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

Tarnowski,Maciej; Tkacz,Marta; Czerewaty,Michał; Poniewierska‑Baran,Agata; Grymuła,Katarzyna; Ratajczak,Mariusz Z.

doi:10.3892/ijo.2015.2906

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

Authors:
- Maciej Tarnowski
- Marta Tkacz
- Michał Czerewaty
- Agata Poniewierska‑Baran
- Katarzyna Grymuła
- Mariusz Z. Ratajczak
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Affiliations: Department of Physiology Pomeranian Medical University, Szczecin, Poland, Stem Cell Biology Program at the James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA
Published online on: February 24, 2015 https://doi.org/10.3892/ijo.2015.2906
Pages: 2241-2250

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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.

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