{'fraction_non_alphanumeric': 0.04150680153470527, 'fraction_numerical': 0.03169066719816633, 'mean_word_length': 4.749069034660556, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Expression Analysis of Circulating miR-22, miR-122, miR-217 and miR-367 as Promising Biomarkers of Acute Lymphoblastic Leukemia

Background Currently, the role of serum-based biomarkers such as microRNAs in cancer diagnosis has been extensively established. This study aimed to determine expression levels of bioinformatically selected miRNAs and whether they can be used as biomarkers or a new therapeutic target in patients with Acute Lymphoblastic Leukemia (ALL). Methods The expression levels of serum miR-22, miR-122, miR-217, and miR-367 in 21 ALL patients and 21 healthy controls were measured using quantitative real-time PCR. The receiver operating characteristic (ROC) curve and the associated area under the curve (AUC) was used to assess candidate miRNAs' diagnostic value as a biomarker. Results The results showed that miR-217 was markedly decreased in patients with ALL compared to controls. Moreover, miR-22, miR-122, and miR-367 were found to be upregulated. Furthermore, ROC analysis showed that serum miR-217 and miR-367 could differentiate ALL patients from the healthy individuals, while miR-22 has approximate discriminatory power that requires further investigation. Collectively, the results suggested that miR-217 may play a tumor suppressor role in ALL, whereas miR-22, miR-122, and miR-367 could function as an oncogene. Overall, miR-22, miR-217, and miR-367 could be considered possible biomarkers for the early diagnosis ALL.


Introduction
Acute lymphoblastic leukemia (ALL) is a neoplastic abnormality of the lymphoid lineage known primarily as a childhood disease, with 75% occurrence in children under the age of 10 years (1). The underlying mechanisms of leukemia are numerous genetic mutations that lead to chromosomal abnormalities and malignant cell division and proliferation composed of immature and dysfunctional lymphocytes (2). Although inappropriate gene function and altered gene expression patterns are a hallmark of ALL, but not enough to cause leukemia and it is most likely caused by interaction between exogenous and endogenous factors, genetic or some hereditary factors, and chance (3).
Careful assessment of prognosis and risk factors and rapid diagnosis is essential for ALL management and successful treatment (1). Due to the lack of sensitivity and speci city of early-stage cancer diagnosis and being aggressive, tumor biopsy-based diagnostic methods are less commonly used to monitor the treatment of patients (4). More recently, the role of bio-uids-based biomarkers and liquid biopsy in cancer diagnosis has been extensively established due to their non-invasive protocols and ease to acquire (5). Since the discovery of the rst miRNA (a small non-coding single-stranded RNA molecule) called lin-4 (Lineage-4) in 1993 (6), numerous studies have shown that inappropriate miRNA expression is associated with many human diseases, including cancer (7,8). Interestingly, they can be as "oncomiRs (oncogenic miRNAs)" or "tumor suppressor miRNAs" and affect the signi cant carcinogenic pathways that are often involved in apoptosis, cell proliferation, angiogenesis, metastasis, and drug resistance (7). In addition, miRNAs could be considered as a biomarker of disease due to their tiny structure and stability under various conditions such as boiling, multiple freeze-thaw cycles, and pH changes (8), as well as secretion into extracellular bio uids including serum and plasma (9), saliva (10) urine, seminal uid, breast milk, cerebrospinal uid, bronchial lavage and also tears (11).
It has recently been shown that miRNA expression pro les vary across different types of leukemia and are associated with survival and prognosis (7). In this study, according to the genes and signaling pathways involved in the ALL, four related miRNAs (MiR-22, miR-122, miR-217, and miR-367) were selected using reliable predictive databases and -previous studies. Despite the therapeutic and diagnostic advances, there is still a frequent failure in ALL. Hence, there is an indispensable need to look at new diagnostic and treatment goals. Thus, this study aimed to assess the potential impact of miRNAs as serum biomarkers and or new therapeutic targets in patients with ALL.

Subjects
The study protocol was approved by the Medical Ethics Committee of Bushehr University of Medical Sciences, Bushehr, Iran (no. IR.BPUMS.REC.1397.059, approved October 22, 2019), and all tests were performed according to the relevant guidelines with prior informed consent from all participants. The current cross-sectional study included 21 patients with newly diagnosed ALL before any administration of chemotherapy, and 21 healthy controls without underlying disease of matched gender and age. Moreover, samples of the same ALL patients were enrolled in the study after one month of routine treatment. The serum of all specimens was separated immediately after blood collection and divided into aliquots to 1.5-mL cryotubes, then stored at -80°C until further use.

Bioinformatics analysis
In the present study, the possible role of miRNAs in ALL was investigated. For this purpose, several genes and signaling pathways involved in developing this disease were identi ed based on the literature. After that, the interaction of these pathways and genes with the expression of miRNAs was searched in databases. For further investigation, the DIANA TOOLS -miR Path v. three databases (http://www.microrna.gr/miRPathv3) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis were employed to predicts the candidate miRNAs targets of a particular function in diseases or pathways, based on three algorithms: Tarbase, MicroT-CDS, and Targetscan. Besides, miRWalk (http://mirwalk.uni-hd.de/) was used to get more information on interactions between miRNAs and target genes. Subsequently, the desired microRNAs were selected focusing on KEGG pathways and based on the results of the predictive sites.

miRNA isolation and cDNA synthesis
In the pre-cDNA synthesis steps, 200 µL serum was utilized to directly withdraw RNA from the serum using Tween-20 buffer as described by Asaga S and Hoon DS (12). Samples were transferred from -80°C to -20°C to prevent shock and kept at room temperature subsequently. They were centrifuged at 15,000 rpm for 10 min, then 10 µL of each sample's supernatant was incubated with 10 µL of Tween-20 buffer for 10 minutes at 70°C in a Dry Bath (Dolphin Co., India). After that, the samples were immediately placed on ice for Tween-20 settling and then centrifuged at 11,000 rpm for 2 minutes. Then, cDNA was synthesized using a reverse transcription kit according to the Oligo-dT method (BonYakhteh Co., Iran) as recommended in the manufacturer's instructions. The purity and quantity of cDNA was assessed spectrophotometrically as 260/280 nm and 260/230 nm ratios, using NanoDrop DS-11 (DeNovix Inc., Wilmington, DE USA).

Quantitative Real-time PCR (qRT-PCR)
The qRT-PCR was performed in duplicate using SYBR Green Master Mix (Yektatajhiz CO., Iran) and speci c primers for miR-22, 122, 217, and 367 (BonYakhteh Co., Iran) by the ABI StepOnePlus RT-PCR system (Applied Biosystems, USA). 0.5 µL of each forward and reverse primer were used for each reaction. The primer sequences are listed in Table.1.
The temperature and schedule were set as one primary denaturation at 95°C for 2 min followed by 45 cycles of ampli cation at 95°C for 5 s, and nally, 60°C for 30 s to con rm ampli cation speci city. Moreover, non-template control (NTC) was used for each primer to ensure the target gene ampli cation and lack of contamination during operation.

Statistical analysis
Data quality control was performed using boxplot to identify and modify outliers for the miRNAs expression levels in controls and patients. The relative quanti cation of target miRNAs expression was calculated using the 2 −ΔΔCt method (13), and cycle thresholds (Ct) values were normalized to miR-17 as an internal control. The Shapiro-Wilk test evaluated the normality of the data distribution. The unpaired T-test or Mann-Whitney test were performed to compare the differences in miRNAs expression between the healthy controls and patients and also paired T-test or Wilcoxon for comparison between the patient's group before and after treatment, using statistical package for social sciences (SPSS) 16 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7 (GraphPad Prism Software, Inc. San Diego CA, USA). Furthermore, the receiver operating characteristic (ROC) curve and the associated area under the curve (AUC) was used to determine diagnostic value of candidate miRNAs' as a biomarker. A p-value less than 0.05 was considered statistically signi cant for each test.

Characteristic of patients
The study population included 21 patients with newly diagnosed ALL, consisting of 13 men (61.9%) and 8 women (38.1%), and 21 age-and gender-matched healthy controls without underlying disease. Table.2 summarizes the demographic and laboratory characteristics of the patients at the time of diagnosis. According to the results, expression of miR-22 was signi cantly higher in the adult group, while there was no signi cant difference in the relative expression levels of other candidate miRNAs between men and women, children and adults, and the ALL subtypes. Moreover, no statistically signi cant correlation was observed between the expression of miRNAs with age, platelets count, white and red blood cell count, and hemoglobin.

Differential expression of serum miRNAs in newly diagnosed ALL patients
Levels of serum miRNAs were assessed by qPCR to evaluate their role in the diagnosis of ALL. As shown in Fig. 1A-C, relative serum miR-22 and miR-367 expression increased signi cantly and up-regulated in the newly diagnosed ALL group compared to control (P=0.01 and 0.002, respectively). Although the serum miR-122 levels showed increased expression, it was not statistically signi cant (P=0.06).
Moreover, we found that the relative levels of serum miR-217 expression were markedly decreased, and it was downregulated in the newly diagnosed ALL group compared to the controls (P<0.0001, Fig. 1D).

The changes in serum miRNAs expression in ALL patients following treatment
One month after the rst routine treatment of the patients, their blood samples were examined to evaluate the changes in the expression level of the miRNAs with the mentioned methods. The results demonstrated that the expression levels of miR-22, miR-122, and miR-367 in the follow-up groups were decreased compared to the rst examination (P=0.05, P=0.78, and P=0.005, respectively, Fig. 2A-C). Interestingly, the expression level of miR-217 was signi cantly higher in the follow-up group than in the preliminary assessment (P=0.01, Fig. 2D).
3.4. The diagnostic performance of candidate miRNAs for ALL patients ROC curve analysis was assessed to determine the speci city and sensitivity of each miRNA as a possible biomarker in the diagnosis and follow-up of ALL patients. To distinguish the newly diagnosed ALL group from the control, an AUC for miR-22, miR-122, miR-217 and miR-367 was found to be 0.7 with a sensitivity of 61% and a speci city of 71% (P=0.03, 95% CI: 0.69-0.94), 0.62 with a sensitivity of 33% and a speci city of 100% (P=0.17, 95% CI: 0.45-0.79), 0.81 with a sensitivity of 95% and a speci city of 61% (P=0, 95% CI: 0.69-0.94) and 0.8 with a sensitivity of 80% and a speci city of 81% (P=0.001, 95% CI: 0.65-0.94); respectively (Fig. 3A-D).

Logistic Regression Results
The data of qPCR were used for binary logistic regression. According to the results of ROC curves, miR-217 and miR-367 were selected to simultaneously evaluate the role of biomarkers due to their appropriate sensitivity and speci city and higher Yuden index in newly diagnosed ALL patients. The mean logit (P) value of the ALL group was found to be 0.83 and signi cantly higher than that of the control group, 0.17 (P<0.0001). The predicted values of logistic regression analysis showed that the combined ROC analysis of these two miRNA revealed an increased AUC value of 0.95, with a sensitivity of 95%, a speci city of 90%, and a Yuden index of 0.85 (P=0, 95% CI: 0.88-1.01; Fig. 5). A logit (P) of 0.50 was used for optimal cutoff by which ALL and controls were distinguished (Table.3).

Discussion
MiRNAs are currently well de ned as small regulatory molecules whose signi cant role is in post-transcriptional regulation of their target genes and play different crucial roles in physiological and pathological cellular processes (14). To date, the expression pro le and dysregulation of microRNAs has been studied in more than a dozen disease, particularly various types of human cancer (15)(16)(17). Since the aberrant expression of serum miRNAs in cancer was for the rst time described (18), their role in tumorigenesis has been highly regarded by researchers. Importantly, miRNAs are effective as biomarkers in classifying and tracing the source tissue of unknown primary and poorly differentiated cancers (19,20). These ndings emphasize the diagnostic value of miRNAs in cancers and also suggest their therapeutic role.
To the best of our knowledge, this study is the rst report on the assessment of serum miR-22, miR-122, miR-217, and miR-367 in ALL and the use of combine miR-217 and miR-367 as biomarkers in this disease. Speci cally, we found that the levels of miR-22, miR-122, and 367 were upregulated in ALL patients compared with controls, and additionally, miR-217 was markedly reduced. The analysis of ROC data revealed that serum miR-217 and miR-367 alone could be effective as a biomarker for early detection of ALL, while miR-22 has approximate discriminatory power that requires further investigation. Moreover, the combination of miR-217 and miR-367 further increased the diagnostic value and could distinguish newly diagnosed ALL patients from healthy controls.
Previous studies have examined the different roles of these miRNAs in various cancers. They have been described as double-faced molecules that can act as both a tumor suppressor and an oncogene. Bar N et al. demonstrated that overexpression of miR-22 can affect the tumor suppressor, i.e., PTEN and its downstream pathways PI3/AKT, thus inhibit AKT proliferative and anti-apoptotic activity (21). Another study by Song et al. reported that miR-22 is a crucial regulator for hematopoiesis maintenance, and it was found to be upregulated in myelodysplastic syndrome and leukemia associated with a poor prognosis. In this study, the TET-2 gene was mentioned as a target of miR-22, which its deletion is involved in hematologic malignancy (22). Given that PTEN activity is lost in many types of cancer, leading to increased cell survival and cell cycle progression (23), as well as the relationship between TET-2 and hematopoietic cell proliferation (24), it seems that miR-22 upregulation may lead to ALL due to its effect on these genes. Conversely, miR-22 was mentioned as a tumor suppressor in a study on acute myeloid leukemia (AML), which affects the MYC pathways (25). Due to the various functions of miRNAs in different tissues, a discrepancy in the results is conceivable. Furthermore, we found that the expression level of miR-22 was not signi cantly related to the patient's sex and laboratory information but was higher in the adult group, which could be related to the severity of ALL in adults. In a similar study on AML, a relationship between miR-22 and sex, platelet count, and disease subtypes was not reported, whereas a signi cant relationship was found with white blood cells and cytogenetic (26). According to the present study results, a marked downregulation of miR-217 was observed that con rmed the tumor suppressor action of miR-217. Moreover, we found a signi cant difference in the expression level of miR-217 in the post-treatment group compared to before treatment, which may be due to the effects of the treatment process. Our results are consistent with a previous study that demonstrated that the expression of miR-217 is decreased in colorectal cancer with the effect of the MAPK pathway, which regulates tumor growth and increases apoptosis (31). Other studies have identi ed AKT3 (32), Runx2 (33), E2F3 (34), and KRAS (35) as targets of miR-217. The interaction of miR-217 and these genes appear to be involved in pathogenicity in ALL. By contrast, in the study by Yebenes et al., miR-217 was mentioned as an oncogene that increases speci cally in the germinal center of lymph nodes. According to this study, miR-217 reduces the expression of genes that respond to DNA damage and its repair and stabilizes the BCL-6 expression (36). Although the ROC analysis in a present study showed that miR-217 is favorable as a biomarker for diagnosing and following up the patients with ALL, it could be more potent in diagnosing. In line with these results, in a study by Yan et al., the ROC analysis revealed that miR-217 could consider as a promising biomarker with high sensitivity and speci city in AML (37).
In the previous study, miR-367 has been identi ed as a regulator of embryonic stem cell self-renewality and pluripotency, which induces the trait of being stemness in the cancer cell and contributes to the progression and spread of cancer (38). Consistent with the present study's data, Kaid et al. showed that miR-367 plays an oncogenic role in medulloblastoma cells by affecting OCT4 transcription factor, leading to poor survival and poor prognosis (39). In another similar study, miR-367 was mentioned as an oncogene that targets tumor suppressor KLF4. It could be blocked at both mRNA and protein levels and its subsequent pathways, BAX and caspase-3, leading to suppress apoptosis (40). Most studies indicated that miR-367 acts primarily as an oncogene. However, contrary to the present study's ndings, it was shown that the expression of miR-367 was signi cantly reduced in gastric cancer tissues (41) and oral carcinoma cells (42).
According to the results of ROC analysis and based on the sensitivity, speci city, and Yuden index, we found that among the studied miRNAs, miR-217 and miR-367 have a more desirable ability to diagnose ALL, and a combination of these two miRNAs could reach a higher sensitivity and speci city. The limitations of our study were; limited access to newly diagnosed ALL patients, small sample sizes, and lack of access to patients' follow-up samples after the treatment process, which was due to the patient's death or referral to out-of-reach medical centers.

Conclusion
In conclusion, our results suggest that miR-217 and miR-367 could consider as potential new biomarkers for ALL diagnosing, as well as a new therapeutic target. Further comprehensive studies are warranted to con rm these results and investigate the possible mechanisms and role of genes associated with these miRNAs in ALL. Data availability:

Abbreviations
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Tables   Table. 1 List of primer sequences used for RT-PCR analysis in this study.  Levels of serum miRNAs were assessed by qPCR to evaluate their role in the diagnosis of ALL. As shown in Fig. 1A-C, relative serum miR-22 and miR-367 expression increased signi cantly and up-regulated in the newly diagnosed ALL group compared to control (P=0.01 and 0.002, respectively). Although the serum miR-122 levels showed increased expression, it was not statistically signi cant (P=0.06). Moreover, we found that the relative levels of serum miR-217 expression were markedly decreased, and it was down-regulated in the newly diagnosed ALL group compared to the controls (P<0.0001, Fig. 1D).

Figure 2
The results demonstrated that the expression levels of miR-22, miR-122, and miR-367 in the follow-up groups were decreased compared to the rst examination (P=0.05, P=0.78, and P=0.005, respectively, Fig. 2A-C). Interestingly, the expression level of miR-217 was signi cantly higher in the follow-up group than in the preliminary assessment (P=0.01, Fig. 2D).