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MicroRNA-101 is repressed by EZH2 and its restoration inhibits tumorigenic features in embryonal rhabdomyosarcoma.

Vella S, Pomella S, Leoncini PP, Colletti M, Conti B, Marquez VE, Strillacci A, Roma J, Gallego S, Milano GM, Capogrossi MC, Bertaina A, Ciarapica R, Rota R - Clin Epigenetics (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.

ABSTRACT

Background: 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 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.

Results: Herein, 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.

Conclusions: Altogether, 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.

No MeSH data available.


Related in: MedlinePlus

MiR-101 and EZH2 levels are inversely expressed in embryonal rhabdomyosarcoma (RMS) patients and cell lines compared to their controls. a Levels of mature miR-101 (left panel) and EZH2 (right panel) were determined by RT-qPCR in primary embryonal rhabdomyosarcoma (eRMS) samples (black and grey bars, respectively) and in normal skeletal muscles (M1-4) used as control tissues (white bars). Values normalized to snoU6 or GAPDH levels (respectively) were expressed as fold increase over M1 control tissue (1 arbitrary unit). b RT-qPCR of miR-101 (left panel) and EZH2 (right panel) in eRMS cell lines (RD, RD18, RUCH2, and JR1; black and grey bars, respectively) and normal skeletal muscle cells (SKMC) cultured in either growth medium (GM) or differentiating medium (DM) (as described in “Methods” section) were normalized to snoU6 or GAPDH levels, respectively, and were expressed as fold increase over SKMC GM cells (1 arbitrary unit). Two independent measurements were done in duplicate
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Fig1: MiR-101 and EZH2 levels are inversely expressed in embryonal rhabdomyosarcoma (RMS) patients and cell lines compared to their controls. a Levels of mature miR-101 (left panel) and EZH2 (right panel) were determined by RT-qPCR in primary embryonal rhabdomyosarcoma (eRMS) samples (black and grey bars, respectively) and in normal skeletal muscles (M1-4) used as control tissues (white bars). Values normalized to snoU6 or GAPDH levels (respectively) were expressed as fold increase over M1 control tissue (1 arbitrary unit). b RT-qPCR of miR-101 (left panel) and EZH2 (right panel) in eRMS cell lines (RD, RD18, RUCH2, and JR1; black and grey bars, respectively) and normal skeletal muscle cells (SKMC) cultured in either growth medium (GM) or differentiating medium (DM) (as described in “Methods” section) were normalized to snoU6 or GAPDH levels, respectively, and were expressed as fold increase over SKMC GM cells (1 arbitrary unit). Two independent measurements were done in duplicate

Mentions: To ascertain whether miR-101 expression is compromised in eRMS, we measured its levels along with those of EZH2 in primary tumors. We noticed that miR-101 was expressed at very low levels in eRMS primary samples compared to normal muscle tissues as controls (mean values: 0.23 ± 0.24 vs 5.7 ± 4.7, respectively) (Fig. 1a, left). Conversely, in line with previous reports [9, 10], EZH2 transcripts were markedly higher in the same group of primary samples compared to controls (mean values: 21.25 ± 8.86 vs 2.87 ± 1.31, respectively) (Fig. 1a, right). Similarly, miR-101 expression was lower in four eRMS cell lines (RD, RD18, JR1, RUCH2) than in differentiated human skeletal muscle cells (SKMC DM) (mean values 1.26 ± 0.49 vs 4.29 ± 0.55, respectively), instead being comparable to the level of miR-101 in proliferating skeletal myoblasts (SKMC GM) (Fig. 1b, left). Moreover, EZH2 mRNA levels were 11.76 ± 2.23 higher in the eRMS cell lines tested compared to SKMC (Fig. 1b, right).Fig. 1


MicroRNA-101 is repressed by EZH2 and its restoration inhibits tumorigenic features in embryonal rhabdomyosarcoma.

Vella S, Pomella S, Leoncini PP, Colletti M, Conti B, Marquez VE, Strillacci A, Roma J, Gallego S, Milano GM, Capogrossi MC, Bertaina A, Ciarapica R, Rota R - Clin Epigenetics (2015)

MiR-101 and EZH2 levels are inversely expressed in embryonal rhabdomyosarcoma (RMS) patients and cell lines compared to their controls. a Levels of mature miR-101 (left panel) and EZH2 (right panel) were determined by RT-qPCR in primary embryonal rhabdomyosarcoma (eRMS) samples (black and grey bars, respectively) and in normal skeletal muscles (M1-4) used as control tissues (white bars). Values normalized to snoU6 or GAPDH levels (respectively) were expressed as fold increase over M1 control tissue (1 arbitrary unit). b RT-qPCR of miR-101 (left panel) and EZH2 (right panel) in eRMS cell lines (RD, RD18, RUCH2, and JR1; black and grey bars, respectively) and normal skeletal muscle cells (SKMC) cultured in either growth medium (GM) or differentiating medium (DM) (as described in “Methods” section) were normalized to snoU6 or GAPDH levels, respectively, and were expressed as fold increase over SKMC GM cells (1 arbitrary unit). Two independent measurements were done in duplicate
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4527101&req=5

Fig1: MiR-101 and EZH2 levels are inversely expressed in embryonal rhabdomyosarcoma (RMS) patients and cell lines compared to their controls. a Levels of mature miR-101 (left panel) and EZH2 (right panel) were determined by RT-qPCR in primary embryonal rhabdomyosarcoma (eRMS) samples (black and grey bars, respectively) and in normal skeletal muscles (M1-4) used as control tissues (white bars). Values normalized to snoU6 or GAPDH levels (respectively) were expressed as fold increase over M1 control tissue (1 arbitrary unit). b RT-qPCR of miR-101 (left panel) and EZH2 (right panel) in eRMS cell lines (RD, RD18, RUCH2, and JR1; black and grey bars, respectively) and normal skeletal muscle cells (SKMC) cultured in either growth medium (GM) or differentiating medium (DM) (as described in “Methods” section) were normalized to snoU6 or GAPDH levels, respectively, and were expressed as fold increase over SKMC GM cells (1 arbitrary unit). Two independent measurements were done in duplicate
Mentions: To ascertain whether miR-101 expression is compromised in eRMS, we measured its levels along with those of EZH2 in primary tumors. We noticed that miR-101 was expressed at very low levels in eRMS primary samples compared to normal muscle tissues as controls (mean values: 0.23 ± 0.24 vs 5.7 ± 4.7, respectively) (Fig. 1a, left). Conversely, in line with previous reports [9, 10], EZH2 transcripts were markedly higher in the same group of primary samples compared to controls (mean values: 21.25 ± 8.86 vs 2.87 ± 1.31, respectively) (Fig. 1a, right). Similarly, miR-101 expression was lower in four eRMS cell lines (RD, RD18, JR1, RUCH2) than in differentiated human skeletal muscle cells (SKMC DM) (mean values 1.26 ± 0.49 vs 4.29 ± 0.55, respectively), instead being comparable to the level of miR-101 in proliferating skeletal myoblasts (SKMC GM) (Fig. 1b, left). Moreover, EZH2 mRNA levels were 11.76 ± 2.23 higher in the eRMS cell lines tested compared to SKMC (Fig. 1b, right).Fig. 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.

ABSTRACT

Background: 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 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.

Results: Herein, 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.

Conclusions: Altogether, 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.

No MeSH data available.


Related in: MedlinePlus