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A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

Jebbawi F, Fayyad-Kazan H, Merimi M, Lewalle P, Verougstraete JC, Leo O, Romero P, Burny A, Badran B, Martiat P, Rouas R - J Transl Med (2014)

Bottom Line: We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes.We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes.These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

View Article: PubMed Central - PubMed

Affiliation: Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles, 121, Boulevard de Waterloo, 1000, Bruxelles, Belgium. Fadi.Jebbawi@ulb.ac.be.

ABSTRACT

Background: Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation.

Methods: We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes.

Results: The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo.

Conclusions: We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

No MeSH data available.


Related in: MedlinePlus

MicroRNA specific activities. (A) MiR-335 negatively regulates luciferase expression in a plasmid coupling its coding sequence with FOXP3 3′UTR. Renilla luciferase reporter assays with constructs holding FOXP3 3′-UTR sequences from the indicated genes were co-transfected into HEK293T cells along with a firefly luciferase transfection control plasmid either alone or together with miR-335. (B) MiR-9 and miR-155 negatively regulate luciferase expression in a plasmid coupling its coding sequence with CTLA-4 3′UTR. Renilla luciferase reporter assays with constructs holding CTLA-4 3′-UTR sequences, wild type or miR-site deleted, were co-transfected into HEK293T cells along with a firefly luciferase transfection control plasmid either alone or together with miR-9, −155. MiR-24 (C) and miR-335 (D) specifically targets GARP 3′UTR and negatively regulate luciferase reporter expression. Renilla and firefly luciferase reporter assays with constructs holding GARP 3′-UTR sequences, wild type or miR-site deleted, were co-transfected into HEK293T cells along with miR-24, −335. Relative luciferase values normalized to transfections without miRNA are shown. Data represent mean ± SD (error bars) of three independent experiments, each performed in triplicate. (*p < 0.05; **, p < 0.01, Student's t test).
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Fig4: MicroRNA specific activities. (A) MiR-335 negatively regulates luciferase expression in a plasmid coupling its coding sequence with FOXP3 3′UTR. Renilla luciferase reporter assays with constructs holding FOXP3 3′-UTR sequences from the indicated genes were co-transfected into HEK293T cells along with a firefly luciferase transfection control plasmid either alone or together with miR-335. (B) MiR-9 and miR-155 negatively regulate luciferase expression in a plasmid coupling its coding sequence with CTLA-4 3′UTR. Renilla luciferase reporter assays with constructs holding CTLA-4 3′-UTR sequences, wild type or miR-site deleted, were co-transfected into HEK293T cells along with a firefly luciferase transfection control plasmid either alone or together with miR-9, −155. MiR-24 (C) and miR-335 (D) specifically targets GARP 3′UTR and negatively regulate luciferase reporter expression. Renilla and firefly luciferase reporter assays with constructs holding GARP 3′-UTR sequences, wild type or miR-site deleted, were co-transfected into HEK293T cells along with miR-24, −335. Relative luciferase values normalized to transfections without miRNA are shown. Data represent mean ± SD (error bars) of three independent experiments, each performed in triplicate. (*p < 0.05; **, p < 0.01, Student's t test).

Mentions: A 249-bp fragment of the 3′-UTR of FOXP3 containing the miR-335 target sequence was cloned into a psiCHECK-1 vector downstream of the Renilla luciferase gene (psiCHECK-UTRwt). In parallel, in the same way we cloned this FOXP3 3′-UTR fragment with a deleted miR-335 target site (psiCHECK-UTRdel). Transient transfections of psiCHECK-UTRwt or psiCHECK-UTRdel in HEK293T cells led to no significant change in reporter luciferase activity when compared with the psiCHECK control vector (Figure 4A). By contrast, co-transfection of HEK293T cells with miR-335 and psiCHECK-UTRwt led to a significant reduction (~47%) in relative reporter luciferase activity, and also for activity compared with the co-transfection of HEK293 T cells with miR-Ctrl and psiCHECK-UTRwt.Figure 4


A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

Jebbawi F, Fayyad-Kazan H, Merimi M, Lewalle P, Verougstraete JC, Leo O, Romero P, Burny A, Badran B, Martiat P, Rouas R - J Transl Med (2014)

MicroRNA specific activities. (A) MiR-335 negatively regulates luciferase expression in a plasmid coupling its coding sequence with FOXP3 3′UTR. Renilla luciferase reporter assays with constructs holding FOXP3 3′-UTR sequences from the indicated genes were co-transfected into HEK293T cells along with a firefly luciferase transfection control plasmid either alone or together with miR-335. (B) MiR-9 and miR-155 negatively regulate luciferase expression in a plasmid coupling its coding sequence with CTLA-4 3′UTR. Renilla luciferase reporter assays with constructs holding CTLA-4 3′-UTR sequences, wild type or miR-site deleted, were co-transfected into HEK293T cells along with a firefly luciferase transfection control plasmid either alone or together with miR-9, −155. MiR-24 (C) and miR-335 (D) specifically targets GARP 3′UTR and negatively regulate luciferase reporter expression. Renilla and firefly luciferase reporter assays with constructs holding GARP 3′-UTR sequences, wild type or miR-site deleted, were co-transfected into HEK293T cells along with miR-24, −335. Relative luciferase values normalized to transfections without miRNA are shown. Data represent mean ± SD (error bars) of three independent experiments, each performed in triplicate. (*p < 0.05; **, p < 0.01, Student's t test).
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Related In: Results  -  Collection

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Fig4: MicroRNA specific activities. (A) MiR-335 negatively regulates luciferase expression in a plasmid coupling its coding sequence with FOXP3 3′UTR. Renilla luciferase reporter assays with constructs holding FOXP3 3′-UTR sequences from the indicated genes were co-transfected into HEK293T cells along with a firefly luciferase transfection control plasmid either alone or together with miR-335. (B) MiR-9 and miR-155 negatively regulate luciferase expression in a plasmid coupling its coding sequence with CTLA-4 3′UTR. Renilla luciferase reporter assays with constructs holding CTLA-4 3′-UTR sequences, wild type or miR-site deleted, were co-transfected into HEK293T cells along with a firefly luciferase transfection control plasmid either alone or together with miR-9, −155. MiR-24 (C) and miR-335 (D) specifically targets GARP 3′UTR and negatively regulate luciferase reporter expression. Renilla and firefly luciferase reporter assays with constructs holding GARP 3′-UTR sequences, wild type or miR-site deleted, were co-transfected into HEK293T cells along with miR-24, −335. Relative luciferase values normalized to transfections without miRNA are shown. Data represent mean ± SD (error bars) of three independent experiments, each performed in triplicate. (*p < 0.05; **, p < 0.01, Student's t test).
Mentions: A 249-bp fragment of the 3′-UTR of FOXP3 containing the miR-335 target sequence was cloned into a psiCHECK-1 vector downstream of the Renilla luciferase gene (psiCHECK-UTRwt). In parallel, in the same way we cloned this FOXP3 3′-UTR fragment with a deleted miR-335 target site (psiCHECK-UTRdel). Transient transfections of psiCHECK-UTRwt or psiCHECK-UTRdel in HEK293T cells led to no significant change in reporter luciferase activity when compared with the psiCHECK control vector (Figure 4A). By contrast, co-transfection of HEK293T cells with miR-335 and psiCHECK-UTRwt led to a significant reduction (~47%) in relative reporter luciferase activity, and also for activity compared with the co-transfection of HEK293 T cells with miR-Ctrl and psiCHECK-UTRwt.Figure 4

Bottom Line: We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes.We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes.These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

View Article: PubMed Central - PubMed

Affiliation: Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles, 121, Boulevard de Waterloo, 1000, Bruxelles, Belgium. Fadi.Jebbawi@ulb.ac.be.

ABSTRACT

Background: Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation.

Methods: We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes.

Results: The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo.

Conclusions: We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

No MeSH data available.


Related in: MedlinePlus