Hypoxia Inducible microRNA-210 Regulates the DIMT1-IRF4 Oncogenetic Axis in Multiple Myeloma

Background: It is known that gene expression patterns in tumor cells are heterogeneous even in the same individual, not only in solid tumors but also in hematological malignancies, including multiple myeloma (MM). MM is characterized by the accumulation of a population of malignant plasma cells within the bone marrow (BM) and microenvironment (endosteal and vascular niches). Hypoxic oxygen stress occurs in the microenvironment, leading to various epigenetic gene alterations. For instance, hypoxia lead to cell cycle arrest, glycolysis, epithelial-mesenchymal transition (EMT)-related machinery, drug resistance and immature phenotype decreasing expression of CD138 and IRF4. Recently, it is reported that a microRNA (miRNA) in exosomes of myeloma treated in hypoxia induces vessel formation and promotes survival of myeloma cells via upregulation of HIF-1. However, to the best of our knowledge, there have been no reports describing expression changes of miRNAs in myeloma cells during hypoxia. The aim of this study was to detect epigenetically regulated miRNA and essential targets that could be affected by hypoxia in MM.

Methods: To identify hypoxia-induced miRNA(s) and their target gene(s), we conducted whole miRNA and mRNA microarray screening using myeloma cell lines (MM.1S, RPMI-8226, KMS12BM, KMS11, and U266) and primary samples (n=4) that were subjected to chronic hypoxia conditions (1% O₂ for 48 hr). We purified CD38⁺⁺ cells (CD38: a marker for activated B-cells and plasma cells) from primary samples, and then conducted functional analysis of target gene/proteins of identified hypoxia inducible miRNA.

Results: We recognized a significant upregulation of microRNA-210 (miR-210) under chronic hypoxia. We explored target genes of miR-210 as well as downregulated genes by hypoxia comprehensively, and we found that five candidate genes including DIMT1, CIAPIN1, TTC13, ARMC1, and NOL12 by TargetScan program. Among these, 18S rRNA base methyltransferase DIMT1, which is necessary for the production of ribosome, was the most likely candidate target of miR-210 in myeloma cells because the product was only directly reduced by miR-210 transduction of myeloma cells. We confirmed significant upregulation of miR-210 and downregulation of DIMT1 in primary samples (n=15) cultured in hypoxia by q-RT-PCR. In the examination of the myeloma patient samples on several GEO data sets, we found that expression of DIMT1 increased significantly with disease progression, and that DIMT1 had a positive correlation of IRF4. Notably, immunohistochemical analysis revealed that DIMT1 was strongly stained at myeloma cells but not other BM cells including various lymphocytes. The knock down of DIMT1 for MM.1S, H929, and KMS11 cell lines lead to apoptosis and downregulation of IRF4. Interestingly, miR-210 transduction also reduces expression of IRF4. Furthermore, we found that pan-HDAC inhibitors (panobinostat and vorinostat) inhibit cell survival via downregulation of the DIMT1-IRF4 axis in myeloma cells.

Conclusion: By screening for targets of hypoxia inducible miR-210, we identified DIMT1 as a novel diagnostic marker and therapeutic target for all molecular subtypes of MM. Our results suggest that the master antiapoptosis regulator might be different between normoxia and hypoxia. In hypoxia, HIFs and/or the HIF-miR-210 axis may convert myeloma cells to an antiapoptotic phenotype. However, in normoxia, DIMT1 was highly expressed in myeloma cells and, herein, activation might convert myeloma cells to the antiapoptotic phenotype.

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