Circulating miR-146a expression as a non-invasive predictive biomarker for acute lymphoblastic leukemia

Dysregulation of non-coding microRNAs during the course of tumor development, invasion and/or progression to the distant organs, makes them a promising candidate marker for the diagnosis of cancer and associated malignancies. This exploratory study aims at evaluating the usefulness of plasma concentration of circulating mir-146a as a non-invasive biomarker for acute lymphoblastic leukemia (ALL). Total RNA including miRNA was isolated from 110 plasma samples of patients (n = 66), healthy controls (n = 24) and follow up (n = 20) cases and reverse transcribed. Relative concentrations were assessed using real-time quantitative PCR and fold-change was calculated by 2−ΔΔCt method. Finally, relative concentrations were correlated to clinicopathological factors. Patients (n = 66) were analyzed to determine fold expression of miR-146a in plasma samples of ALL. Before chemotherapy, pediatric (n = 42) and adult (n = 24) showed overexpression of miR-146a compared with healthy controls (P < 0.0001). There was no effect of age and gender on mir-146a expression in plasma. mirR-146a expression was independent of clinical and hematological features. Moreover, miR-146a levels in plasma of paired samples (n = 20) after treatment showed significant decrease in expression (P < 0.001). Expression of plasma miR-146a may be utilized as non-invasive marker to diagnose and predict prognosis in pediatric and adult patients with ALL. Moreover predicted targets may be utilized for ALL therapy in future.

www.nature.com/scientificreports/ in Pakistan. The discovery of non-coding RNAs called microRNAs (miRNAs) opened a new horizon to discover novel diagnostic and prognostic markers for several malignant disorders including leukemia 9 . Studies on expression profiling of miRNA have shown its role in the development of ALL [10][11][12] . A single miRNA can regulate the expression of more than one gene, playing role in different stages of hematopoiesis and deregulation at any level may lead to leukemia 10 . miRNAs help to establish accurate classification of leukemia and to determine origin of the tumor of different types of leukemia 13 . miRNA is highly stable in both fresh and stored samples of plasma, making it the most potential blood-based biomarker of diagnostic and prognostic significance 11 . Several studies have shown that there are distinct microRNAs for specific cancer types that differentiate between malignant and its adjacent normal tissue 6 . miR-146a has been reported to play a key role in immune system, inflammation, myeloproliferation and oncogenesis 14 . As an oncogene its up-regulation may alter hematopoiesis leading to the development of leukemia 13,15 . There are few reports available in the literature on the role of miR-146a in ALL.
A study reported the up-regulation of miR-146a in ALL samples of children 16 . Another report described its down-regulation in adult AML patient samples 17 . The miR-146a has shown an important role in hematopoiesis, predominantly in T-cell development affecting proliferation and apoptosis of NB4 cells 18 . Some abnormal phenotypes in hematopoietic system were found in a mice deficient with miR-146a that indicates its potential role in hematopoiesis 19 . The overexpression of miR-146a has been reported in stem cell lines including monocytic and lymphocytic lineage after bone marrow transplant 17 . A study presented microarray results of miRNA profiling on bone marrow biopsy and peripheral blood samples of childhood ALL. After validation through qPCR, up-regulation of miR-146a was revealed in bone marrow biopsy but its overexpression in blood samples was not found 20 . A study on 49 bone marrow samples reported miR-146a overexpression in childhood cases of ALL 21 . Most of these studies reported up-regulation of miR-146a in bone marrow samples of ALL. The studies reporting its expression in plasma samples are limiting. The present research was conducted to validate whether miR-146a levels in plasma of ALL patients were overexpressed or otherwise and analyze the plasma miR-146a expression levels after treatment to evaluate its potential as a biomarker for ALL. The altered expression in the current study provides the basis to utilize circulating miR-146 s as a novel predictive marker for diagnosis and prognosis of adult as well as pediatric-ALL.

Methods
Patients and clinical samples. Whole blood samples were collected in EDTA tubes from a total of 110 study subjects including patients (n = 66), controls (n = 24) and follow up cases (n = 20). Patients were diagnosed based on morphology, cytogenetic and immunophenotype and characterized according to WHO classification criteria 22 . The patients registered at INMOL hospital, Lahore, Pakistan from January, 2013 to November, 2016 were included in the study. The patients who were newly diagnosed were designated as "cases" and those with any kind of infectious diseases were excluded to determine accurate representation of the study. The cases monitored after four cycles of chemotherapy and nominated as "follow up cases" were also included to evaluate remission status after treatment. Complete remission was considered with bone marrow having less than 5 blasts percentage. The control samples were obtained from healthy subjects as "normal control" that has no malignancy and infectious disease. Patient's history, clinical features, hematological and other laboratory parameters, date of diagnosis and start of treatment were recorded on a pre-designed performa.
Plasma isolation. From whole blood, plasma was isolated within 2-4 h after collection by two step centrifugation at 4 °C, first for 5 min at 2500×g and second for 2 min at 3500×g. The plasma was separated in RNase free 1.5 mL tubes considering recommended procedures for working on cell free miRNA 23 . Then, aliquots (each of 250 µL) were prepared for isolation of miRNA and down-stream process. The prepared aliquots were stored at − 80 °C till further analysis. miRNA isolation from plasma. Plasma was thawed on ice and processed to isolate miRNA according to manufacturer's instruction provided in the miRNeasy serum/plasma kit from Qiagen (Hilden, Germany). The syn-cel-miR-39 (synthetic caenorhabditis elegans mir-39) from miRNeasy serum/plasma spike-In control (Qiagen, Hilden, Germany) was added to each denatured sample as exogenous control for normalization of sampleto-sample variation during the RNAisolation 24,25 . The elution containing miRNA was stored at − 20 °C without delay for downstream process. The quality and quantity of RNAwas evaluated using NanoDrop by Thermo Scientific (USA). Quantification of RNAincluding miRNA was performed at 260 nm using RNase free water as a blank. The samples with absorbance ≥ 2 were considered for further analysis.
cDNA synthesis from RNA including miRNA. cDNA was synthesized by reversely transcribed isolated RNA including miRNA using master mix from miScript II RT kit from Qiagen (Hilden, Germany) using the protocols followed by the manufacturer. RNA with concentration of 100 ng (used as a template) was added to the master mix containing nucleic mix (10 × miScript) HiSpec buffer (5 × miScript), reverse transcriptase and RNase free water. Reaction was carried out in a total of 20 µL volume using thermo cycler from BioRad®. Reaction conditions for reverse transcription were first incubation of 60 min at 37 °C followed by second incubation of 5 min at 95 °C. The synthesized cDNA was carefully stored in microtubes at -20 °C for further use.
Quantitative real time polymerase chain reaction (qPCR). miRNA quantification was performed using relative quantitative method of qPCR. SYBR green dye from Qiagen (Hilden, Germany) was used to measure relative quantification of miRNA from plasma using protocols provided with the kit. Primer assays (10 × miScript) were purchased from Qiagen (Hilden, Germany) that contained forward sequences. Universal primer (available with SYBR green PCR kit) was used as a reverse primer for qPCR assays. The sequences of www.nature.com/scientificreports/ primers and their accession numbers were listed in Supplementary Table S1. Reactions were run in duplicate using real time thermal cycler, CFX96 from BioRad. Reference gene, hsa-mir-16, was used as a normalizer 26 and NTC (no template control) was run as a negative control in each run to control for contamination. Normalized fold change was calculated using Livak 2 −ΔΔCt method 27 that utilizes ΔCt (Ct miR-146a -Ct miR- 16) and ΔΔCt (Ct miR-146a -Ct miR-16 ) mean of the patients -(Ct miR-146a -Ct miR-16 ) mean of the controls.
Target prediction analysis of miR-146a. The total 3367 targets of miR-146a were retrieved from miRNA target database target scan (release 7.2) 28 . These targets were evaluated based on their target score and those with highest score were analyzed with MetaCore and STRING (Version 10.5) to search for targets related to ALL.
Statistical analysis. The data was analyzed using statistical tools like GraphPad prism software (version 7) and SPSS for windows (version 21). For validation of the results, Mann Whitney test, ANOVA (analysis of variance) and student's t test was applied depending upon different variables. Kruskal Wallis analyses was performed to compare multiple groups. The diagnostic value of miR-146a was evaluated through ROC curves produced by MedCalc (version 15.8). The potential of miR-146a as a prognostic factor was determined using Univariate analysis. Non-parametric spearman's correlation analyses were performed to evaluate clinical significance of miR-146a in ALL. The P-values obtained were two-tailed that considered significant at 0.05. Targets of miR-146a were determined through target predicting software, targetScan, and network and pathway was generated using gene ontology (GO) analysis including STRING and MetaCore software.
Ethical approval and consent to participate. The study was approved by the ethical review board of the University of the Punjab, Lahore, Pakistan with approval No. 284/15. The study design was constructed according to the guidelines of the declaration of Helsinki to conduct research on human beings. The samples were taken after informed consent form all the study participants.

Results
Characteristics of study subjects. Overall 110 study subjects were included in the study comprising of patients (n = 66), normal controls (n = 24) and follow up cases (n = 20 Circulating miR-146a is up-regulated in ALL. To explore potential role of circulating miR-146a in the development of ALL, we determined its normalized fold expression in plasma samples of patients and controls by relative quantification using real time PCR. Fold change calculated by 2 −ΔΔCt method revealed significant up-regulation of miR-146a in patients of ALL (33.46 ± 15.84) B-ALL (34.74 ± 16.19) and T-ALL (32.22 ± 15.66), compared with controls (1.36 ± 1.03) with a P-value of < 0.0001 (Fig. 1A). However, there was no significant difference of expression in ALL subtype, and childhood and adults in ALL, B-ALL, and T-ALL (P > 0.05) ( Fig. 2A1-C1). Moreover, no gender differences were observed in all the subtypes of ALL (P > 0.05) ( Fig. 2A2-C2). However, when categorized according to WHO risk classification, patients of ALL with high risk showed higher mean expression levels of mir-146a, compared with standard risk patients (P = 0.0469) (Fig. 2A3). The categorization based on risk also showed statistically significant differences in T-ALL (Fig. 2C3) although these differences were not significant in B-ALL (Fig. 2B3). We found that levels of plasma miR-146a in ALL patients were independent of subtype, age and gender. Furthermore, correlation analysis between age and miR-146a was insignificant (P < 0.05) that confirmed its importance as a diagnostic marker for childhood as well as adult ALL (Fig. 3A).
Plasma miR-146a levels are independent of hematological parameters. A remarkable feature of a diagnostic marker is that it should be independent of clinical relevance and show no evident change with clinical and hematological parameters. To identify this aspect, correlation analysis was performed between miR-146a levels and hematological parameters. We could not detect any significant correlation with hemoglobin, RBC's, WBC's, lymphocytes, neutrophils, platelets and peripheral blood blasts (P < 0.05) (Fig. 3B-H). The insignificant correlation proved that miR-146a levels in plasma were not affected by hematological parameters and could not influence its significance to use as a diagnostic marker for ALL.
Plasma miR-146a as a diagnostic marker for ALL. To evaluate diagnostic potential of plasma miR-146a in ALL, ROC (receiver operating curve) analyses were performed that showed area under curve (AUC) of 1 for ALL (95% CI 0.960 to 1.00) having 100% sensitivity, 100% specificity (P < 0.0001) (Fig. 1C). For B-and T-ALL, AUC of 1 was observed (95% CI 0.935 to 1.00) showing 100% sensitivity and 100% specificity (P < 0.0001). www.nature.com/scientificreports/ Plasma miR-146a as a prognostic marker for ALL. To evaluate prognostic impact of miR-146a in plasma of ALL, expression levels were analyzed before and after complete rounds of chemotherapeutic treatment in paired samples. It was noted that mean expression of miR-146a before chemotherapy was 33.46 ± 15.84, 34.74 ± 16.19 and 32.33 ± 15.66 in patients of ALL, B-ALL and T-ALL, respectively. Interestingly, these levels significantly decreased after treatment with a P value of < 0.0001 (Fig. 1B). Mean fold expression in male patients of ALL was 32.95 ± 15.74 that reduced to 0.8865 ± 0.754 showing significant difference (P < 0.0001) (Fig. 4A). Similarly, plasma miR-146a expression in females with ALL was 35.55 ± 16.68 that decreased to 1.61 ± 1.196 Table 1. Baseline characteristics of participants, demographic and clinical history of the patients enrolled in the study. The values are n = number and/or mean ± standard deviation (minimum-maximum). The data tabulated is analyzed by SPSS for windows (version 21).

Parameter Normal (n = 24) Pre-treatment (n = 20) Post-treatment (n = 20) P-value
Potential predictive targets of miR-146a. miR-146a targets a number of tumor suppressor genes that have shown their role in the pathogenesis of ALL. We first, selected targets of highest score (≥ 0.90) through targetScan and then analyzed these targets by STRING as shown (Fig. 5A). The genes predicted in the network were involved in cell cycle, proliferation, cell growth, development, DNA replication and repair mechanism and cell death. Further, we constructed a hypothetical pathway of these targets on MetaCore software for predicting novel targets for miR-146a (Fig. 5B). The round shape highlighted with red color showed the up-regulated targets that include

Discussion
There has been an emerging interest in miRNA to explore its potential as a predictive marker for diagnosis and prognosis of several hematological diseases including ALL. In the previous studies, miR-146a has shown a major role in the hematopoiesis, predominantly in T-cell development influencing proliferation and apoptosis of NB4 cells 18 . Another study developed a mice deficient with miR-146a and found some abnormal phenotypes in hematopoietic system indicating its potential role in hematopoiesis 19 . Aberrant miR-146a expression has been studied in ALL. A single report described decreased expression of miR-146a in blood and bone marrow of children with T-ALL 29 . All other reports showed up-regulation of miR-146a in bone marrow. We performed our experiment on plasma samples isolated from whole blood to determine whether miR-146a levels in plasma were overexpressed or otherwise. The targets with score > 0.9 and P < 1.0e−16 were selected to achieve highest confidence (B) Pathway analysis performed using MetaCore showed up-regulated targets genes of mir-146a highlighted with round shape (red). The thickness of lines showed the extent of interaction with the target molecules. www.nature.com/scientificreports/ In the current work, we, first, found up-regulation of miR-146a in plasma samples of childhood and adult cases of ALL, compared with healthy subjects as normal controls. Previously, overexpression of miR-146a has been reported in stem cell lines including monocytic and lymphocytic lineage after bone marrow transplant 17 . miR-146a has shown oncogenic role in childhood ALL as it has shown up-regulation in pediatric samples of ALL 16 . A study presented microarray results of miRNA profiling on bone marrow biopsy and peripheral blood samples of childhood ALL. After validation through qPCR revealed up-regulation of miR-146a in bone marrow biopsy but could not find its overexpression in blood samples 30 . A study on 49 bone marrow samples reported miR-146a overexpression in childhood cases of ALL 21 . We were able to determine its expression both in children as well as adults with ALL and its subtypes including B-and T-cell. Another study reported down-regulation of miR-146a expression in adult AML patients 17 that enhanced disease status by regulating TRAF6 involved in NF-κB pathway 31 .
Further, we analyzed clinical relevance of miR-146a with hematological parameters that showed no significant correlation with WBC's, RBC's, hemoglobin, platelets, neutrophils and lymphocytes. On the basis of these results, it may be suggested that miR-146a levels were not affected by the clinical features. These results are in agreement with the previous finding that also reported no clinical association with WBC's, hemoglobin and platelets count in ALL 32 .
The most striking feature of the current research was determination of miR-146a expression levels after treatment in paired samples of ALL. We were able to find significant reduction in miR-146a expression after chemotherapy demonstrating its prognostic significance in plasma samples of pediatrics and adults. Similar effects of treatment were reported on bone marrow samples by Duyu et al., who described post-treatment decrease in miR-146a expression in childhood ALL 30 . Association of poor survival with overexpression of miR-146a in adults of ALL has been described 33 . Moreover, favorable treatment outcome in ALL patients was related to miR-146a down-regulation 34 . The significant reduction in expression of miR-146a in pre-and post-treatment plasma samples indicated its prognostic significance and it was suggested that plasma miR-146a has a potential to predict treatment response and can be utilized as a prognostic marker of pediatric and adult ALL. miRNA may act as an oncogenic or tumor suppressor based on the target gene regulated by miRNA 13 . miR-146a is a dual nature miRNA showing both tumor suppressor and oncogenic properties in different types of leukemia. Studies reported its role as a tumor suppressor by regulating STAT1, an apoptotic factor, and Bcl-XL, an antiapoptotic factor, promoting apoptosis in jurkat cells in ALL cell lines 35 . It has been reported to target TRAF and IRAK6 that are regulatory genes of immune response 36 . Previously, the targets for mir-146a i.e., TRAF6, IRAK1 31 , and IRAK2 37 , were reported to be involved in immune system. We found the role of these targets in ALL that may serve as potential targets for treatment of ALL.

Conclusion
We concluded that miR-146a up-regulation in plasma may be utilized as a novel, non-invasive marker for diagnosis of ALL in childhood and adults. Furthermore, treatment response may be monitored through altered miR-146a expression levels that may improve prognosis of pediatric and adult ALL. The non-invasive diagnosis and prognosis may overcome poor overall survival of patients with ALL and good treatment outcome. miR-146a target prediction may also pave a way to understand the mechanisms in the regulation of gene expression establishing more effective targets for ALL therapy. Therefore, plasma miR-146a may be employed as a promising marker for diagnosis, prognosis and treatment of adult as well as pediatric ALL.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.