title: 5‑Azacytidine inhibits human rhabdomyosarcoma cell growth by downregulating insulin‑like growth factor 2 expression and reactivating the H19 gene product miR‑675, which negatively affects insulin‑like growth factors and insulin signaling.
abstract: Insulin-like growth factor 2 (IGF2) and 1 (IGF1) and insulin (INS) promote proliferation of rhabdomyosarcoma (RMS) cells by interacting with the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor (INSR). Loss of imprinting (LOI) by DNA hypermethylation at the differentially methylated region (DMR) for the IGF2‑H19 locus is commonly observed in RMS cells and results in an increase in the expression of proliferation-promoting IGF2 and downregulation of proliferation-inhibiting non-coding H19 miRNAs. One of these miRNAs, miR‑675, has been reported in murine cells to be a negative regulator of IGF1R expression. To better address the role of IGF2 and 1, as well as INS signaling in the pathogenesis of RMS and the involvement of LOI at the IGF2‑H19 locus, we employed the DNA demethylating agent 5‑azacytidine (AzaC). We observed that AzaC‑mediated demethylation of the DMR at the IGF2‑H19 locus resulted in downregulation of IGF2 and an increase in the expression of H19. This epigenetic change resulted in a decrease in RMS proliferation due to downregulation of IGF2 and, IGF1R expression in an miR‑675‑dependent manner. Interestingly, we observed that miR‑675 not only inhibited the expression of IGF1R in a similar manner in human and murine cells, but we also observed its negative effect on the expression of the INSR. These results confirm the crucial role of LOI at the IGF2‑H19 DMR in the pathogenesis of RMS and are relevant to the development of new treatment strategies.
authors.affiliation: Department of Physiology Pomeranian Medical University, Szczecin, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Tarnowski
authors.affiliation: Department of Physiology Pomeranian Medical University, Szczecin, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Tkacz
authors.affiliation: Department of Physiology Pomeranian Medical University, Szczecin, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Czerewaty
authors.affiliation: Department of Physiology Pomeranian Medical University, Szczecin, Poland.
authors.forename: Agata
authors.initials: A
authors.lastname: Poniewierska-Baran
authors.affiliation: Department of Physiology Pomeranian Medical University, Szczecin, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Grymuła
authors.affiliation: Stem Cell Biology Program at the James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA.
authors.forename: Mariusz Z
authors.initials: MZ
authors.lastname: Ratajczak
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000502:physiology; D001374:Azacitidine / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000502:physiology; D019175:DNA Methylation; D015536:Down-Regulation; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D018392:Genomic Imprinting / Q000502:physiology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis*; D035683:MicroRNAs / Q000096:biosynthesis*; D062085:RNA, Long Noncoding / Q000187:drug effects; D011972:Receptor, Insulin / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012208:Rhabdomyosarcoma / Q000473:pathology*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; C094335:H19 long non-coding RNA; C035170:IGF2 protein, human; D007328:Insulin; C548379:MIRN675 microRNA, human; D035683:MicroRNAs; D062085:RNA, Long Noncoding; D007335:Insulin-Like Growth Factor II; D011972:Receptor, Insulin; D001374:Azacitidine