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EVI1 controls proliferation in acute myeloid leukaemia through modulation of miR-1-2.

Gómez-Benito M, Conchillo A, García MA, Vázquez I, Maicas M, Vicente C, Cristobal I, Marcotegui N, García-Ortí L, Bandrés E, Calasanz MJ, Alonso MM, Odero MD - Br. J. Cancer (2010)

Bottom Line: However, there is no analysis of miRNAs profiles associated with EVI1 overexpression in humans.Our data showed that EVI1 expression was significantly correlated with the expression of miR-1-2 and miR-133-a-1 in established cell lines and in patient samples.This study contributes to further understand the transcriptional networks involving transcription factors and miRNAs in AML.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology, Center for Applied Medical Research, University of Navarra, Navarra, Pamplona 31008, Spain.

ABSTRACT

Background: The EVI1(ecotropic virus integration site 1) gene codes for a zinc-finger transcription factor, whose transcriptional activation leads to a particularly aggressive form of acute myeloid leukaemia (AML). Although, EVI1 interactions with key proteins in hematopoiesis have been previously described, the precise role of this transcription factor in promoting leukaemic transformation is not completely understood. Recent works have identified specific microRNA (miRNA) signatures in different AML subgroups. However, there is no analysis of miRNAs profiles associated with EVI1 overexpression in humans.

Methods: We performed QT-RT-PCR to assess the expression of 250 miRNAs in cell lines with or without EVI1 overexpression and in patient samples. We used ChIP assays to evaluated the possible binding of EVI1 binding to the putative miRNA promoter. Proliferation of the different cell lines transfected with the anti- or pre-miRs was quantified by MTT.

Results: Our data showed that EVI1 expression was significantly correlated with the expression of miR-1-2 and miR-133-a-1 in established cell lines and in patient samples. ChIP assays confirmed that EVI1 binds directly to the promoter of these two miRNAs. However, only miR-1-2 was involved in abnormal proliferation in EVI1 expressing cell lines.

Conclusions: Our data showed that EVI1 controls proliferation in AML through modulation of miR-1-2. This study contributes to further understand the transcriptional networks involving transcription factors and miRNAs in AML.

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Related in: MedlinePlus

EVI1 controls proliferation through miR-1-2 modulation. (A) Schematic representation of EVI1-binding sites and PCR design in the putative promoter of miR-1-2 and miR-133a-1. (B) The PCR amplification of EVI1-immunoprecipitated DNA with the oligos no. 1 and oligos no. 2. Amplification of negative control (no DNA), input chromatin (input) and mock-immunoprecipitated chromatin (no Ab) were carried out as controls. (C) Quantitative real-time PCR was performed with oligos no. 1. Input chromatin was used as a normaliser. Error bars represent standard deviation. Results from a representative experiment, out of three, are shown. (D) Expression levels of mRNA EVI1, miR-1-2 and miR-133a-1 in cells treated with DMSO 1% or ATRA 1 μ relative to P19 control cells, β-actin and snRNA U6B levels were used as normalisers for mRNA and miRNAs levels, respectively. (E) Quantification of mRNA EVI1, miR-1-2, and miR-133a-1 in P19 transfected with a scramble or an EVI1 siRNA, during the ATRA treatment. (F) HEL and HL-60 cells were respectively transfected with premiRs or anti-miRs, as indicated, and cell proliferation was measured at different time points. Untreated cells day 0 was given an arbitrary value of 1 and all the values were normalised with this group. Abbreviation: a.u.=arbitrary unit.
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fig2: EVI1 controls proliferation through miR-1-2 modulation. (A) Schematic representation of EVI1-binding sites and PCR design in the putative promoter of miR-1-2 and miR-133a-1. (B) The PCR amplification of EVI1-immunoprecipitated DNA with the oligos no. 1 and oligos no. 2. Amplification of negative control (no DNA), input chromatin (input) and mock-immunoprecipitated chromatin (no Ab) were carried out as controls. (C) Quantitative real-time PCR was performed with oligos no. 1. Input chromatin was used as a normaliser. Error bars represent standard deviation. Results from a representative experiment, out of three, are shown. (D) Expression levels of mRNA EVI1, miR-1-2 and miR-133a-1 in cells treated with DMSO 1% or ATRA 1 μ relative to P19 control cells, β-actin and snRNA U6B levels were used as normalisers for mRNA and miRNAs levels, respectively. (E) Quantification of mRNA EVI1, miR-1-2, and miR-133a-1 in P19 transfected with a scramble or an EVI1 siRNA, during the ATRA treatment. (F) HEL and HL-60 cells were respectively transfected with premiRs or anti-miRs, as indicated, and cell proliferation was measured at different time points. Untreated cells day 0 was given an arbitrary value of 1 and all the values were normalised with this group. Abbreviation: a.u.=arbitrary unit.

Mentions: miR-1-2 and miR-133a-1 are clustered together in the same loci at chromosome 18 (Liu et al, 2007), suggesting that their transcription might be regulated by similar mechanisms. To elucidate whether EVI1 acts as a transcription factor for these two microRNAs, we examine their putative promoters using bioinformatics prediction tools. Interestingly, there were several binding sites for EVI1 in the upstream regions of both miR-1-2 and miR-133a-1 (Figure 2A). ChIP assays showed that EVI1 was bound only to the region upstream miR-1-2, but not upstream miR-133a-1 (Figure 2B). The occupancy of this site by EVI1 was also confirmed by qRT–PCR (Figure 2C).


EVI1 controls proliferation in acute myeloid leukaemia through modulation of miR-1-2.

Gómez-Benito M, Conchillo A, García MA, Vázquez I, Maicas M, Vicente C, Cristobal I, Marcotegui N, García-Ortí L, Bandrés E, Calasanz MJ, Alonso MM, Odero MD - Br. J. Cancer (2010)

EVI1 controls proliferation through miR-1-2 modulation. (A) Schematic representation of EVI1-binding sites and PCR design in the putative promoter of miR-1-2 and miR-133a-1. (B) The PCR amplification of EVI1-immunoprecipitated DNA with the oligos no. 1 and oligos no. 2. Amplification of negative control (no DNA), input chromatin (input) and mock-immunoprecipitated chromatin (no Ab) were carried out as controls. (C) Quantitative real-time PCR was performed with oligos no. 1. Input chromatin was used as a normaliser. Error bars represent standard deviation. Results from a representative experiment, out of three, are shown. (D) Expression levels of mRNA EVI1, miR-1-2 and miR-133a-1 in cells treated with DMSO 1% or ATRA 1 μ relative to P19 control cells, β-actin and snRNA U6B levels were used as normalisers for mRNA and miRNAs levels, respectively. (E) Quantification of mRNA EVI1, miR-1-2, and miR-133a-1 in P19 transfected with a scramble or an EVI1 siRNA, during the ATRA treatment. (F) HEL and HL-60 cells were respectively transfected with premiRs or anti-miRs, as indicated, and cell proliferation was measured at different time points. Untreated cells day 0 was given an arbitrary value of 1 and all the values were normalised with this group. Abbreviation: a.u.=arbitrary unit.
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Related In: Results  -  Collection

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fig2: EVI1 controls proliferation through miR-1-2 modulation. (A) Schematic representation of EVI1-binding sites and PCR design in the putative promoter of miR-1-2 and miR-133a-1. (B) The PCR amplification of EVI1-immunoprecipitated DNA with the oligos no. 1 and oligos no. 2. Amplification of negative control (no DNA), input chromatin (input) and mock-immunoprecipitated chromatin (no Ab) were carried out as controls. (C) Quantitative real-time PCR was performed with oligos no. 1. Input chromatin was used as a normaliser. Error bars represent standard deviation. Results from a representative experiment, out of three, are shown. (D) Expression levels of mRNA EVI1, miR-1-2 and miR-133a-1 in cells treated with DMSO 1% or ATRA 1 μ relative to P19 control cells, β-actin and snRNA U6B levels were used as normalisers for mRNA and miRNAs levels, respectively. (E) Quantification of mRNA EVI1, miR-1-2, and miR-133a-1 in P19 transfected with a scramble or an EVI1 siRNA, during the ATRA treatment. (F) HEL and HL-60 cells were respectively transfected with premiRs or anti-miRs, as indicated, and cell proliferation was measured at different time points. Untreated cells day 0 was given an arbitrary value of 1 and all the values were normalised with this group. Abbreviation: a.u.=arbitrary unit.
Mentions: miR-1-2 and miR-133a-1 are clustered together in the same loci at chromosome 18 (Liu et al, 2007), suggesting that their transcription might be regulated by similar mechanisms. To elucidate whether EVI1 acts as a transcription factor for these two microRNAs, we examine their putative promoters using bioinformatics prediction tools. Interestingly, there were several binding sites for EVI1 in the upstream regions of both miR-1-2 and miR-133a-1 (Figure 2A). ChIP assays showed that EVI1 was bound only to the region upstream miR-1-2, but not upstream miR-133a-1 (Figure 2B). The occupancy of this site by EVI1 was also confirmed by qRT–PCR (Figure 2C).

Bottom Line: However, there is no analysis of miRNAs profiles associated with EVI1 overexpression in humans.Our data showed that EVI1 expression was significantly correlated with the expression of miR-1-2 and miR-133-a-1 in established cell lines and in patient samples.This study contributes to further understand the transcriptional networks involving transcription factors and miRNAs in AML.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology, Center for Applied Medical Research, University of Navarra, Navarra, Pamplona 31008, Spain.

ABSTRACT

Background: The EVI1(ecotropic virus integration site 1) gene codes for a zinc-finger transcription factor, whose transcriptional activation leads to a particularly aggressive form of acute myeloid leukaemia (AML). Although, EVI1 interactions with key proteins in hematopoiesis have been previously described, the precise role of this transcription factor in promoting leukaemic transformation is not completely understood. Recent works have identified specific microRNA (miRNA) signatures in different AML subgroups. However, there is no analysis of miRNAs profiles associated with EVI1 overexpression in humans.

Methods: We performed QT-RT-PCR to assess the expression of 250 miRNAs in cell lines with or without EVI1 overexpression and in patient samples. We used ChIP assays to evaluated the possible binding of EVI1 binding to the putative miRNA promoter. Proliferation of the different cell lines transfected with the anti- or pre-miRs was quantified by MTT.

Results: Our data showed that EVI1 expression was significantly correlated with the expression of miR-1-2 and miR-133-a-1 in established cell lines and in patient samples. ChIP assays confirmed that EVI1 binds directly to the promoter of these two miRNAs. However, only miR-1-2 was involved in abnormal proliferation in EVI1 expressing cell lines.

Conclusions: Our data showed that EVI1 controls proliferation in AML through modulation of miR-1-2. This study contributes to further understand the transcriptional networks involving transcription factors and miRNAs in AML.

Show MeSH
Related in: MedlinePlus