method, where DCT ¼ (CT mirna CT U6RNA ). We also tested the expression of the target gene in six common precursor B-cell ALL samples relative to the normal control by qrt-pcr. For analysis of CNTFR, NNAT, and GNG12 expression in Jurkat cells after mir- 708 mimics or inhibitor transfection, the mirna negative control (mir-nc) transfection group was taken as the calibrator with DCT ¼ (CT target gene CT GAPDH ). Customized primers were designed using the Primer Express software (Applied Biosystems). Primer sequences used are listed in Supplemental Table I. Plasmid Construction and Seed Mutagenesis The 3 -UTRs of CNTFR, NNAT, and GNG12 were amplified from synthetic CNTFR, NNAT, and GNG12 cdna clones (Origene, Rockville). Mutants of the CNTFR 3 -UTR were generated on each of two mir-708 target recognition sites (seed sequences) by site mutation [14]. The primers are shown in Supplemental Table I. The wild-type CNTFR, NNAT, and GNG12, as well as mutated 3 -UTRs of CNTFR M1 and M2 were inserted into the pcdna3.1(þ)- luciferase vector using Xba1 restriction sites. Cell Transfection and Dual-Luciferase Reporter Assay To experimentally verify whether CNTFR, NNAT, and GNG12 are targets of mir-708 in vivo, we performed dual-luciferase reporter assays. HEK-293 cells were transfected with recombinant pcdna3.1-(þ)-luciferase vector containing the 3 -UTR of the target gene and mir-708 mimics, inhibitors, or scrambled oligonucleotides (mirna negative control; 40 nm) using Lipofectamine 2000 (Invitrogen) together with the prenilla-tk vector as an internal control. Luciferase activity was measured 36 hours after transfection using a dual-luciferase assay kit (Promega, WI). Cells were lysed in 100 ml of lysis buffer. About 10 ml ofthe lysate was mixed with 50 ml of the substrate solution and immediately measured using an Ascent FL (Thermofisher, MD) in 96-well plates. The mir-708 mimics, inhibitor, and mir-nc were purchased from Invitrogen. Data were normalized to renilla activity: M ¼ (F1/R1 þ F2/R2 þ F3/R3)/(F1 /R1 þ F2 / R2 þ F3 /R3 ). F and R represent the firefly luciferase and renilla activities, respectively, in the experimental groups. F and R stand for the firefly luciferase and renilla activities, respectively, in control groups. Western Blot To verify the regulation of mir-708 in CNTFR, NNAT, and GNG12, Jurkat cells were transfected with mir-708 mimics or mir-nc using Lipofectamine Cells were rinsed with ice-cold phosphate buffered saline (ph 7.4) and lysed with radioimmunoprecipitation assay lysis buffer 48 hours after transfection. Lysates were incubated in ice for 30 minutes and then centrifugated at 12,000 rpm and 4 C for 20 minutes. Equal amounts of CNTFR proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. The membranes were incubated with antibodies against CNTFR, NNAT, GNG12, or GAPDH (Abcam, Inc., Cambridge, MA) at 4 C overnight. Antibody binding was assessed by incubation with horseradish peroxidase-conjugated secondary antibodies (Beyotime, Shanghai, China). Chemiluminescence was detected using an ECL Plus immunoblotting detection system (Beyotime). Bioinformatics Putative mrna targets of mir-708 and its feasible target seed sequences were predicted by three algorithms, namely, miranda ( Memorial Sloan-Kettering Cancer Center), PicTar ( and TargetScan ( Statistical Analysis All data are presented as mean SD of three separate experiments. SPSS 17.0 was used for statistical analysis. Homogeneity test of variance was done by conducting the Levene test. The expression levels and M values of CNTFR, NNAT, and GNG12 in the sample and control groups were compared by t-test. Analysis of mirna-708 and mirna-181b expression in different clinical-types was performed by t-test. The mean levels of expression of mirnas were compared between sample and control

4 mir-708 and Its Targets in Childhood B-ALL 2063 Fig. 1. mirna expression in childhood ALL as validated by qrt-pcr. (A C) Differential expression of mirnas between common B-ALL and the control group was validated by stem-loop RT-PCR. (D E) Expression levels of mir-708 or mir-181b in different clinical subtypes of ALL and normal hematopoietic cells determined by stem-loop RT-PCR. (A) MiR-210, (B) mir-27a, (C) mir-345. Normal, healthy control; SR, standard-risk common precursor B-cell ALL; IR, intermediate-risk common precursor B-cell ALL; HR, high-risk common precursor B-cell ALL ( P < 0.05). groups using the Mann Whitney U-test. Differences were considered statistically significant at probability level less than 0.05 (P < 0.05). Two-tailed tests were used for univariate comparisons. Graphs were plotted using the Graphpad Prism 5 software. RESULTS Identification of mirnas Specifically Expressed in Common Precursor B-Cell ALL The results of microarray analyses revealed 11 differentially regulated mirnas between common precursor B-cell ALL and normal samples. Of the 11 mirnas, eight were upregulated and three were downregulated in common precursor B-cell ALL samples. Each mirna had at least twofold difference between the two samples and a q-value < mir-708, mir-210, mi-181b, mir-345, mir-324-5p, and mir-125b were the six most upregulated mirnas whereas mir-23a, mir-27a, and mir-23b were the three downregulated ones. Then, we quantified the levels of the 11 significantly dysregulated mirnas by performing stem-loop RT-PCR on the common precursor B-cell ALL samples. Real-time PCR confirmed that, of the upregulated mirnas, expression of mir-708, mir-210, and mir-181b but not of mir-324-5p, mir-125b, and mir-345 were markedly upregulated. mir-345, and mir-27a had downregulated expression (Table II; Fig. 1A E). In addition, we studied mirnas differentially expressed in different clinical types of common precursor B-cell ALL. Interestingly, mir-708 and mir- 181b exhibited higher expression in high-risk (HR) common precursor B-cell ALL compared to standard-risk (SR) and intermediate-risk (IR) ALL (Fig. 1A and B). CNTFR, NNAT, and GNG12 Are the Targets of mir- 708 Among the aberrantly expressed mirnas, mir-708 is dysregulated the most significantly, suggesting that it may have a vital function in the pathogenesis and progression of common precursor B-cell ALL. mir-708 is located on chromosome 11q14.1 and is encoded in the intron 1 of Odd Oz/ten-m homolog 4 genes, which are regulated by the CCAAT enhancer binding protein in vertebrates. Human mir-708 genes encode mir-708-5p and mir-708-3p, but the construct we generated only contained the mir-708-5p transcript (Fig. 2A). To further study the regulatory mechanism of mir-708 in ALL, we performed a KEGG pathway analysis of mir-708 using the bioinformatics tools PicTar, miranda, and TargetScan. Candidate mir-708 targets were pooled from these three databases, and CNTFR, NNAT, and GNG12 were identified as those with the highest predicted context score percentiles. To validate the three predicted targets of mir-708, we constructed combined firefly luciferase reporters that contained

5 2064 Xue et al. Fig. 2. mir-708 can inhibit reporter activity and regulate the expression of tumor suppressor genes. (A) Sequence of the human mir-708 stem-loop and mature structures. (B) Results of luciferase reporter assays to determine the effects of mir-708 on the 3 -UTRs of the indicated genes. mir NC, mir-708 negative control ( P < 0.05). (C E) Quantitative RT-PCR (qrt-pcr) analyses of gene expression in common precursor B-cell ALL and normal hematopoietic cells. Points shown indicate the range and mean of the measurement as fold change compared with normal bone marrows ( P < 0.05). the entire wild-type 3 -UTR of the target genes. The results show that co-transfection of mir-708 mimics and the luciferase vector with the 3 -UTRs of CNTFR, NNAT, and GNG12 reduced luciferase activity to 20%, 89%, and 28%, respectively, compared with cotransfection with mir-nc alone (P < 0.05). Application of the mir-708 inhibitor eliminated the inhibition of wild-type CNTFR, NNAT, and GNG12 3 -UTR reporter activity, and the relative luciferase activity increased to 102%, 757%, and 65% (P < 0.05) compared with co-transfection with the mir-708 mimic group (Fig. 2B). Next, we assayed CNTFR, NNAT, and GNG12 mrna expression in six high-risk common precursor B-cell ALL samples and five normal samples by qrt-pcr. CNTFR and NNAT, but not GNG12, were found to be statistically significantly downregulated in the experimental group (Fig. 2C E). This suggests that mir-708 may act on its target genes via different mechanisms. One possible scenario is that mir-708 regulates GNG12 expression by translational inhibition with no effect at the mrna level. By contrast, mir-708 may specifically target CNTFR and NNAT mrna for degradation. Overexpression of microrna-708 Inhibits the Expression of CNTFR, NNAT, and GNG12 To confirm the function of mir-708 in leukemia cell lines, we transfected mir-708 mimics and a scramble control in Jurkat cells, and analyzed the protein levels of the three target genes by Western blot. Downregulation of CNTFR, NNAT, and GNG12 protein levels were found in mir-708 mimic-transfected cells compared with scrambled RNA-transfected cells (Fig. 3A). This indicates that mir-708 expression is inversely correlated with CNTFR, NNAT, and GNG12 protein levels and that mir-708 overexpression may regulate leukemia cell biological functions by suppressing the translation of CNTFR, NNAT, and GNG12 mrna. To obtain further insights into the expression of the target genes, we performed qrt-pcr in Jurkat cells transfected as described above. CNTFR and NNAT mrna were expressed at low levels compared with the control group, whereas no statistical differences were observed between the experimental group and the control group in terms of GNG12 mrna expression (Fig. 3B D). This finding further supports the idea that mir-708 may regulate the three target genes via different mechanisms. Repression of CNTFR by mir-708 Via a Conserved Binding Site in the 3 -UTR of CNTFR mrna mirnas function by forming a mirna/mrna duplex through binding to their respective target sites. The binding sites of mirnas in the 3 -UTR of target genes are usually evolutionarily conserved and perfectly match the mirna sequence [15]. In order to identify the binding sites of mir-708 within its target genes, the predicted seed region from positions 2 to 8 nt and the anchor A of the target

6 mir-708 and Its Targets in Childhood B-ALL 2065 Fig. 3. Dysregulation of mir-708 specifically represses the expression of key proteins. (A) Jurkat cells were transfected with mir-708 inhibitor, mirna-nc or mir-708 mimics. GAPDH expression served as loading controls. (B D) Expression of predicted target genes were determined by quantitative RT-PCR of the RNA extracted from transfected Jurkat cells using the standard 2 DDCT method. Expression of genes in each sample is reported relative to the untransfected control group ( P < 0.05). genes were obtained from TargetScan 5.1. Almost all sites were completely conserved in different species (Fig. 4A and B). However, two potential binding sites of mir-708 were found in the 3 -UTR of CNTFR mrna; one site is located in the bp region and the other is located in the bp region. We further examined the specificity of CNTFR regulation by mir- 708 by mutating two highly conserved seeding sites in the CNTFRderived 3 -UTR of the luciferase reporter, yielding M1 and M2 mutants. The repression effects observed for the modified mir-708 mimic were essentially abrogated in the presence of a mutated seed Figure 4. mir-708 can inhibit reporter activity through the bp region of the 3 -UTR of CNTFR mrna. (A and B) Schematic representation of the interaction of mir-708 with the 3 -UTR of its target genes. (C) Each predicted MRE 3 -UTR was inserted into a pcdna3.1 (þ)-luciferase vector immediately downstream of the firefly luciferase gene. HEK-293 cells were co-transfected with the pcdna3.1 (þ)-3 UTR luciferase reporter and mirna as indicated. At 48 hours post-transfection, cells were harvested for luciferase assay. Asterisks indicate significant difference between mir-708 and the control ( P < 0.05).

7 2066 Xue et al. region. Changes in the bp region did not influence the interaction between mir-708 and the CNTFR recombinant plasmid M2, and the relative luciferase activity decreased to 51%. However, mutation of the bp region strongly suppressed the inhibitory effects compared with the wild-type 3 -UTR (Fig. 4C). These results demonstrate that mir-708 acts on CNTFR by binding to the bp sequence region of its 3 -UTR. DISCUSSION An increasing body of evidence has highlighted the link between aberrant mirna expression and hematopoietic cancer. mirnas are appealing novel therapeutic targets for the treatment of leukemia [16]. In our study, we employed mirna microarray analysis and stem-loop qrt-pcr to analyze common precursor B- cell ALL samples and found that mir-708, mir-210, and mir-181b are upregulated while mir-27b and mir-345 are downregulated in those samples. RT-PCR analysis of novel dysregulation in mirnas also confirm the differential expression of mir-708 and mir-181b between high-risk common precursor B-cell ALL and the two other clinical types of common precursor B-cell ALL. This suggests that dysregulation of mir-708 and mir-181b have important roles in the pathogenesis and progression of high-risk common precursor B-cell ALL. However, our previous study on the aberrant regulation of mirnas in pre-b ALL (presence of cytoplasmic immunoglobulin, precursor B-cell acute lymphocytic leukemia) revealed differences in mirna expression between the various immunological classifications of leukemia. Compared with common precursor B-cell ALL, mir-519e, mir-222, and mir-339 were over expressed and mir-373-5p, mir-485-3p, and mir-451 were minimally expressed in pre-b ALL [17]. Our studies confirm that mirna expression levels and functions highly depend on the cellular context of leukemia, including its hematopoietic lineage, presence of genomic translocations and clinical type [18]. mirnas can direct different biological processes, including development, differentiation, and hematopoiesis, by regulating the expression of target genes. Han et al. [19] identified differential expression of mirna-708 between relapse and complete remission (CR) ALL patients, and showed that mir-708 can target FOXO3 to promote the occurrence of B-ALL and chronic granulocytic leukemia (CML). This suggests that mir-708 may play an important role in mediating the high relapse risk of ALL. Consistent with this interpretation, we found that mir-708 is the most dysregulated mirna in high-risk common precursor B-cell ALL and may be crucial in the regulation of this disease. In order to verify the function of mir-708 in high-risk common precursor B-cell ALL, we attempted to identify mir-708 target genes using a combination of computational prediction and expression profiling. Consequently, we identified three targets of mir-708, namely CNTFR, NNAT, and GNG12. The CNTFR gene encodes a member of the type 1 cytokine receptor family, which is the ligand-specific component of a tripartite receptor for ciliary neurotrophic factor. Binding of ciliary neurotrophic factor to the receptor recruits other transmembrane coreceptors, namely gp130 and the leukemia inhibitory factor (LIF) receptor [20]. LIF belongs to the IL-6 cytokine family and is involved in the induction of hematopoietic differentiation in normal and myeloid leukemia cells [20]. Downregulation of CNTFR may result in loss of control of LIF hematopoietic differentiation functionality. CNTFR can also facilitate JAK/STAT signal transduction, which is closely related to leukemia cell proliferation. WHI-P131, a Jak3 specific inhibitor, can induce apoptosis of Nalm- 6 (B-ALL cell line) and suppress Nalm-6 and, Molt-3 cell proliferation [21]. Loss of CNTFR may activate JAK/STAT signal transduction, leading to massive leukemia cell proliferation and apoptosis. Therefore, we speculate that mir-708 exerts its oncogenic effects in common precursor B-cell ALL by suppressing LIF function and activating the JAK/STAT signal pathway. The NNAT gene is imprinted and actively transcribed from the paternally inherited allele. It maps to human chromosomes 20q11.2 q12, a region frequently deleted in myeloid disorders and lost in 9% of leukemia patients [22]. Steven et al. [23] revealed that NNAT hypermethylation occurs in the majority of pediatric leukemia, resulting in the down regulation of NNAT. Furthermore, loss of NNAT or other oncogenes located in 20q11.2 q12 indirectly caused by NNAT deletion is likely associated with pediatric leukemogenesis [22]. Overexpression of mir-708 may cause the deletion of chromosomes 20q11.2 q12 and downregulate the function of other oncogenes by inhibiting NNAT expression, and thus contribute to the pathogenesis of common precursor B-cell ALL. The GNG12 gene encodes a G protein, g12, which participates in MAPK signal transduction and affects transcription factor activity. GNG12 gene is highly conserved, as there are human, rat, cow, frog, chicken, and zebrafish homologs. It is known that GNG12 is an important signaling component of the inflammatory cascade [24]. More research is needed to elucidate the role of GNG12 in hematopoiesis. The functions of mirnas should be closely related to their targets. Currently, it is believed that mirnas change a cell s gene expression profile on multiple levels, including direct repression of target genes, indirect competitive endogenous RNAs (cernas), and alternative splicing of mrnas [25]. We infer that mir-708 regulates GNG12 expression by translational inhibition with no effect at the mrna level. By contrast, mir- 708 may directly contribute to the degradation of CNTFR and NNAT mrna. In summary, our study identified specific mirna signatures associated with childhood common precursor B-cell ALL and suggests that expression level of mir-708 has an essential role in high-risk common precursor B-cell ALL. We also demonstrate that mir-708 regulates CNTFR, NNAT, and GNG12 and may thus contribute to pathogenesis of leukemia via inhibition of LIF and activation of the JAK/STAT and MAPK signal transduction pathways, thereby, initiating leukemia development and influencing the biological functions of leukemia cells. Future research endeavors could elucidate the precise regulatory mechanisms by which mir-708 exerts its functions and determine the signaling pathways targeted by mir-708 in common-all. REFERENCES 1. Pui CH, Campana D, Evans WE. Childhood acute lymphoblastic leukemia Current status and future perspectives. Lancet Oncol 2010;2: Uzunel M, Jaksch M, Mattsson M, et al. Minimal residual disease detection after allogeneic stem cell transplantation is correlated to relapse in patients with acute lymphoblastic leukaemia. Br J Hematol 2003;122: Coustan SE, Sancho J, Hancock ML, et al. Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia. Blood 2000;96: Chen CZ, Li L, Lodish HF, Bartel DP. MicroRNAs modulate hematopoietic lineage differentiation. Science 2004;303: Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004;116: Esquela-Kerscher A, Slack FJ. Oncomirs-microRNAs with a role in cancer. Nat Rev Cancer 2006;6:

8 mir-708 and Its Targets in Childhood B-ALL Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-rna genes mir15 and mir16 at 13q14 in chronic lymphocytic leukemia. Natl Acad Sci 2002;99: Pedro PM, Mona N, Frank JS. OncomiR addiction in an in vivo model of microrna-21 induced pre-bcell lymphoma. Nature 2010;467: Mavrakis KJ, Van Der Meulen J, Wolfe AL, et al. A cooperative microrna-tumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL). Nat Genet 2011;43: Schotte D, Akbari Moqadam F, Lange-Turenhout EAM, et al. Discovery of new micrornas by small RNAome deep sequencing in childhood acute lymphoblastic leukemia. Leukemia 2011;25: Schotte D, Pieters R, Den Boer ML. MicroRNAs in acute leukemia: From biological players to clinical contributors. Leukemia 2012;26: Gao C, Zhao XX, Li WJ, et al. Clinical features, early treatment responses, and outcomes of pediatric acute lymphoblastic leukemia in China with or without specific fusion transcripts: A single institutional study of 1,004 patients. Am J Hematol 2012;87: Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of micrornas by stem-loop RT-PCR. Nucl Acids Res 2005;33:e Zheng L, Baumann U, Reymond JL. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucl Acids Res 2004;32:e Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microrna targets. Cell 2005;120: Kotani A, Ha D, Hsieh J, et al. mir-128b is a potent glucocorticoid sensitizer in MLL-AF4 acute lymphocytic leukemia cells and exerts cooperative effects with mir-221. Blood 2009;114: Ju XL, Li D, Shi Q, et al. Differential microrna expression in childhood B-cell precursor acute lymphoblastic leukemia. Pediatr Hematol Oncol 2009;26: Zhang H, Luo XQ, Zhang P, et al. MicroRNA patterns associated with clinical prognostic parameters and CNS relapse prediction in pediatric acute leukemia. PLoS ONE 2009;4:e Han BW, Feng DD, Li ZG, et al. A set of mirnas that involve in the pathways of drug resistance and leukemic stem-cell differentiation is associated with the risk of relapse and glucocorticoid response in childhood ALL. Hum Mol Genet 2011;20: Nancy YI, Steven HN, Teri GB, et al. CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130. Cell 1992;69: David W, Sternberg DW, Gilliland DG. The role of signal transducer and activatorof transcription factors in leukemogenesis. J Clin Oncol 2004;22: Evans HK, Wylie AA, Murphy SK, et al. The neuronatin gene resides in a micro-imprinted domain on human chromosome 20q11.2. Genomics 2010;77: StevenJK, Joshua P, Alison W, et al. Hypermethylation of the imprinted NNAT locus occurs frequently in pediatric acute leukemia. Carcinogenesis 2002;23: Kelley CL, Michael PD, Maciej L, et al. Gng12 is a novel negative regulator of LPS-induced inflammation in the microglial cell line BV-2. Inflamm Res 2010;59: Karreth FA, Tay Y, Perna D, et al. In vivo identification of tumor-suppressive PTEN cernas in an oncogenic BRAF-induced mouse model of melanoma. Cell 2011;147: SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher s web-site. Table SI Primers Used in Stem-Loop RT-PCR and Reverse Transcription PCR