The Expression of Different Micrornas in Iraqi Patients with Childhood Acute Leukemia and Their Association to C/EBP-Β Serum Level

Leukemia is the most common cancer in children which causes death despite the high survival rate. Therefore, new methods are required to find a suitable therapy. A small RNA called microRNAs (miRNAs) is used as a biomarker for cancer diagnosis and early prognostic evaluation. Expression levels of three miRNAs from the 3' arm (miR-142-3p, miR-223-3p and miR-146-3p) were detected in serum samples from 30 acute leukemic children and from 30 healthy individuals by using qPCR. The miR-142-3p and miR-146-3p profiles were significantly downregulated (P=0.0010 and 0.0012, respectively), while miR-223 was found to be significantly upregulated (P= 0.0044) in the pateints. Serum level of C/EBP-β (CCAATenhancer-binding protein) was also determined by enzyme-linked immunosorbent assay (ELISA) and showed a significantly higher level in the patients (p≤0.0001). However, this higher enhancer level is probably not the reason for the abovementioned differences in miRNAs expression, as indicated by the weak correlation values with the three miRNA molecules (0.5737, 0.6625 and 0.7769, respectively). In conclusion, the fold change of miR-142-3p, miR-223-3p and miR146-3p in serum could be potentially used as an early indicator of the occurrence of acute leukemia in children.


Introduction
Highly sensitive and specific biomarkers are required for early diagnosis of leukemia in order to enable better decisions for treatment [1]. Functional investigations showed that miRNAs play crucial roles in cell proliferation, apoptosis, immune response, and tumorigenesis [2]. Specific miRNA molecules demonstrated high functional values in different leukemia subtypes, including those of diagnostic management and prognostic assessment [3]. Induction of oncogenes and negative regulation of tumor suppressor genes are the responsible mechanisms for relapse after the change in miRNAs expression. Hence, testing miRNA expression can enhance an efficient early evaluation of leukemic patients [4]. A biomarker that is used in screening the progress of leukemia, e.g. through the usage of miRNA expression, can provide an indication of relapse-free survival or as an overall survival [5]. A previous study demonstrated that the expression level of miR-142-3p has about 90% sensitivity and 100% specificity in peripheral blood mononuclear cells in acute myeloid leukemia (AML) when compared to the control, which justified its employment as a biomarker [6]. In another study, this miRNA showed low level of expression in acute lymphoid leukemia (ALL) in comparison with AML [7]. miR-146a is highly expressed in children with ALL and AML; therefore, it could be used to significantly differentiate these cases from those of chronic lymphoid leukemia (CLL) and AML in adult. The same study revealed that, despite that ALL patients at the age group of 1-14 year could cure with the current available treatments, one third of them underwent miRNA profiling continue to relapse after one year of follow up. The expression of several miRNAs, such as miR-7, miR-216, and miR-100, miR-486, miR-191, miR-150, miR-487, and miR-342 could distinguish between cases of relapse and complete remission (CR) in the patients [8]. The possible association between serum levels of CEBP-β and fold change in the expression of miRNAs in the patients has been scarcely investigated in the literature. Normally, the expression of miR-142 and miR-223 decreases during hematopoietic cells proliferation. Some studies showed that the miR-223, miR-142 and CEBP-β are involved in a regulatory pathway during hematopoiesis and that C/EBPβ increases at later stages of differentiation. It was also shown that miR-223 can upregulate the expression of miR-142 through CEBP-β [9,10].
The aim of this study is to determine the expression of different miRNAs (miR-142-3p, miR-22-3p3 and miR-146a-3p) in Iraqi patients with childhood acute leukemia along with their potential use as diagnostic biomarkers. A second aim is to evaluate the relationship between serum level of C/EBP and the fold change in the expression of these miRNAs in the pateints.

Materials and Methods Serum samples from leukemia patients
Family history of leukemia, age, gender, and other data were recorded. Whole blood samples were collected in special test tubes for serum separation. A total of sixty serum samples were collected, including thirty from healthy children and thirty from children diagnosed with acute leukemia who attended the acute leukemia unit/ Children's Central Teaching Hospital/ Baghdad (from December 2018 to March 2019). Serum was collected in 2ml tubes by allowing the blood to coagulate at room temperature for 30 minutes followed by centrifugation for 5 minutes at 1600 rpm. Finally, serum was stored at -0 C for further analysis.

Extraction procedure of miRNAs
MiRNAs were extracted from serum samples by suing the kits purchased from Qiagen/ USA. After thawing, 200μl of serum was mixed with the denaturation buffer. Nuclease-free water was added (50-100 μl) for elution, according to manufacturer's instruction. Finally, miRNA was stored at -80°C in DEPC-treated water after determining the concentration by NanoDrop-5000 spectrophotometer (Nano Drop Technologies, USA).

Reverse Transcription
The TaqMan miRNA reverse transcription kit (Applied Biosystems, USA) was used for the achievement of the reverse transcription reaction. The reaction mixture contains 10 ng of RNA extract, 100 mM of each deoxynucleotide triphosphate, RT buffer, 50 U/L of reverse transcriptase, 20 U/mL of RNase inhibitor, miRNAs (15 ul), nuclease-free water, and specific TaqMan primers (miR-142-3p, miR-223-3p, miR-146a-3p and endogenous control -RNU48)] (Applied Biosystems, USA). The reaction mixture was incubated at 42 °C for 30 min then at 85 °C for 5 min and hold at 4 °C by using Eppendorf/Hamburg/Germany RT-PCR.

Statistical analysis
ANOVA (one-way analysis of variance) was performed to test whether the differences between groups were significant or not. Statistical significance was defined as p ≤ 0.05. Data were expressed as mean ± standard deviation and statistical analysis was carried out using Graph Pad Prism version 6 (Graph Pad Software Inc., La Jolla, Ca, USA). Significant variation between acute leukemic and normal individuals was determined by student's T -test. R value was used to determine significant correlation between gene expression and CEBP-B concentration.

Results and Discussion
The patients group consisted of thirty patients diagnosed with acute leukemia, including ALL and AML. The number of Children with ALL was 20 and that for patients with AML was 10 ( Figure-1), including 16 males with a mean age of 5.25 ± 2. 8 and 14 females with a mean age of 6.53 ± 3.09. These data are in agreement with those of Ruiz-Delgado and colleagues who showed that ALL represents the most common malignancy in pediatrics, the age range of patients with ALL was 2-9 years, and there was a slight predominance in males [11]. Also, Obaid et al., (2010) found that AML incidence was higher in adults than children [12], while Mohammed and colleagues showed that ALL incidence was higher in children (72%), with 51.6% in males and 49% in females [13]. The healthy group (control) in the present study consisted of 17 males with a mean age of 7.73 ± 2.76 years and 13 females with a mean age of 6.19 ± 1.95 years. The distribution of the age and gender groups among children with acute leukemia is presented in Figure-2, which reveals that the frequency of the age group of 1-5 years in male patients with leukemia was higher than that in females, a similar result to that shown by Ismael and Hassan [14].

miRNAs expression levels in patients with acute leukemia
The expression of miRNAs found in the circulating blood is either due to death and lyses of tumor cells or the release by tumor cells into blood vessels [15].

MiR-223-3p
The results of the present study revealed that children with acute leukemia had an elevated level of the expression of miR-223-3p, as presented in Figure-(3A). This over-expression was associated with a significant difference in comparison with the levels in healthy children (P= 0.0044). However, the difference in the expression level in ALL as compared to AML pateints was not significant (p ≤ 0.05) (Fig.3B). In hematopoietic tissues, miR-223-3p was shown to be highly expressed and its level is increased during WBCs and RBCs development [16]. In addition, the phenotype of this molecule was demonstrated to be possibly changed in hematological and solid cancers [17]. Many studies reported that miR-223 tends to be upregulated in several cancers, as in ovarian cancer tissues, advanced pathological stage gastric cancer, and non-small cell lung cancer cases [18,19,20]. Also, upregulation of this miRNA can distinguish early stage pancreatic adenocarcinoma from chronic pancreatitis [21], whereas it is highly deregulated in metastasis and advanced clinical stages in the Iraqi Journal of Science, 2020, Vol. 61, No. 11, pp: 2879-2887 0882 serum of patients with osteosarcoma and colorectal cancer [22,23]. The high serum level of this miRNA in lymphoma B cell was shown to correlate with good survival in comparison to low level [24,25].

Mir-142-3p
mir-142-3p expression was examined in 30 serum samples in children with acute leukemia compared to 30 normal serum samples in healthy children by qPCR using TaqMan probes. mir-142-3p showed low expression in acute leukemia patients in comparison to healthy children, as presented in Figure-(4A.) This downregulation was significant (p= 0.0010). More investigation showed no differences in mir-142-3p expression in serum of patients with AML compared with ALL (p ≤ 0.01) (Fig. 4B). mir-142-3p has a role in cancer progression related to cell cycle and invasion, regulating cell migration and apoptosis [2,26,27]. The downregulation was reported in both solid cancer and leukemia [28,29]. Low expression was reported in solid cancers, such as hepatic cancer, with poor prognosis [30,31]. It also has a role in growth inhibition in lung cancer and acute lymphoblastic leukemia through inhibiting TGFβ-1 receptor and MLL-AF4 oncogene, respectively [32,33]. This miRNA was also reported to be downregulated in AML [6].

MiR-146a-3p
As shown Figure-(5A), the expression of miR-146a-3p was significantly downregulated (P=0.0012)in children with acute leukemia in comparison to healthy donors. . Swellam and El-Khazragy showed similar results; they found that ALL children had a lower expression level in WBCs in comparison to the healthy group [34]. miR-146a was reported to be highly expressed in bone marrow monocytes in cases of AML [35]. Our results also showed no significant difference in the expression of miR-146a-3p between AML and ALL patients (P≤ 0.05) (Figure-5B). The expression level of miR-146a was reported to be positively correlated to the clinical outcomes [36]. Low expression was also found in prostate, gastric and glioma malignancies [37,38,39].

Detection of C/EBP-β level
There are many targets for each miRNA [40]. CEBP-β is an important transcriptional regulator which causes changes in gene expression and affects hematopoiesis in a regulatory network targeting other miRNAs [41]. The level of C/EBP-β in pediatric acute leukemia and the control group was detected by ELISA. Our results showed a highly significant difference in the level of C/EBP-β (P < 0.0001) in the patients in comparison to healthy children, as presented in Figure-(6A). Also, the difference in the level of C/EBP-β in serum of children with AML and ALL was significant (P ≤ 0.0001) (Figure-6B).

The correlation between C/EBPβ and miRNAs expression levels in serum samples from Iraqi children diagnosed with acute leukemia and healthy children
For further examination, we tested whether we could find an association between C/EBPβ and miR-142-3p, miR-146a-3p and miR-223-3p expression in the serum of pediatrics with acute leukemia. The test was conducted on 20 serum samples from children with acute leukemia and 10 normal serum samples from healthy children. Our results indicated that the high levels of C/EBPβ were not sufficient to induce the down-regulation of the expression of miR-142-3p, miR-146a-3p and the upregulation of miR-223-3p in serum samples, as indicated by the weak correlation values (R= 0.5737, 06625 and 0.7769, respectively), as shown in Figure-7 (A, B,C). To reach a final conclusions about the relation between C\EBPβ and miRNAs, the sample size needs to be increased. Also, there is probably a need to find the effects of other factors like LMO2-L/-S isoforms (LIM domain only 2) and the relation of other C/EBP family members, such as alpha, gama,…etc. with miRNAs in cancers.

Conclusions
The serum expression levels of miR-142-3p, miR-223-3p and miR-146-3p can act as useful indicators for early detection of pediatric acute leukemia. Advanced research is needed to find the association between these miRNAs and the C/EBP-β level in acute leukemia.