title: Multiple myeloma cells alter the senescence phenotype of bone marrow mesenchymal stromal cells under participation of the DLK1-DIO3 genomic region.
abstract: BACKGROUND
  Alterations and senescence in bone marrow mesenchymal stromal cells of multiple myeloma patients (MM-BMMSCs) have become an important research focus. However the role of senescence in the pathophysiology of MM is not clear.
  
  
  METHODS
  Correlation between senescence, cell cycle and microRNA expression of MM-BMMSCs (n = 89) was analyzed. Gene expression analysis, copy number analysis and methylation specific PCR were performed by Real-Time PCR. Furthermore, cyclin E1, cyclin D1, p16 and p21 genes were analyzed at the protein level using ELISA. Cell cycle and senescence were analyzed by FACS. MiRNA transfection was performed with miR-485-5p inhibitor and mimic followed by downstream analysis of senescence and cell cycle characteristics of MM-BMMSCs. Results were analyzed by Mann-Whitney U test, Wilcoxon signed-rank test and paired t-test depending on the experimental set up.
  
  
  RESULTS
  MM-BMMSCs displayed increased senescence associated β-galactosidase activity (SA-βGalA), cell cycle arrest in S phase and overexpression of microRNAs. The overexpressed microRNAs miR-485-5p and miR-519d are located on DLK1-DIO3 and C19MC, respectively. Analyses revealed copy number accumulation and hypomethylation of both clusters. KMS12-PE myeloma cells decreased SA-βGalA and influenced cell cycle characteristics of MM-BMMSCs. MiR-485-5p was significantly decreased in co-cultured MM-BMMSCs in connection with an increased methylation of DLK1-DIO3. Modification of miR-485-5p levels using microRNA mimic or inhibitor altered senescence and cell cycle characteristics of MM-BMMSCs.
  
  
  CONCLUSIONS
  Here, we show for the first time that MM-BMMSCs have aberrant methylation and copy number of the DLK1-DIO3 and C19MC genomic region. Furthermore, this is the first study pointing that multiple myeloma cells in vitro reduce both the senescence phenotype of MM-BMMSCs and the expression of miR-223 and miR-485-5p. Thus, it is questionable whether senescence of MM-BMMSCs plays a pathological role in active multiple myeloma or is more important when cell interaction with myeloma cells is inhibited. Furthermore, we found that MiR-485-5p, which is located on the DLK1-DIO3 cluster, seems to participate in the regulation of senescence status and cell cycle characteristics of MM-BMMSCs. Thus, further exploration of the microRNAs of DLK1-DIO3 could provide further insights into the origin of the senescence state and its reversal in MM-BMMSCs.
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany. rimma.berenstein@charite.de.
authors.forename: Rimma
authors.initials: R
authors.lastname: Berenstein
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany. olga.blau@charite.de.
authors.forename: Olga
authors.initials: O
authors.lastname: Blau
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany. axel.nogai@charite.de.
authors.forename: Axel
authors.initials: A
authors.lastname: Nogai
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany. marlies.waechter@charite.de.
authors.forename: Marlies
authors.initials: M
authors.lastname: Waechter
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany. ekaterina.slonova@charite.de.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Slonova
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Helios Clinic Berlin-Buch, Berlin, Germany. martin.schmidt-hieber@charite.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Schmidt-Hieber
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany. annegret.kunitz@charite.de.
authors.forename: Annegret
authors.initials: A
authors.lastname: Kunitz
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany. antonio.pezzutto@charite.de.
authors.forename: Antonio
authors.initials: A
authors.lastname: Pezzutto
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany. Bernd.doerken@charite.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Doerken
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany. Igor.blau@charite.de.
authors.forename: Igor Wolfgang
authors.initials: IW
authors.lastname: Blau
mesh_terms: D002453:Cell Cycle; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D016922:Cellular Senescence; D018920:Coculture Techniques; D056915:DNA Copy Number Variations; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D035683:MicroRNAs / Q000235:genetics*; D009101:Multiple Myeloma / Q000235:genetics / Q000473:pathology*
chemical_list: D035683:MicroRNAs; C525067:MIR223, human; C556501:MIRN485 microRNA, human; C546194:MIRN519 microRNA, human