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Abstract
BackgroundRhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma arising from myogenic precursors that have lost their capability to differentiate into skeletal muscle. The polycomb-group protein EZH2 is a Lys27 histone H3 methyltransferase that regulates the balance between cell proliferation and differentiation by epigenetically silencing muscle-specific genes. EZH2 is often over-expressed in several human cancers acting as an oncogene. We previously reported that EZH2 inhibition induces cell cycle arrest followed by myogenic differentiation of RMS cells of the embryonal subtype (eRMS). MiR-101 is a microRNA involved in a negative feedback circuit with EZH2 in different normal and tumor tissues. To that, miR-101 can behave as a tumor suppressor in several cancers by repressing EZH2 expression. We, therefore, evaluated whether miR-101 is de-regulated in eRMS and investigated its interplaying with EZH2 as well as its role in the in vitro tumorigenic potential of these tumor cells.ResultsHerein, we report that miR-101 is down-regulated in eRMS patients and in tumor cell lines compared to their controls showing an inverse pattern of expression with EZH2. We also show that miR-101 is up-regulated in eRMS cells following both genetic and pharmacological inhibition of EZH2. In turn, miR-101 forced expression reduces EZH2 levels as well as restrains the migratory potential of eRMS cells and impairs their clonogenic and anchorage-independent growth capabilities. Finally, EZH2 recruitment to regulatory region of miR-101-2 gene decreases in EZH2-silenced eRMS cells. This phenomenon is associated to reduced H3K27me3 levels at the same regulatory locus, indicating that EZH2 directly targets miR-101 for repression in eRMS cells.ConclusionsAltogether, our data show that, in human eRMS, miR-101 is involved in a negative feedback loop with EZH2, whose targeting has been previously shown to halt eRMS tumorigenicity. They also demonstrate that the re-induction of miR-101 hampers the tumor features of eRMS cells. In this scenario, epigenetic dysregulations confirm their crucial role in the pathogenesis of this soft tissue sarcoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0107-z) contains supplementary material, which is available to authorized users.
Highlights
Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma arising from myogenic precursors that have lost their capability to differentiate into skeletal muscle
MiR-101 expression was lower in four embryonal RMS (eRMS) cell lines (RD, RD18, JR1, RUCH2) than in differentiated human skeletal muscle cells (SKMC differentiating medium (DM)), instead being comparable to the level of miR-101 in proliferating skeletal myoblasts (SKMC Growth Medium (GM)) (Fig. 1b, left)
To analyze whether miR-101 expression was affected by enhancer of zeste homologue 2 (EZH2) modulation in eRMS, RD, JR1, and RD18 cell lines were silenced for EZH2 using either a pool of oligo small interfering RNA (siRNA) or oligo siRNA targeting the 5′UTR region of EZH2 mRNA, both previously validated (Additional file 1: Figures S1A and S1B) [11], and the expression of miR-101 together with that of other microRNAs known to be modulated by EZH2 in RMS, such as miR-214 and miR-29b [3, 8], was evaluated
Summary
Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma arising from myogenic precursors that have lost their capability to differentiate into skeletal muscle. The polycomb-group protein EZH2 is a Lys histone H3 methyltransferase that regulates the balance between cell proliferation and differentiation by epigenetically silencing muscle-specific genes. We and others recently reported that EZH2 is markedly expressed in RMS primary specimens and cell lines compared to their normal counterparts [9, 10] and that inhibition of EZH2 represents a promising pro-differentiation therapeutic strategy in eRMS [11]. Our data demonstrate that EZH2 inhibits miR-101 expression in eRMS cells by direct gene targeting. These results suggest a negative feedback loop between miR-101 and EZH2 in eRMS cells and point on miR-101 as a potential anticancer microRNA
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