Search

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with large heterogeneity at the clinical and molecular levels. As diagnostic procedures shift from bone marrow biopsies towards less invasive techniques, circulating small noncoding RNAs (sncRNAs) have become of particular interest as potential novel noninvasive biomarkers of the disease. We aimed to characterize the expression profiles of circulating sncRNAs of MDS patients and to search for specific RNAs applicable as potential biomarkers. We performed small RNA-seq in paired samples of total plasma and plasma-derived extracellular vesicles (EVs) obtained from 42 patients and 17 healthy controls and analyzed the data with respect to the stage of the disease, patient survival, response to azacitidine, mutational status, and RNA editing. Significantly higher amounts of RNA material and a striking imbalance in RNA content between plasma and EVs (more than 400 significantly deregulated sncRNAs) were found in MDS patients compared to healthy controls. Moreover, the RNA content of EV cargo was more homogeneous than that of total plasma, and different RNAs were deregulated in these two types of material. Differential expression analyses identified that many hematopoiesis-related miRNAs (e.g., miR-34a, miR-125a, and miR-150) were significantly increased in MDS and that miRNAs clustered on 14q32 were specifically increased in early MDS. Only low numbers of circulating sncRNAs were significantly associated with somatic mutations in the SF3B1 or DNMT3A genes. Survival analysis defined a signature of four sncRNAs (miR-1237-3p, U33, hsa_piR_019420, and miR-548av-5p measured in EVs) as the most significantly associated with overall survival (HR = 5.866, p < 0.001). In total plasma, we identified five circulating miRNAs (miR-423-5p, miR-126-3p, miR-151a-3p, miR-125a-5p, and miR-199a-3p) whose combined expression levels could predict the response to azacitidine treatment. In conclusion, our data demonstrate that circulating sncRNAs show specific patterns in MDS and that their expression changes during disease progression, providing a rationale for the potential clinical usefulness of circulating sncRNAs in MDS prognosis. However, monitoring sncRNA levels in total plasma or in the EV fraction does not reflect one another, instead, they seem to represent distinctive snapshots of the disease and the data should be interpreted circumspectly with respect to the type of material analyzed.

The DLK1–DIO3 region contains a large miRNA cluster, the overexpression of which has previously been associated with myelodysplastic syndromes (MDS). To reveal whether this overexpression is epigenetically regulated, we performed an integrative analysis of miRNA/mRNA expression and DNA methylation of the regulatory sequences in the region (promoter of the MEG3 gene) in CD34+ bone marrow cells from the patients with higher-risk MDS and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), before and during hypomethylating therapy with azacytidine (AZA). Before treatment, 50% of patients showed significant miRNA/mRNA overexpression in conjunction with a diagnosis of AML-MRC. Importantly, increased level of MEG3 was associated with poor outcome. After AZA treatment, the expression levels were reduced and were closer to those seen in the healthy controls. In half of the patients, we observed significant hypermethylation in a region preceding the MEG3 gene that negatively correlated with expression. Interestingly, this hypermethylation (when found before treatment) was associated with longer progression-free survival after therapy initiation. However, neither expression nor methylation status were associated with future responsiveness to AZA treatment. In conclusion, we correlated expression and methylation changes in the DLK1–DIO3 region, and we propose a complex model for regulation of this region in myelodysplasia.