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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 colony formation and anchorage-independent growth capabilities in eRMS cells. RD (a) and JR1 (b) cells were infected with pS-pre-miR-101 or control pS- retrovirus and, 72 h later, seeded to examine their clonogenic capability 2 weeks post seeding (see “Methods” section). Histograms depict the number of colonies per plate from four independent experiments. Representative pictures of stained colonies were shown on the right. RD (c) and JR1 (d) cells, infected as in (a) and (b), were seeded on soft agar for an anchorage-independent growth assay. Colonies were visible. Histograms depict the number of colonies per plate after 4 weeks of incubation, calculated as means ± SD from four independent experiments. Columns, means; bars, SD. *P < 0.05, **P < 0.01 (Student’s t-test)
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Fig5: MiR-101 over-expression reduces colony formation and anchorage-independent growth capabilities in eRMS cells. RD (a) and JR1 (b) cells were infected with pS-pre-miR-101 or control pS- retrovirus and, 72 h later, seeded to examine their clonogenic capability 2 weeks post seeding (see “Methods” section). Histograms depict the number of colonies per plate from four independent experiments. Representative pictures of stained colonies were shown on the right. RD (c) and JR1 (d) cells, infected as in (a) and (b), were seeded on soft agar for an anchorage-independent growth assay. Colonies were visible. Histograms depict the number of colonies per plate after 4 weeks of incubation, calculated as means ± SD from four independent experiments. Columns, means; bars, SD. *P < 0.05, **P < 0.01 (Student’s t-test)

Mentions: As a negative regulator of proliferation and migration, miR-101 is predicted to reduce tumorigenicity of eRMS cells. To test whether miR-101 restoration might restrain the clonogenic ability of eRMS cells, we performed colony formation assays with RD and JR1 cells over-expressing miR-101. As reported in Fig. 5a,b, miR-101 over-expression reduced of 30 % of the ability to form colonies in both RD and JR1 cells. Then, we evaluated the capability of eRMS cell lines over-expressing miR-101 to grow as colonies in soft agar in an anchorage-independent manner, indicative of malignant transformation and considered an in vitro surrogate of the in vivo tumorigenicity testing. As shown in Fig. 5c, d, miR-101 over-expression reduced the formation of colonies in soft agar of about 50 % in both RD and JR1 cells. Consistently, miR-101 over-expressing RD18 cells showed 50 % EZH2 down-regulation associated to cell cycle slow-down (5.4 ± 0.6 % increase of cells in the G1 phase and 14 ± 2 and 3.4 ± 0.6 % decrease in S and G2 phases, respectively) and a more modest but significant reduction of colony formation of about 20 and 15 % on either in culture dishes or soft agar (Additional file 4: Figure S4). Taken together, these results indicate that restoration of miR-101 in eRMS exerts an antitumor effect in vitro.Fig. 5


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 colony formation and anchorage-independent growth capabilities in eRMS cells. RD (a) and JR1 (b) cells were infected with pS-pre-miR-101 or control pS- retrovirus and, 72 h later, seeded to examine their clonogenic capability 2 weeks post seeding (see “Methods” section). Histograms depict the number of colonies per plate from four independent experiments. Representative pictures of stained colonies were shown on the right. RD (c) and JR1 (d) cells, infected as in (a) and (b), were seeded on soft agar for an anchorage-independent growth assay. Colonies were visible. Histograms depict the number of colonies per plate after 4 weeks of incubation, calculated as means ± SD from four independent experiments. Columns, means; bars, SD. *P < 0.05, **P < 0.01 (Student’s t-test)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig5: MiR-101 over-expression reduces colony formation and anchorage-independent growth capabilities in eRMS cells. RD (a) and JR1 (b) cells were infected with pS-pre-miR-101 or control pS- retrovirus and, 72 h later, seeded to examine their clonogenic capability 2 weeks post seeding (see “Methods” section). Histograms depict the number of colonies per plate from four independent experiments. Representative pictures of stained colonies were shown on the right. RD (c) and JR1 (d) cells, infected as in (a) and (b), were seeded on soft agar for an anchorage-independent growth assay. Colonies were visible. Histograms depict the number of colonies per plate after 4 weeks of incubation, calculated as means ± SD from four independent experiments. Columns, means; bars, SD. *P < 0.05, **P < 0.01 (Student’s t-test)
Mentions: As a negative regulator of proliferation and migration, miR-101 is predicted to reduce tumorigenicity of eRMS cells. To test whether miR-101 restoration might restrain the clonogenic ability of eRMS cells, we performed colony formation assays with RD and JR1 cells over-expressing miR-101. As reported in Fig. 5a,b, miR-101 over-expression reduced of 30 % of the ability to form colonies in both RD and JR1 cells. Then, we evaluated the capability of eRMS cell lines over-expressing miR-101 to grow as colonies in soft agar in an anchorage-independent manner, indicative of malignant transformation and considered an in vitro surrogate of the in vivo tumorigenicity testing. As shown in Fig. 5c, d, miR-101 over-expression reduced the formation of colonies in soft agar of about 50 % in both RD and JR1 cells. Consistently, miR-101 over-expressing RD18 cells showed 50 % EZH2 down-regulation associated to cell cycle slow-down (5.4 ± 0.6 % increase of cells in the G1 phase and 14 ± 2 and 3.4 ± 0.6 % decrease in S and G2 phases, respectively) and a more modest but significant reduction of colony formation of about 20 and 15 % on either in culture dishes or soft agar (Additional file 4: Figure S4). Taken together, these results indicate that restoration of miR-101 in eRMS exerts an antitumor effect in vitro.Fig. 5

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