To gain insights into a possible role of microRNAs in myeloproliferative neoplasms, we performed short RNA massive sequencing and extensive bioinformatic analysis in the JAK2V617F-mutated SET2 cell line. Overall, 652 known mature miRNAs were detected, of which 21 were highly expressed, thus being responsible of most of miRNA-mediated gene repression. microRNA putative targets were enriched in specific signaling pathways, providing information about cell activities under massive posttranscriptional regulation. The majority of miRNAs were mixtures of sequence variants, called isomiRs, mainly because of alternative, noncanonical processing of hairpin precursors. We also identified 78 novel miRNAs (miRNA*) derived from known hairpin precursors. Both major and minor (*) forms of miRNAs were expressed concurrently from half of expressed hairpins, highlighting the relevance of miRNA* and the complexity of strand selection bias regulation. Finally, we discovered that SET2 cells express a number of miRNA-offset RNAs (moRNAs), short RNAs derived from genomic regions flanking mature miRNAs. We provide novel data about the possible origin of moRNAs, although their functional role remains to be elucidated. Overall, this study shed light on the complexity of microRNA-mediated gene regulation in SET2 cells and represents the basis for future studies in JAK2V617F-mutated cellular models.