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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 forced expression as well as EZH2 pharmacological inhibition reduces eRMS cell migration. RD (a) and JR1 (c) cells were infected with pS-pre-miR-101 or control pS- retrovirus. Twenty-four hours post-infection cells were seeded on inserts and lived to reach the confluence for 48 h, when the inserts were removed. Cells were imaged at 0 and 24 h or 36 h after the insert removal. RD (b) and JR1 (d) were treated with DZNep (5 μM) or vehicle (i.e., water, referred as untreated condition: UN) for 72 h and then inserts were removed and cells were imaged as in (a) and (b). Representative phase contrast microscopy images of the migration assays at 0 and 24 h or 36 h after gap creation were shown. Dashed lines indicate the boundary of the edges of the wound at 0 and 24 h or 36 h. The histograms depict the measurements of the total area between the wound edges of the scratch from at least five random fields per scratch from two separate experiments, expressed as fold change over control pS- (a and c, 1 arbitrary unit) or untreated (UN) (b and d, 1 arbitrary unit) samples. Columns, means; bars, SD. *P < 0.05 (Student’s t-test)
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Fig4: MiR-101 forced expression as well as EZH2 pharmacological inhibition reduces eRMS cell migration. RD (a) and JR1 (c) cells were infected with pS-pre-miR-101 or control pS- retrovirus. Twenty-four hours post-infection cells were seeded on inserts and lived to reach the confluence for 48 h, when the inserts were removed. Cells were imaged at 0 and 24 h or 36 h after the insert removal. RD (b) and JR1 (d) were treated with DZNep (5 μM) or vehicle (i.e., water, referred as untreated condition: UN) for 72 h and then inserts were removed and cells were imaged as in (a) and (b). Representative phase contrast microscopy images of the migration assays at 0 and 24 h or 36 h after gap creation were shown. Dashed lines indicate the boundary of the edges of the wound at 0 and 24 h or 36 h. The histograms depict the measurements of the total area between the wound edges of the scratch from at least five random fields per scratch from two separate experiments, expressed as fold change over control pS- (a and c, 1 arbitrary unit) or untreated (UN) (b and d, 1 arbitrary unit) samples. Columns, means; bars, SD. *P < 0.05 (Student’s t-test)

Mentions: The miR-101 tumor-suppressive activities have been also related to its ability to negatively modulate tumor cell migration [22–24]. Therefore, we decide to evaluate the effects of miR-101 over-expression on the migratory potential of eRMS cells in a wound healing assay. The 24-h migration rate of pS-pre-miR-101-infected cells was reduced of about 40 and 30 % for RD and JR1 cells, respectively, compared to pS- cells (Fig. 4a,c). To determine whether EZH2 might be involved in their migratory capability, eRMS cells were treated with DZNep and the migration rate measured. As observed for miR-101 over-expression, EZH2 pharmacological inhibition reduced eRMS cell migration (70 and 35 % reduction for RD and JR1 cells, respectively) (Fig. 4b,d). In agreement with the effects of DZNep on miR-101 expression reported in Fig. 2b, DZNep-treated RD cells showed a down-regulation of the miR-101 target gene N-Myc (Additional file 3: Figure S3B). Altogether, these findings suggest that miR-101 and EZH2 regulate the migration of eRMS cells in an opposite manner.Fig. 4


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 forced expression as well as EZH2 pharmacological inhibition reduces eRMS cell migration. RD (a) and JR1 (c) cells were infected with pS-pre-miR-101 or control pS- retrovirus. Twenty-four hours post-infection cells were seeded on inserts and lived to reach the confluence for 48 h, when the inserts were removed. Cells were imaged at 0 and 24 h or 36 h after the insert removal. RD (b) and JR1 (d) were treated with DZNep (5 μM) or vehicle (i.e., water, referred as untreated condition: UN) for 72 h and then inserts were removed and cells were imaged as in (a) and (b). Representative phase contrast microscopy images of the migration assays at 0 and 24 h or 36 h after gap creation were shown. Dashed lines indicate the boundary of the edges of the wound at 0 and 24 h or 36 h. The histograms depict the measurements of the total area between the wound edges of the scratch from at least five random fields per scratch from two separate experiments, expressed as fold change over control pS- (a and c, 1 arbitrary unit) or untreated (UN) (b and d, 1 arbitrary unit) samples. Columns, means; bars, SD. *P < 0.05 (Student’s t-test)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4527101&req=5

Fig4: MiR-101 forced expression as well as EZH2 pharmacological inhibition reduces eRMS cell migration. RD (a) and JR1 (c) cells were infected with pS-pre-miR-101 or control pS- retrovirus. Twenty-four hours post-infection cells were seeded on inserts and lived to reach the confluence for 48 h, when the inserts were removed. Cells were imaged at 0 and 24 h or 36 h after the insert removal. RD (b) and JR1 (d) were treated with DZNep (5 μM) or vehicle (i.e., water, referred as untreated condition: UN) for 72 h and then inserts were removed and cells were imaged as in (a) and (b). Representative phase contrast microscopy images of the migration assays at 0 and 24 h or 36 h after gap creation were shown. Dashed lines indicate the boundary of the edges of the wound at 0 and 24 h or 36 h. The histograms depict the measurements of the total area between the wound edges of the scratch from at least five random fields per scratch from two separate experiments, expressed as fold change over control pS- (a and c, 1 arbitrary unit) or untreated (UN) (b and d, 1 arbitrary unit) samples. Columns, means; bars, SD. *P < 0.05 (Student’s t-test)
Mentions: The miR-101 tumor-suppressive activities have been also related to its ability to negatively modulate tumor cell migration [22–24]. Therefore, we decide to evaluate the effects of miR-101 over-expression on the migratory potential of eRMS cells in a wound healing assay. The 24-h migration rate of pS-pre-miR-101-infected cells was reduced of about 40 and 30 % for RD and JR1 cells, respectively, compared to pS- cells (Fig. 4a,c). To determine whether EZH2 might be involved in their migratory capability, eRMS cells were treated with DZNep and the migration rate measured. As observed for miR-101 over-expression, EZH2 pharmacological inhibition reduced eRMS cell migration (70 and 35 % reduction for RD and JR1 cells, respectively) (Fig. 4b,d). In agreement with the effects of DZNep on miR-101 expression reported in Fig. 2b, DZNep-treated RD cells showed a down-regulation of the miR-101 target gene N-Myc (Additional file 3: Figure S3B). Altogether, these findings suggest that miR-101 and EZH2 regulate the migration of eRMS cells in an opposite manner.Fig. 4

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