The Potential of Extracellular Vesicle Derived microRNAs As a Biomarker in Acute Myeloid Leukemia

Abstract

Purpose MicroRNAs (miRNAs) are small non-coding RNA molecules that target messenger RNAs (mRNAs) for cleavage or translational repression, and function in RNA silencing and post-transcriptional regulation of gene expression. Extracellular vesicle (EV)-derived miRNAs are miRNAs present in EVs, and compared to non-EV-encapsulated miRNAs, EV-encapsulated miRNAs could be more valid as biomarkers because they are highly protected from degradation. Several studies have shown that EV-derived miRNAs may be involved in the pathogenesis of acute myeloid leukemia (AML) and can be used as biomarkers for the early detection of AML recurrence or prognosis. However, most of these studies were conducted based on laboratory research (cell line-based or animal studies) and used relatively low purity EV isolation methods, such as sequential ultracentrifugation or precipitation. In this study, we isolated miRNA in EVs from the bone marrow (BM) blood of patients with AML using a high-purity EV isolation method and attempted to validate its potential as a biomarker.

Methods BM blood (serum or plasma) from 32 patients with AML was collected at the time of AML diagnosis, and EVs were isolated using size-exclusion chromatography (EXo-I, Exopert, South Korea). Total RNA was isolated from each sample using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany). Libraries were prepared using SMARTer smRNA for Illumina kit (Takara Bio, Shiga, Japan), and sequencing was performed using Illumina HiSeq 2500 (Illumina, San Diego, CA, USA). Following sequence alignment, known and novel small RNAs were retrieved using the miRDeep2 software algorithm. All data processing and visualization were conducted using R 4.0.3 (http://www.r-project.org).

Results A total of 965 EV-derived miRNAs were identified in all samples. We analyzed the expression levels of these EV-derived miRNAs between the favorable (n=10) and unfavorable (intermediate and adverse, n=22) risk groups based on the 2017 European LeukemiaNet recommendations and identified 32 differentially expressed EV-derived miRNAs in the unfavorable risk group (23 downregulated and 11 upregulated). Validation was performed by qPCR analysis (top ten significantly downregulated miRNAs and all upregulated miRNAs in the unfavorable risk group), and 20 out of a total of 21 selected EV-derived miRNAs were confirmed to be present in the samples. To evaluate the potential of selected miRNAs as biomarkers, we investigated the correlation of these miRNAs with risk stratification and the survival of patients with AML using information from the TCGA database. Among the downregulated miRNAs in the unfavorable risk group, hsa-mir-181b and hsa-mir-143 were downregulated and correlated with unfavorable risk and shortening the overall survival. In the case of upregulated miRNAs in the unfavorable risk group, hsa-mir-188 and hsa-mir-501 were upregulated and correlated with unfavorable risk but not survival. In the miRNA-mRNA network analysis, hsa-mir-181b, hsa-mir-143, hsa-mir-188, and hsa-mir-501 were related to PLA2G4A, SCHIP1, RAB27B, DDIT4, LGALSL, CALCRL, PDE7B, GLIS3, STOX2, and ADAMTS3, all of which were significantly correlated with the survival of patients with AML in TCGA database analysis.

Conclusion Overall, the findings of this study revealed that EV-derived miRNAs may serve as biomarkers for risk stratification and prognosis in AML. However, further studies are required to validate these EV-derived miRNAs in other cohorts before their implementation in clinical practice.