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Integrated mRNA-microRNA profiling of human NK cell differentiation identifies MiR-583 as a negative regulator of IL2Rγ expression.

Yun S, Lee SU, Kim JM, Lee HJ, Song HY, Kim YK, Jung H, Park YJ, Yoon SR, Oh SR, Kim TD, Choi I - PLoS ONE (2014)

Bottom Line: The overexpression of miR-583 had an inhibitory effect on NK cell differentiation.In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3' UTR.Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.

ABSTRACT
Natural killer (NK) cells are innate immune effector cells that protect against cancer and some viral infections. Until recently, most studies have investigated the molecular signatures of human or mouse NK cells to identify genes that are specifically expressed during NK cell development. However, the mechanism regulating NK cell development remains unclear. Here, we report a regulatory network of potential interactions during in vitro differentiation of human NK cells, identified using genome-wide mRNA and miRNA databases through hierarchical clustering analysis, gene ontology analysis and a miRNA target prediction program. The microRNA (miR)-583, which demonstrated the largest ratio change in mature NK cells, was highly correlated with IL2 receptor gamma (IL2Rγ) expression. The overexpression of miR-583 had an inhibitory effect on NK cell differentiation. In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3' UTR. Therefore, we show that miR-583 acts as a negative regulator of NK cell differentiation by silencing IL2Rγ. Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.

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Human miR-583 downregulates NK cell differentiation.(a) Differentiating cells were transfected with miR-143, a miR-583 mimic or negative control miRNA. NK cell populations (CD56+CD3− cells) were analyzed by FACS 10 days after transfection. *, p<0.1. (b) The expression level of miR-583 was decreased during NK cell development as shown by qRT-PCR. The results are shown as the mean expression values normalized against stage 2 (CD34+CD117+CD94−). *, p<0.1. (c) NK cell populations (CD56+CD3− cells) were analyzed by FACS after miR-583 transfection at regular intervals during NK cell differentiation. The absolute numbers of differentiated NK cells are shown in the parentheses (×105). *, p<0.01 **, p<0.001. The data are representative of three independent experiments performed using three different UCB samples and represent the mean values ± S.E.M. of duplicates.
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pone-0108913-g005: Human miR-583 downregulates NK cell differentiation.(a) Differentiating cells were transfected with miR-143, a miR-583 mimic or negative control miRNA. NK cell populations (CD56+CD3− cells) were analyzed by FACS 10 days after transfection. *, p<0.1. (b) The expression level of miR-583 was decreased during NK cell development as shown by qRT-PCR. The results are shown as the mean expression values normalized against stage 2 (CD34+CD117+CD94−). *, p<0.1. (c) NK cell populations (CD56+CD3− cells) were analyzed by FACS after miR-583 transfection at regular intervals during NK cell differentiation. The absolute numbers of differentiated NK cells are shown in the parentheses (×105). *, p<0.01 **, p<0.001. The data are representative of three independent experiments performed using three different UCB samples and represent the mean values ± S.E.M. of duplicates.

Mentions: To investigate the biological effects of miRNAs on NK cell development, miR-583 and miR-143 were validated as regulators of NK cell differentiation by targeting IL2Rγ. Although IL-15 receptor-mediated signaling is important for NK cell differentiation, it is not known whether miRNAs regulate IL-15 receptor expression during NK cell differentiation. To evaluate whether overexpression of miRNAs caused the selective reduction of IL2Rγ we transfected synthetic miRNA mimics into differentiating NK cells (0 day). We transfected synthetic miR-583 mimics into differentiating NK cells (0 day). Then, the medium was changed to differentiation medium containing human IL-15 (30 ng/ml). The introduction of miR-583 mimics led to an approximately 2-fold decrease in the percentage of mature CD56+CD3− NK cell by the 10 d after transfection compared with the mimic control (Fig. 5a and 5c). In contrast, the introduction of a miR-143 mimic resulted in similar percentages of mature CD56+CD3− NK cells and mimic controls (Fig. 5a). Thus, we focused on miR-583 as a regulator of NK cell differentiation via the IL-15 signaling pathway.


Integrated mRNA-microRNA profiling of human NK cell differentiation identifies MiR-583 as a negative regulator of IL2Rγ expression.

Yun S, Lee SU, Kim JM, Lee HJ, Song HY, Kim YK, Jung H, Park YJ, Yoon SR, Oh SR, Kim TD, Choi I - PLoS ONE (2014)

Human miR-583 downregulates NK cell differentiation.(a) Differentiating cells were transfected with miR-143, a miR-583 mimic or negative control miRNA. NK cell populations (CD56+CD3− cells) were analyzed by FACS 10 days after transfection. *, p<0.1. (b) The expression level of miR-583 was decreased during NK cell development as shown by qRT-PCR. The results are shown as the mean expression values normalized against stage 2 (CD34+CD117+CD94−). *, p<0.1. (c) NK cell populations (CD56+CD3− cells) were analyzed by FACS after miR-583 transfection at regular intervals during NK cell differentiation. The absolute numbers of differentiated NK cells are shown in the parentheses (×105). *, p<0.01 **, p<0.001. The data are representative of three independent experiments performed using three different UCB samples and represent the mean values ± S.E.M. of duplicates.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196775&req=5

pone-0108913-g005: Human miR-583 downregulates NK cell differentiation.(a) Differentiating cells were transfected with miR-143, a miR-583 mimic or negative control miRNA. NK cell populations (CD56+CD3− cells) were analyzed by FACS 10 days after transfection. *, p<0.1. (b) The expression level of miR-583 was decreased during NK cell development as shown by qRT-PCR. The results are shown as the mean expression values normalized against stage 2 (CD34+CD117+CD94−). *, p<0.1. (c) NK cell populations (CD56+CD3− cells) were analyzed by FACS after miR-583 transfection at regular intervals during NK cell differentiation. The absolute numbers of differentiated NK cells are shown in the parentheses (×105). *, p<0.01 **, p<0.001. The data are representative of three independent experiments performed using three different UCB samples and represent the mean values ± S.E.M. of duplicates.
Mentions: To investigate the biological effects of miRNAs on NK cell development, miR-583 and miR-143 were validated as regulators of NK cell differentiation by targeting IL2Rγ. Although IL-15 receptor-mediated signaling is important for NK cell differentiation, it is not known whether miRNAs regulate IL-15 receptor expression during NK cell differentiation. To evaluate whether overexpression of miRNAs caused the selective reduction of IL2Rγ we transfected synthetic miRNA mimics into differentiating NK cells (0 day). We transfected synthetic miR-583 mimics into differentiating NK cells (0 day). Then, the medium was changed to differentiation medium containing human IL-15 (30 ng/ml). The introduction of miR-583 mimics led to an approximately 2-fold decrease in the percentage of mature CD56+CD3− NK cell by the 10 d after transfection compared with the mimic control (Fig. 5a and 5c). In contrast, the introduction of a miR-143 mimic resulted in similar percentages of mature CD56+CD3− NK cells and mimic controls (Fig. 5a). Thus, we focused on miR-583 as a regulator of NK cell differentiation via the IL-15 signaling pathway.

Bottom Line: The overexpression of miR-583 had an inhibitory effect on NK cell differentiation.In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3' UTR.Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.

ABSTRACT
Natural killer (NK) cells are innate immune effector cells that protect against cancer and some viral infections. Until recently, most studies have investigated the molecular signatures of human or mouse NK cells to identify genes that are specifically expressed during NK cell development. However, the mechanism regulating NK cell development remains unclear. Here, we report a regulatory network of potential interactions during in vitro differentiation of human NK cells, identified using genome-wide mRNA and miRNA databases through hierarchical clustering analysis, gene ontology analysis and a miRNA target prediction program. The microRNA (miR)-583, which demonstrated the largest ratio change in mature NK cells, was highly correlated with IL2 receptor gamma (IL2Rγ) expression. The overexpression of miR-583 had an inhibitory effect on NK cell differentiation. In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3' UTR. Therefore, we show that miR-583 acts as a negative regulator of NK cell differentiation by silencing IL2Rγ. Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.

Show MeSH
Related in: MedlinePlus