Circulating miR-92a, miR-143 and miR-342 in Plasma are Novel Potential Biomarkers for Acute Myeloid Leukemia

MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional gene expression regulators. The expression profiling of miRNAs has already entered into cancer clinics as diagnostic and prognostic biomarkers to assess tumor initiation, progression and response to treatment in cancer patients. Recent studies have opened the way for the use of circulating miRNAs as non-invasive diagnosis and prognosis of Acute myeloid leukemia (AML). The aim of this study was to identify plasma miR-92a, miR-143 and miR-342 expression signatures in AML patients to introduce new markers for establishing AML diagnosis and prognosis. Blood samples were collected from 65 AML patients and 50 controls. The expression of three target miRNAs (miR-92a, miR-143 and miR-342) was measured using quantitative real-time PCR method. Plasma levels of miR-92a, miR-143 and miR-342 were significantly lower in AML patients in comparison with control group. Receiver operator characteristic (ROC) analysis revealed that the sensitivity and specificity values of miR-92a were 81.5% and 94%, respectively, with a cut-off value of 0.704. The sensitivity and specificity values of miR-143 were 87.7% and 80%, respectively, with a cut-off value of 0.65. The sensitivity and specificity values of miR-342 were 75.4% and 90%, respectively, with a cut-off value of 0.479. Our findings suggest that plasma miR-92a, miR-143 and miR-342 could be promising novel circulating biomarkers in clinical detection of AML.

. MiRNAs are involved in numerous physiological and biological functions such as proliferation, differentiation, organogenesis, embryogenesis, and apoptosis (7). Moreover, various diseases have shown aberrant expression and dysregulation of miRNAs, for example, cancer, lung, renal and hepatic diseases (8,9).
Dysregulation of miRNAs plays a major role in oncogenesis through the abnormal activation of oncogenes and the silencing of tumor suppressor genes. Therefore, several studies suggested the therapeutic targeting of specific miRNAs as a potential modality of suppressing tumors (10).
Furthermore, expression profiles of miRNAs in plasma and serum have been used as non-invasive biomarkers to categorize specific types of cancers (11). Recent studies have highlighted the aberrant expression profiles of various miRNAs in different tissues including plasma in AML patients. Hence, plasma miRNAs represent novel biomarkers and valuable tools in diagnosing AML and estimating prognosis of AML patients (12,13).
Several studies reported that miR-92a, miR-143 and miR-342 were dysregulated in different AML cell lines and tumor samples (14)(15)(16)(17). Thus, studying the expression profile of these miRNAs in plasma presents new non-invasive biomarkers for this type of hematologic malignancy. In the present study, we examined alterations in the expression of miR-92a, miR-143 and miR-342 in plasma samples from AML patients and healthy volunteers to determine the significance of these miRNAs as diagnostic tools for AML. We also analyzed the correlation between the expression of these miRNAs and the clinicopathological features of AML.

Patients and samples
The present study was conducted on 65 plasma samples provided by AML patients, who were clinically diagnosed with AML and confirmed by bone marrow aspiration and biopsy at Tanta  Table 1. Blood (10 ml) was drawn on Ethylene diamine tetra acetic acid (EDTA) from an antecubital vein in AML patients or healthy control subjects using standard percutaneous venipuncture. Plasma was separated by centrifugation at 3000 rpm for 10 min at 4 °C, and then recentrifuged at 5000 rpm for 5 min to obtain cell-free plasma, which was stored at −80 C until use. Only non-hemolyzed plasma samples were used in our study, as haemolysis affects miRNA levels. Hemolysis was detected by spectral analysis at 541 nm (19,20).

Plasma miRNA assay
Total RNA with preserved miRNAs was

Statistical analysis
Comparisons of quantitative variables were performed using the nonparametric Mann-Whitney U test when comparing AML group and control group. The P-values less than 0.05 were considered as statistically significant. Statistical comparison among AML subgroups was performed by one-way analysis of variance (Anova). When a significant overall p value (P<0.05) was present, differences between individual means were tested using Fisher's least-significant differences (LSD).
Spearman's rank correlation coefficient was also performed to find the correlations between the expression of miRNAs and other variables, e.g. age, gender, and FAB classification. Receiver operator characteristic (ROC) curves were derived and areaunder-the curve (AUC) analysis was performed to

Expression levels of miRNAs
The data presented in Figure 1 shows that there is a significant decrease in the expression levels of miR-92a, miR-143 and miR-342 in AML patients compared with the control group (P< 0.001). The ratios of the plasma miRNAs in AML patients to healthy controls are displayed in AML subtypes showed significant decrease in the three miRNAs expression levels compared to healthy normal subjects ( Figure 2). As only one AML case with M7 classification was recruited, therefore, group M7 was not included in the post hoc tests because there was a standard deviation of this group.
In addition, no significant correlations were  found between the expression levels of any of the three miRNAs and age, gender, and FAB classification in AML patients, except for miR-92a.

Discussion
In AML patients, early detection and timely intervention can increase survival chances.  In conclusion, this study shows a reduction in miR-92a, miR-143 and miR-342 levels in plasma of AML patients. The combination of the plasma markers miR-92a, miR-143 and miR-342 represents a very promising screening test for AML diagnosis.
Although testing with these markers reached high sensitivity and specificity for AML prediction, there is a need to compare the results with other types of cancer to clarify whether these miRNAs are useful as specific markers for AML.