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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 is directly targeted by EZH2 in RD cells. a, b Two independent ChIP assays on RD cells 72 h after EZH2 or CTR siRNA transfection showing the recruitment of EZH2 and histone H3 trimethylation on Lys27 (H3K27me3) levels on miR-101-2 promoter region and MCK regulatory regions. SMAD6 was the negative control gene. Rabbit IgG was used as a negative immunoprecipitation control. Histograms represent the percent of immunoprecipitated material relative to input DNA of the two independent experiments. c mRNA levels (RT-qPCR) of pri-miR-101-2 in RD cells 72 h after EZH2 siRNA treatment were normalized to GAPDH levels and expressed as fold increase over CTR siRNA. d Proposed model depicting the interplay between EZH2 and miR-101 in both normal myogenic differentiation (left) and eRMS (right). In muscle cells, when myogenesis is triggered, miR-101 is upregulated due to the lowering of EZH2 expression. Then, miR-101 directly inhibits EZH2 expression thus enforcing its own expression, driving late skeletal muscle differentiation. In eRMS, this circuit is dysregulated due to EZH2 over-expression, which leads to miR-101 down-regulation, thus maintaining the cells in an undifferentiated and proliferative state
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Fig6: MiR-101 is directly targeted by EZH2 in RD cells. a, b Two independent ChIP assays on RD cells 72 h after EZH2 or CTR siRNA transfection showing the recruitment of EZH2 and histone H3 trimethylation on Lys27 (H3K27me3) levels on miR-101-2 promoter region and MCK regulatory regions. SMAD6 was the negative control gene. Rabbit IgG was used as a negative immunoprecipitation control. Histograms represent the percent of immunoprecipitated material relative to input DNA of the two independent experiments. c mRNA levels (RT-qPCR) of pri-miR-101-2 in RD cells 72 h after EZH2 siRNA treatment were normalized to GAPDH levels and expressed as fold increase over CTR siRNA. d Proposed model depicting the interplay between EZH2 and miR-101 in both normal myogenic differentiation (left) and eRMS (right). In muscle cells, when myogenesis is triggered, miR-101 is upregulated due to the lowering of EZH2 expression. Then, miR-101 directly inhibits EZH2 expression thus enforcing its own expression, driving late skeletal muscle differentiation. In eRMS, this circuit is dysregulated due to EZH2 over-expression, which leads to miR-101 down-regulation, thus maintaining the cells in an undifferentiated and proliferative state

Mentions: Since EZH2 down-regulation by either gene silencing or pharmacological inhibition induces miR-101 up-regulation (Fig. 2), we asked whether EZH2 might directly repress the expression of miR-101 in eRMS. To test this hypothesis, we performed chromatin immunoprecipitation (ChIP) experiments upon EZH2 silencing in RD and JR1 cells testing the occupancy of EZH2 on the promoter of miR-101-2 that codifies for the miR-101 precursor pri-miR-101-2 from which we derived the pre-miR-101-2 vector used for over-expression experiments [25, 26]. As shown in two independent experiments reported in Fig. 6a, b, the promoter of the miR-101-2 was occupied by EZH2 in RD cells and upon EZH2 siRNA, in accordance with EZH2 binding reduction to the promoter, also the level of H3K27me3 resulted strikingly reduced. Similar results were obtained in JR1 cells (Additional file 5: Figure S5). EZH2 silencing induced the up-regulation of the pri-miR-101-2 in RD (Fig. 6c) and in JR1 (Additional file 5: Figure S5B) cells further corroborating the de-repression effect of EZH2 depletion.Fig. 6


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 is directly targeted by EZH2 in RD cells. a, b Two independent ChIP assays on RD cells 72 h after EZH2 or CTR siRNA transfection showing the recruitment of EZH2 and histone H3 trimethylation on Lys27 (H3K27me3) levels on miR-101-2 promoter region and MCK regulatory regions. SMAD6 was the negative control gene. Rabbit IgG was used as a negative immunoprecipitation control. Histograms represent the percent of immunoprecipitated material relative to input DNA of the two independent experiments. c mRNA levels (RT-qPCR) of pri-miR-101-2 in RD cells 72 h after EZH2 siRNA treatment were normalized to GAPDH levels and expressed as fold increase over CTR siRNA. d Proposed model depicting the interplay between EZH2 and miR-101 in both normal myogenic differentiation (left) and eRMS (right). In muscle cells, when myogenesis is triggered, miR-101 is upregulated due to the lowering of EZH2 expression. Then, miR-101 directly inhibits EZH2 expression thus enforcing its own expression, driving late skeletal muscle differentiation. In eRMS, this circuit is dysregulated due to EZH2 over-expression, which leads to miR-101 down-regulation, thus maintaining the cells in an undifferentiated and proliferative state
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Related In: Results  -  Collection

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Fig6: MiR-101 is directly targeted by EZH2 in RD cells. a, b Two independent ChIP assays on RD cells 72 h after EZH2 or CTR siRNA transfection showing the recruitment of EZH2 and histone H3 trimethylation on Lys27 (H3K27me3) levels on miR-101-2 promoter region and MCK regulatory regions. SMAD6 was the negative control gene. Rabbit IgG was used as a negative immunoprecipitation control. Histograms represent the percent of immunoprecipitated material relative to input DNA of the two independent experiments. c mRNA levels (RT-qPCR) of pri-miR-101-2 in RD cells 72 h after EZH2 siRNA treatment were normalized to GAPDH levels and expressed as fold increase over CTR siRNA. d Proposed model depicting the interplay between EZH2 and miR-101 in both normal myogenic differentiation (left) and eRMS (right). In muscle cells, when myogenesis is triggered, miR-101 is upregulated due to the lowering of EZH2 expression. Then, miR-101 directly inhibits EZH2 expression thus enforcing its own expression, driving late skeletal muscle differentiation. In eRMS, this circuit is dysregulated due to EZH2 over-expression, which leads to miR-101 down-regulation, thus maintaining the cells in an undifferentiated and proliferative state
Mentions: Since EZH2 down-regulation by either gene silencing or pharmacological inhibition induces miR-101 up-regulation (Fig. 2), we asked whether EZH2 might directly repress the expression of miR-101 in eRMS. To test this hypothesis, we performed chromatin immunoprecipitation (ChIP) experiments upon EZH2 silencing in RD and JR1 cells testing the occupancy of EZH2 on the promoter of miR-101-2 that codifies for the miR-101 precursor pri-miR-101-2 from which we derived the pre-miR-101-2 vector used for over-expression experiments [25, 26]. As shown in two independent experiments reported in Fig. 6a, b, the promoter of the miR-101-2 was occupied by EZH2 in RD cells and upon EZH2 siRNA, in accordance with EZH2 binding reduction to the promoter, also the level of H3K27me3 resulted strikingly reduced. Similar results were obtained in JR1 cells (Additional file 5: Figure S5). EZH2 silencing induced the up-regulation of the pri-miR-101-2 in RD (Fig. 6c) and in JR1 (Additional file 5: Figure S5B) cells further corroborating the de-repression effect of EZH2 depletion.Fig. 6

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