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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 over-expression reduces EZH2 levels and cell proliferation in eRMS cells. RT-qPCR analysis of mature a miR-101 and b EZH2 in RD and JR1 cells 72 h post infection with pS-pre-miR-101 or control pS- retrovirus. Data were normalized using snoU6 and GAPDH levels respectively and expressed as fold increase over control (pS-, 1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t-test). c Western blots showing EZH2 and p21Cip1 levels in RD and JR1 cells 72 h post infection with pS-pre-miR-101 or control pS- retrovirus. Total α-tubulin and GAPDH were used as loading controls. Representative of three independent experiments. d Flow cytometry analysis after propidium iodide (PI) staining 72 h post infection with pS-pre-miR-101 or control pS- retrovirus on RD and JR1 cells was performed. Ten thousand events per sample were acquired. The histogram depicts the fold change of cells in the G1, S, and G2 phases after normalization to the percentage of GPF-positive cells for each sample. Results are means ± SD of two independent experiments
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Fig3: MiR-101 over-expression reduces EZH2 levels and cell proliferation in eRMS cells. RT-qPCR analysis of mature a miR-101 and b EZH2 in RD and JR1 cells 72 h post infection with pS-pre-miR-101 or control pS- retrovirus. Data were normalized using snoU6 and GAPDH levels respectively and expressed as fold increase over control (pS-, 1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t-test). c Western blots showing EZH2 and p21Cip1 levels in RD and JR1 cells 72 h post infection with pS-pre-miR-101 or control pS- retrovirus. Total α-tubulin and GAPDH were used as loading controls. Representative of three independent experiments. d Flow cytometry analysis after propidium iodide (PI) staining 72 h post infection with pS-pre-miR-101 or control pS- retrovirus on RD and JR1 cells was performed. Ten thousand events per sample were acquired. The histogram depicts the fold change of cells in the G1, S, and G2 phases after normalization to the percentage of GPF-positive cells for each sample. Results are means ± SD of two independent experiments

Mentions: Next, we investigated in vitro whether miR-101 could regulate EZH2 expression in eRMS cells, as reported for other types of human cancers [12, 13]. We obtained an about 6-fold increase of miR-101 expression by infecting RD and JR1 cells with a GFP-coding retroviral vector expressing the pre-miR-101-2 form (pS-pre-miR-101) [19] (Fig. 3a and Additional file 2: Figure S2 for the efficiency of infection). Over-expression of miR-101 in eRMS cell lines induced a 30 % down-regulation of EZH2 mRNA and reduced protein levels compared to cells infected with an empty retrovirus (pS-) (Fig. 3b,c). Moreover, forced expression of miR-101 for 72 h resulted in the up-regulation of protein levels of the cyclin-dependent kinase inhibitor p21Cip1 (Fig. 3c). Therefore, we sought to evaluate whether miR-101 ectopic over-expression might affect eRMS cell proliferation. As reported in Fig. 3d, miR-101 over-expression determined a cell cycle G1/S blockade in RD cells whose percentage in G1 phase increased by 10 ± 3 % while in S and G2 phases decreased by 13 ± 2 and 2 ± 0.8 %, respectively, compared to pS- cells (Fig. 3d). These results are similar to those previously published by our group on RD cells after EZH2 silencing [11]. Interestingly, in JR1 cells in which miR-101 has been over-expressed, we noticed a cell cycle blockade in G2 phase (11.2 ± 1.8 % of increase), compared to pS- cells (Fig. 3d). Of note, the transcript levels of the oncogene N-Myc, a recognized miR-101 target gene in cancer [20] and involved in the aggressiveness of RMS [21], were markedly reduced in miR-101-over-expressing RD cells (Additional file 3: Figure S3A), confirming a targeted effect of miR-101 forced expression also in our setting. Altogether, these data suggest a reciprocal regulation between EZH2 and miR-101 in eRMS cells and indicate that miR-101 induction hampers their proliferative potential.Fig. 3


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 over-expression reduces EZH2 levels and cell proliferation in eRMS cells. RT-qPCR analysis of mature a miR-101 and b EZH2 in RD and JR1 cells 72 h post infection with pS-pre-miR-101 or control pS- retrovirus. Data were normalized using snoU6 and GAPDH levels respectively and expressed as fold increase over control (pS-, 1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t-test). c Western blots showing EZH2 and p21Cip1 levels in RD and JR1 cells 72 h post infection with pS-pre-miR-101 or control pS- retrovirus. Total α-tubulin and GAPDH were used as loading controls. Representative of three independent experiments. d Flow cytometry analysis after propidium iodide (PI) staining 72 h post infection with pS-pre-miR-101 or control pS- retrovirus on RD and JR1 cells was performed. Ten thousand events per sample were acquired. The histogram depicts the fold change of cells in the G1, S, and G2 phases after normalization to the percentage of GPF-positive cells for each sample. Results are means ± SD of two independent experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: MiR-101 over-expression reduces EZH2 levels and cell proliferation in eRMS cells. RT-qPCR analysis of mature a miR-101 and b EZH2 in RD and JR1 cells 72 h post infection with pS-pre-miR-101 or control pS- retrovirus. Data were normalized using snoU6 and GAPDH levels respectively and expressed as fold increase over control (pS-, 1 arbitrary unit). Columns, means; bars, SD. Results from three independent experiments are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t-test). c Western blots showing EZH2 and p21Cip1 levels in RD and JR1 cells 72 h post infection with pS-pre-miR-101 or control pS- retrovirus. Total α-tubulin and GAPDH were used as loading controls. Representative of three independent experiments. d Flow cytometry analysis after propidium iodide (PI) staining 72 h post infection with pS-pre-miR-101 or control pS- retrovirus on RD and JR1 cells was performed. Ten thousand events per sample were acquired. The histogram depicts the fold change of cells in the G1, S, and G2 phases after normalization to the percentage of GPF-positive cells for each sample. Results are means ± SD of two independent experiments
Mentions: Next, we investigated in vitro whether miR-101 could regulate EZH2 expression in eRMS cells, as reported for other types of human cancers [12, 13]. We obtained an about 6-fold increase of miR-101 expression by infecting RD and JR1 cells with a GFP-coding retroviral vector expressing the pre-miR-101-2 form (pS-pre-miR-101) [19] (Fig. 3a and Additional file 2: Figure S2 for the efficiency of infection). Over-expression of miR-101 in eRMS cell lines induced a 30 % down-regulation of EZH2 mRNA and reduced protein levels compared to cells infected with an empty retrovirus (pS-) (Fig. 3b,c). Moreover, forced expression of miR-101 for 72 h resulted in the up-regulation of protein levels of the cyclin-dependent kinase inhibitor p21Cip1 (Fig. 3c). Therefore, we sought to evaluate whether miR-101 ectopic over-expression might affect eRMS cell proliferation. As reported in Fig. 3d, miR-101 over-expression determined a cell cycle G1/S blockade in RD cells whose percentage in G1 phase increased by 10 ± 3 % while in S and G2 phases decreased by 13 ± 2 and 2 ± 0.8 %, respectively, compared to pS- cells (Fig. 3d). These results are similar to those previously published by our group on RD cells after EZH2 silencing [11]. Interestingly, in JR1 cells in which miR-101 has been over-expressed, we noticed a cell cycle blockade in G2 phase (11.2 ± 1.8 % of increase), compared to pS- cells (Fig. 3d). Of note, the transcript levels of the oncogene N-Myc, a recognized miR-101 target gene in cancer [20] and involved in the aggressiveness of RMS [21], were markedly reduced in miR-101-over-expressing RD cells (Additional file 3: Figure S3A), confirming a targeted effect of miR-101 forced expression also in our setting. Altogether, these data suggest a reciprocal regulation between EZH2 and miR-101 in eRMS cells and indicate that miR-101 induction hampers their proliferative potential.Fig. 3

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