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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

Inhibition of EZH2 restores endogenous miR-101 levels in eRMS cells. a RT-qPCR analysis of mature forms of miR-214, miR-29b, and miR-101 in RD, JR1, and RD18 cells 72 h post EZH2 siRNA transfection (RD were transfected with SMART pool siRNA EZH2 (asterisks), JR1 and RD18 were transfected with siRNA targeting 5′-UTR of EZH2, see “Methods” section). Data normalized using snoU6 and expressed as fold increase over a non-targeting siRNA (CTR siRNA, 1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05 (Student’s t-test). b MiR-101 level in RD, JR1, and RD18 cells daily treated for 72 h with either S-adenosyl-L-homocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) (5 μM) or vehicle (i.e., water, referred as untreated condition: UN), normalized using snoU6 and expressed as fold increase over UN (1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05 (Student’s t-test)
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Fig2: Inhibition of EZH2 restores endogenous miR-101 levels in eRMS cells. a RT-qPCR analysis of mature forms of miR-214, miR-29b, and miR-101 in RD, JR1, and RD18 cells 72 h post EZH2 siRNA transfection (RD were transfected with SMART pool siRNA EZH2 (asterisks), JR1 and RD18 were transfected with siRNA targeting 5′-UTR of EZH2, see “Methods” section). Data normalized using snoU6 and expressed as fold increase over a non-targeting siRNA (CTR siRNA, 1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05 (Student’s t-test). b MiR-101 level in RD, JR1, and RD18 cells daily treated for 72 h with either S-adenosyl-L-homocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) (5 μM) or vehicle (i.e., water, referred as untreated condition: UN), normalized using snoU6 and expressed as fold increase over UN (1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05 (Student’s t-test)

Mentions: To analyze whether miR-101 expression was affected by EZH2 modulation in eRMS, RD, JR1, and RD18 cell lines were silenced for EZH2 using either a pool of oligo siRNAs 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. As shown in Fig. 2a, EZH2 knockdown in eRMS cells increases the expression of miR-29b, miR-214, and miR-101 as soon as 72 h after siRNA transfection. Interestingly, miR-101 was the most up-regulated in RD and RD18 cells, showing an about 4-fold increase compared with cells transfected with a control non-targeting siRNA (CTR siRNA). Similarly, treatment with DZNep, the prototype of EZH2 inhibitors [11] which induces EZH2 degradation (Additional file 1: Figure S1C), resulted in the up-regulation of miR-101 with respect to cells treated with vehicle (about 3.5-, 1.5-, and 5-fold increase in RD, JR1, and RD18, respectively) (Fig. 2b). These results suggest that miR-101 and EZH2 are inversely expressed in eRMS and indicate EZH2 as a repressor of miR-101 in eRMS cells.Fig. 2


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)

Inhibition of EZH2 restores endogenous miR-101 levels in eRMS cells. a RT-qPCR analysis of mature forms of miR-214, miR-29b, and miR-101 in RD, JR1, and RD18 cells 72 h post EZH2 siRNA transfection (RD were transfected with SMART pool siRNA EZH2 (asterisks), JR1 and RD18 were transfected with siRNA targeting 5′-UTR of EZH2, see “Methods” section). Data normalized using snoU6 and expressed as fold increase over a non-targeting siRNA (CTR siRNA, 1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05 (Student’s t-test). b MiR-101 level in RD, JR1, and RD18 cells daily treated for 72 h with either S-adenosyl-L-homocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) (5 μM) or vehicle (i.e., water, referred as untreated condition: UN), normalized using snoU6 and expressed as fold increase over UN (1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05 (Student’s t-test)
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Fig2: Inhibition of EZH2 restores endogenous miR-101 levels in eRMS cells. a RT-qPCR analysis of mature forms of miR-214, miR-29b, and miR-101 in RD, JR1, and RD18 cells 72 h post EZH2 siRNA transfection (RD were transfected with SMART pool siRNA EZH2 (asterisks), JR1 and RD18 were transfected with siRNA targeting 5′-UTR of EZH2, see “Methods” section). Data normalized using snoU6 and expressed as fold increase over a non-targeting siRNA (CTR siRNA, 1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05 (Student’s t-test). b MiR-101 level in RD, JR1, and RD18 cells daily treated for 72 h with either S-adenosyl-L-homocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) (5 μM) or vehicle (i.e., water, referred as untreated condition: UN), normalized using snoU6 and expressed as fold increase over UN (1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05 (Student’s t-test)
Mentions: To analyze whether miR-101 expression was affected by EZH2 modulation in eRMS, RD, JR1, and RD18 cell lines were silenced for EZH2 using either a pool of oligo siRNAs 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. As shown in Fig. 2a, EZH2 knockdown in eRMS cells increases the expression of miR-29b, miR-214, and miR-101 as soon as 72 h after siRNA transfection. Interestingly, miR-101 was the most up-regulated in RD and RD18 cells, showing an about 4-fold increase compared with cells transfected with a control non-targeting siRNA (CTR siRNA). Similarly, treatment with DZNep, the prototype of EZH2 inhibitors [11] which induces EZH2 degradation (Additional file 1: Figure S1C), resulted in the up-regulation of miR-101 with respect to cells treated with vehicle (about 3.5-, 1.5-, and 5-fold increase in RD, JR1, and RD18, respectively) (Fig. 2b). These results suggest that miR-101 and EZH2 are inversely expressed in eRMS and indicate EZH2 as a repressor of miR-101 in eRMS cells.Fig. 2

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