Background
Leukemia is one of the leading causes of cancer death worldwide. Acute myeloid leukemia (AML), one type of malignant diseases, arises from myeloid progenitor cells that are arrested at early stages of differentiation. Chronic myeloid leukemia (CML) is a clonal disorder in which cells of the myeloid lineage undergo massive clonal expansion. Although the recent advancement in understanding and treatment of AML and CML has remarkably improved the cure rate over the past decade, a number of patients still die of these diseases. This highlights the need for more thorough knowledge of these two leukemias.
Recently, microRNAs (miRNAs), a class of non-coding RNA, were found to play important roles in various fundamental biological processes, such as cell proliferation, apoptosis, differentiation and signaling pathway, which are accomplished by silencing specific target genes through translational repression or direct mRNA degradation[
1‐
3]. Studies demonstrated that about 50% of annotated human miRNAs are located at fragile sites and genomic regions involved in cancers[
4]. Some miRNAs are involved in cancer regulation and are considered as oncogenes or tumor suppressors[
5]. The expression profiles of miRNAs could be linked to disease diagnosis, therapeutic response and prognosis[
6‐
10]. The first finding linking miRNAs and leukemia was that adult patients with chronic lymphocytic leukemia often have deletions or downregulation of miR-15 and miR-16 at 13q14[
11]. Up to date, an increasing number of studies have revealed that the differentiation of AML lineages is regulated by miRNAs, which have key roles in hematopoiesis[
12,
13].
Our previous miRNA profiling analysis showed the expression of miR-99a in pediatric-onset AML (FAB M1-M3) was 3.81 times higher at average than that in normal donors[
14], suggesting miR-99a may be involved in the progression of pediatric myeloid leukemia. To confirm this, we performed further investigation to assess the expression of miR-99a in childhood AML and CML, and the function of miR-99a in these diseases.
Methods
Clinical samples
A total of 88 bone marrow samples were enrolled in this study. The samples taken by bone marrow puncture were from 68 pediatric acute myeloid leukemia patients (including 41 samples before therapy, 23 samples with complete remission and 4 samples with relapse), 8 chronic myeloid leukemia patients (including 4 samples before therapy and 4 samples with complete remission) and 12 pediatric controls from the First and Second Affiliated Hospital of Sun Yat-Sen University. The newly diagnosed AML patients included 6 with M1, 17 with M2, 10 with M3, 4 with M4 and 4 with M5. The AML patients with complete remission (CR) included 1 with M1, 6 with M2, 6 with M3, 6 with M4 and 4 with M5. The relapsed AML patients were 4 with M2. Patients
’ characteristics are shown in Additional file
1: Tables S1 and S2. Written informed consent for biological studies was obtained and the study was approved by the Ethics Committee of the affiliated hospitals of Sun Yat-Sen University.
Cell culture and RNA/protein isolation
Human HL60 (acute myeloid leukemia cell line) and K562 cells (chronic myeloid leukemia cell line) were cultured in RPMI 1640 medium (Invitrogen). HEK-293 T, the human embryonic kidney cell line, was grown in Dulbec-co’s modified Eagle’s medium (Invitrogen). Both cultures were supplemented with 10% fetal bovine serum (fetal bovine serum, Australia) and sodium pyruvate, and cultured at 37°C in a humidified atmosphere consisting of 5% CO2. Total RNA and protein were isolated from clinic samples with Trizol (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer.
Quantitative real-time PCR analysis for miR-99a expression
Quantitative real-time reverse transcriptase PCR (qRT-PCR) was performed to detect miR-99a expression. Briefly, 0.2 μg of small RNA extracted from cell samples was reverse-transcribed to cDNA using M-MLV reverse transcriptase (Promega) and amplified with specific designed miRNA RT-primers and PCR amplification primers (Sangon, Shanghai, China). Sequences of all the primers are shown in Additional file
1: Table S3. The expression level of each miRNA was measured using the 2-DeltaDeltaCt method.
MTT assay
K562 and HL60 cells were respectively plated at 1 × 104 per well. The cells were transfected with 100 nM miR-99a mimics/NC (miR-99a/scrambled oligonucleotides) or inhibitor-miR-99a/NC using Lipofectamine 2000 (Invitrogen) following manufacturer’s recommendation and were then incubated for 24 h, 48 h and 72 h, respectively. Next, the cells were incubated with Dye Solution (15 μL) for another 4 h until purple precipitate was visible. Lastly, after 100 μL Stop Mix was added, the cells were left at room temperature in the dark for 2 h and the absorbance was recorded.
Apoptosis assay
K562 and HL60 cells were transfected with miR-99a mimics/NC or inhibitor-miR-99a/NC (100 nM) using Lipofectamine 2000 as mentioned above. The cells were collected at 48 h, 72 h and 96 h post transfection respectively. The cells were centrifuged and resuspended in 500 μl of staining solution (containing annexin V fluorescein and propidium iodide in HEPES buffer) (annexin V: FITC apoptosis detection kit; BD PharMingen, San Diego, CA). After incubation at room temperature for 15 min, cells were analyzed by flow cytometry.
Target genes prediction and vector constructs
The potential targets of miR-99a were predicted by means of TargetScan (
http://www.targetscan.org/) and PICTAR (
http://pictar.mdc-berlin.de/) software. In order to reduce the number of false positives, only putative target genes predicted by both programs were accepted. The vectors of pre-miR-99a and the potential targets predicted (TRIB2 and CTDSPL) were constructed. Briefly, the primers of miR-99a were designed to amplify pre-miR-99a by PCR from genomic DNA. The amplified products were ligated into the PCD6.2 vector (Promega, Madison, WI). Ecological forms and mutants of the potential targets of miR-99a, designed by TargetScan and generated by annealing, were ligated into the pGL3 vector or the psi-CHECK-2 vector (Promega, Madison, WI). Proper insertions were all confirmed by DNA sequencing. All the primers were synthesized (Sangon, Shanghai, China) and the information is available in Additional file
1: Table S4.
Cell transfections and Luciferase assays
HEK-293 T cells were grown in 24-well plates at a density of 1 × 105 cells per well in 0.5 ml of complete growth medium and allowed HEK-293 T cells to adhere overnight. K562 cells were grown in 24-well plates at a density of 1 × 106 cells per well. 0.1 μg of pre-miR-99a and the potential targets vectors were transfected into HEK-293 T cells using Lipofectamine 2000 (Invitrogen) and were transfected into K562 cells by electroblotting (Ambion, Austin, Texas) respectively in growth medium according to manufacturer’s recommendation. After 24-48 h, the transfected cells were harvested for Dual-luciferase reporter transfection assay. Similarly, 100 nM miR-99a mimics/NC duplex or inhibitor/inhibitor-NC were used for transfection.
Western immunoblotting
K562 and HL60 cells were treated as indicated in the figures and lysed in RIPA buffer (Pierce, Rockford, IL, USA) with protease and phosphatase inhibitors (Roche, Beijing, China). The protein of bone marrow, K562 and HL60 cells was quantified using the BCA protein assay (Pierce, Rockford, Illinois). Protein (30 μg) was loaded onto a 12% SDS–PAGE gel then transferred onto nitrocellulose. The membrane was blocked for 2 h in Tris-buffered saline Tween-20 (TBST) containing 2% bovine serum albumin, and cleaved parp was incubated with rabbit anti-CTDSPL (1:10000, Cell Signaling, Danvers, MA) and mouse anti-TRIB2 (1:5000, Abcam, Invitrogen, USA) overnight at 4°C. After incubation with HRP-conjugated secondary anti-mouse or anti-rabbit (ABR, Golden, Colorado) at room temperature for 1 h, blots were then developed according to ECL Substrate (Pierce) following manufacturer’s instructions. Protein was normalized with β-actin (Sigma St. Louis, Missouri), Tublin (Cell Signaling Technology, USA) and GAPDH (Cell Signaling Technology, USA), and measured by densitometry by two independent researchers.
Statistical analysis
T test, ANOVA and non-parametric rank sum test were performed using SPSS16.0 statistical software. A Fisher r-to-z transformation was carried out to calculate a probability level (P value). Student’s t test was performed to assay the statistical significance.
Discussion
Leukemia is the commonest childhood malignant disease. With the rapid development of modern combination chemotherapy and hematopoietic stem cell transplantation, 5-year event-free survival for pediatric acute lymphoblastic leukemia (ALL) has been improved to rates as high as 80%[
20,
21]. However, the prognosis of pediatric AML is still poor, with long-term survival rates of about 50% to 65%[
22]. The overall survival of CML was recently reported to be up to 80% at 8 years of follow-up in respondent patients due to the introduction of imatinib (a tyrosine kinase inhibitor)[
23], there still remains a subset of patients who fail the treatment. It is therefore of significance to clarify the molecular mechanisms of these two diseases for further improving survival rate. For a long time, the pathogenesis researches of AML and CML mainly focus on chromosome abnormalities and protein coding genes. Recently, more and more studies indicated that abnormal expressions of relevant miRNAs may promote tumors. Their abnormal expressions are closely related to the incidence, development, treatment response and prognosis of leukemia[
11,
24‐
28]. Although some miRNA expression signatures associated with types and cytogenetics of leukemia have been addressed, there has been no report on miR-99a expressional and functional study in pediatric AML and CML so far.
In this study, we found that the expression of miR-99a increased significantly not only in childhood patients with AML (M1-M5) but also in those with CML, decreased obviously in CR patients with these two myeloid leukemias, and increased again in relapsed patients with AML-M2 analyzed. Furthermore, MTT assay showed that the proliferation of K562 and HL60 cells was effectively promoted by miR-99a, and apoptosis experiment demonstrated that the apoptosis of K562 and HL60 cells was suppressed by miR-99a. These results illustrate that miR-99a may function as an oncogene, which contributes to the generation and development of both AML and CML in children. Finally, dual-luciferase reporter transfection assay and western blot analysis on clinical samples and leukemia cell lines further supported that miR-99a played a potential oncogene role by targeting CTDSPL and TRIB2 in most pediatric myeloid leukemia patients.
CTDSPL gene exhibits tumor suppressor gene activity. It has been reported that CTDSPL protein plays the role of phosphatase, regulating cells growth and differentiation, and expresses significantly low in major epithelial malignancies[
15]. In leukemia cell lines and 24% of patients with acute lymphoblastic leukemia, CTDSPL promoter is highly methylated, which promotes the occurrence of leukemia[
29]. A study further revealed that RBSP3, also denoted as HYA22 and CTDSPL, is involved in the regulation of cell growth and differentiation, and frequent mutations in this gene are detected in human hematopoietic cell lines[
16]. The tumor suppressor property of CTDSPL is related to its ability to remove the phosphate group from serine 807 and 811, and induce the formation of the RB-E2F1 complex[
30]. The 'RB’ pathway has a critical role in both cell physiology and tumorigenic transformation via distinct molecular mechanisms.
TRIB2, like some other genes, has distinctive roles in different biological conditions. On the one hand, TRIB2, a pseudokinase, may function as an oncogene and cooperates with HoxA9 to accelerate the onset of AML in mice by binding COP1 and C/EBP-α and leading to degradation of C/EBP-α[
31‐
33]. On the other hand, TRIB2 may serve as a tumor suppressor gene. A resent report showed that forced expression of PITX1 in JURKAT cells prompted deregulation of genes involved in T-cell development including TRIB2. Leukemic activation of PITX1 was observed in a subset of early-staged T-ALL by inhibiting T-cell development[
34]. TRIB2 is also a pro-apoptotic molecule and activates Bax gene to induce apoptosis in hematopoietic cells through degradation of MCL-1[
35]. Its tumour suppressor activity may be abrogated in a proportion of AML patients, which may lead to enhanced cell survival and therefore contribute to pathogenesis of the disease[
36].
MiR-99a has been addressed to be involved in the tumorigenesis of several cancers. MiR-99a may play different roles in different tumors. Wong and Wszolek reported that miR-99a expresses notably high in untreated patients with tongue squamous cell carcinoma and non-invasive urothelial cancer, suggesting that miR-99a exhibits oncogenic activity[
37,
38]. However, Yamada et al. addressed that the expression of miR-99a deceased in onset patients with lung cancer, implying that miR-99a may behave as a tumor suppressor gene[
39]. In this study, we find that miR-99a plays a potential oncogenic role in pediatric AML and CML through targeting CTDSPL and TRIB2. All the above studies elucidate that the roles of certain miRNAs are completely distinctive in different biological conditions, which is also supported by some other studies[
18,
19,
40‐
43]. All these highlight the importance of thoroughly studying and comprehensively expounding the expression and function of miRNAs in different tumors as well as the same tumor in adults and children.
It is noted that in this study there was a relatively larger number of clinical samples which were tested for the expressions of miR-99a, CTDSPL and TRIB2 compared with previous studies. The results revealed that miR-99a may target CTDSPL and TRIB2 more convincingly from a statistical point of view. However, the results also showed that CTDSPL or TRIB2 protein and miR-99a exhibited opposite expression trends in most the patients, but not in all the patients, indicating that CTDSPL and TRIB2 genes are not the only target genes of miR-99a. Therefore, there may be other potential undiscovered target genes, which is in line with our present knowledge. As a single miRNA could regulate several different genes and the same gene could also be regulated by several miRNAs[
44,
45], the regulation of miRNA forms a complex network. The complicated interactions of relevant miRNAs contribute to the occurrence and development of leukemia.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LDZ performed experiments, analyzed the data and wrote the manuscript; XJL, YNL JW and XLZ performed a part of western blot; ZYK and LBH provided samples for the analysis; YQC, HZ and XQL designed experiments and edited the manuscript. All authors critically reviewed the manuscript. All authors read and approved the final manuscript.