Tumor suppressor microRNAs are downregulated in myelodysplastic syndrome with spliceosome mutations

// Derya Aslan 1 , Christian Garde 2 , Mette Katrine Nygaard 3 , Alexandra Sogaard Helbo 1 , Konstantinos Dimopoulos 1 , Jakob Werner Hansen 1 , Marianne Tang Severinsen 3 , Marianne Bach Treppendahl 1 , Lene Dissing Sjo 4 , Kirsten Gronbaek 1 , Lasse Sommer Kristensen 1 1 Department of Hematology, Rigshospitalet, Copenhagen, Denmark 2 Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark 3 Department of Hematology, Aalborg University Hospital, Aalborg, Denmark 4 Department of Pathology, Rigshospitalet, Copenhagen, Denmark Correspondence to: Lasse Sommer Kristensen, e-mail: lasse.sommer.kristensen@regionh.dk , lasse@biomed.au.dk Keywords: myelodysplastic syndrome, microRNAs, spliceosome mutations, RT-qPCR, high-resolution melting Received: August 23, 2015      Accepted: January 19, 2016      Published: February 02, 2016 ABSTRACT Spliceosome mutations are frequently observed in patients with myelodysplastic syndromes (MDS). However, it is largely unknown how these mutations contribute to the disease. MicroRNAs (miRNAs) are small noncoding RNAs, which have been implicated in most human cancers due to their role in post transcriptional gene regulation. The aim of this study was to analyze the impact of spliceosome mutations on the expression of miRNAs in a cohort of 34 MDS patients. In total, the expression of 76 miRNAs, including mirtrons and splice site overlapping miRNAs, was accurately quantified using reverse transcriptase quantitative PCR. The majority of the studied miRNAs have previously been implicated in MDS. Stably expressed miRNA genes for normalization of the data were identified using GeNorm and NormFinder algorithms. High-resolution melting assays covering all mutational hotspots within SF3B1 , SRSF2 , and U2AF1 ( U2AF35 ) were developed, and all detected mutations were confirmed by Sanger sequencing. Overall, canonical miRNAs were downregulated in spliceosome mutated samples compared to wild-type ( P = 0.002), and samples from spliceosome mutated patients clustered together in hierarchical cluster analyses. Among the most downregulated miRNAs were several tumor-suppressor miRNAs, including several let-7 family members, miR-423, and miR-103a. Finally, we observed that the predicted targets of the most downregulated miRNAs were involved in apoptosis, hematopoiesis, and acute myeloid leukemia among other cancer- and metabolic pathways. Our data indicate that spliceosome mutations may play an important role in MDS pathophysiology by affecting the expression of tumor suppressor miRNA genes involved in the development and progression of MDS.