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Signal transducer and activator of transcription-3 induces microRNA-155 expression in chronic lymphocytic leukemia.

Li P, Grgurevic S, Liu Z, Harris D, Rozovski U, Calin GA, Keating MJ, Estrov Z - PLoS ONE (2013)

Bottom Line: Of the two putative binding sites, STAT3-siRNA reduced the luciferase activity of the construct containing the 700-709 bp STAT3 binding site, suggesting that this site is involved in STAT3-induced transcription.Finally, STAT3-small hairpin RNA downregulated miR-155 gene expression, suggesting that constitutively activated STAT3 binds to the miR-155 gene promoter.Together, these results suggest that STAT3 activates miR-155 in CLL cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.

ABSTRACT
MicroRNA (miR) abnormalities play a key role in the pathogenesis of chronic lymphocytic leukemia (CLL). High levels of miR-155 have been detected in human neoplasms, and overexpression of miR-155 has been found to induce lymphoma in mice. High levels of miR-155 were detected in CLL cells and STAT3, which is known to induce miR-21 and miR-181b-1 expression, is constitutively activated in CLL. Given these findings, we hypothesized that STAT3 induces miR-155. Sequence analysis revealed that the miR-155 promoter harbors two putative STAT3 binding sites. Therefore, truncated miR-155 promoter constructs and STAT3 small interfering RNA (siRNA) were co-transfected into MM1 cells. Of the two putative binding sites, STAT3-siRNA reduced the luciferase activity of the construct containing the 700-709 bp STAT3 binding site, suggesting that this site is involved in STAT3-induced transcription. Electrophoretic mobility shift assay confirmed that STAT3 bound to the miR-155 promoter in CLL cells, and chromatin immunoprecipitation and luciferase assay confirmed that STAT3 bound to the 700-709 bp but not the 615-624 bp putative STAT3 binding site in CLL cells. Finally, STAT3-small hairpin RNA downregulated miR-155 gene expression, suggesting that constitutively activated STAT3 binds to the miR-155 gene promoter. Together, these results suggest that STAT3 activates miR-155 in CLL cells.

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STAT3 binds to the miR-155 promoter.A, Left panel: ChIP of CLL cell nuclear protein revealed that STAT3 co-immunoprecipitated with the DNA of the STAT3 target genes STAT3, c-Myc, p21/WAF1, and VEGF-C, but not that of the control gene RPL30. Right panel: Anti-STAT3 antibodies co-immunoprecipitated with the DNA of probe 1 but not that of probes 2 or 3, suggesting that STAT3 binds to the putative STAT3 binding site 1. In both experiments, immunoglobulin G1 (IgG) was used as an isotypic control. B, ChIP of CLL cells incubated without or with 30 ng/ml rhIL-6. Using primer 1, we found that the amount of STAT3 DNA that was co-immunoprecipitated with anti-STAT3 antibodies from CLL cells incubated with IL-6 was higher than that from control cells, suggesting that IL-6 enhances the binding of STAT3 to the miR-155 promoter. C. EMSA performed using the cells of two CLL patients demonstrated that CLL cell nuclear protein binds to miR-155– biotinylated (labeled) DNA probe 1 and that excess cold, unlabeled probe attenuates this binding. No significant binding was obtained with labeled probe 2. D, EMSA performed using the cells of the same two CLL patients demonstrated that CLL cell nuclear protein binds to miR-155–labeled DNA probe 1 and that excess cold, unlabeled probe attenuates this binding. The addition of anti-phosphoserine–STAT3 and anti-STAT3 antibodies revealed a similar effect, suggesting that STAT3 binds the miR-155 promoter at binding site 1.
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pone-0064678-g003: STAT3 binds to the miR-155 promoter.A, Left panel: ChIP of CLL cell nuclear protein revealed that STAT3 co-immunoprecipitated with the DNA of the STAT3 target genes STAT3, c-Myc, p21/WAF1, and VEGF-C, but not that of the control gene RPL30. Right panel: Anti-STAT3 antibodies co-immunoprecipitated with the DNA of probe 1 but not that of probes 2 or 3, suggesting that STAT3 binds to the putative STAT3 binding site 1. In both experiments, immunoglobulin G1 (IgG) was used as an isotypic control. B, ChIP of CLL cells incubated without or with 30 ng/ml rhIL-6. Using primer 1, we found that the amount of STAT3 DNA that was co-immunoprecipitated with anti-STAT3 antibodies from CLL cells incubated with IL-6 was higher than that from control cells, suggesting that IL-6 enhances the binding of STAT3 to the miR-155 promoter. C. EMSA performed using the cells of two CLL patients demonstrated that CLL cell nuclear protein binds to miR-155– biotinylated (labeled) DNA probe 1 and that excess cold, unlabeled probe attenuates this binding. No significant binding was obtained with labeled probe 2. D, EMSA performed using the cells of the same two CLL patients demonstrated that CLL cell nuclear protein binds to miR-155–labeled DNA probe 1 and that excess cold, unlabeled probe attenuates this binding. The addition of anti-phosphoserine–STAT3 and anti-STAT3 antibodies revealed a similar effect, suggesting that STAT3 binds the miR-155 promoter at binding site 1.

Mentions: Because the luciferase activity analysis suggested that STAT3 binds to site 1 in MM1 cells, we sought to determine whether STAT3 binds to site 1 of the miR-155 promoter in CLL cells. ChIP revealed that anti-STAT3 antibodies co-immunoprecipitated the DNA of the STAT3-regulated genes STAT3, c-Myc, VEGF-C, and p21/WAF1 but not the DNA of the control gene RPL30 (Figure 3A, left panel). Anti-STAT3 antibodies also co-immunoprecipitated the DNA of primer 1, which harbored the STAT3 binding site 1, but not the DNA of primer 2, which harbored the STAT3 binding site 2, or the DNA of primer 3, the negative control (Fig. 3A, right panel), suggesting that STAT3 binds to site 1 of the miR-155 promoter.


Signal transducer and activator of transcription-3 induces microRNA-155 expression in chronic lymphocytic leukemia.

Li P, Grgurevic S, Liu Z, Harris D, Rozovski U, Calin GA, Keating MJ, Estrov Z - PLoS ONE (2013)

STAT3 binds to the miR-155 promoter.A, Left panel: ChIP of CLL cell nuclear protein revealed that STAT3 co-immunoprecipitated with the DNA of the STAT3 target genes STAT3, c-Myc, p21/WAF1, and VEGF-C, but not that of the control gene RPL30. Right panel: Anti-STAT3 antibodies co-immunoprecipitated with the DNA of probe 1 but not that of probes 2 or 3, suggesting that STAT3 binds to the putative STAT3 binding site 1. In both experiments, immunoglobulin G1 (IgG) was used as an isotypic control. B, ChIP of CLL cells incubated without or with 30 ng/ml rhIL-6. Using primer 1, we found that the amount of STAT3 DNA that was co-immunoprecipitated with anti-STAT3 antibodies from CLL cells incubated with IL-6 was higher than that from control cells, suggesting that IL-6 enhances the binding of STAT3 to the miR-155 promoter. C. EMSA performed using the cells of two CLL patients demonstrated that CLL cell nuclear protein binds to miR-155– biotinylated (labeled) DNA probe 1 and that excess cold, unlabeled probe attenuates this binding. No significant binding was obtained with labeled probe 2. D, EMSA performed using the cells of the same two CLL patients demonstrated that CLL cell nuclear protein binds to miR-155–labeled DNA probe 1 and that excess cold, unlabeled probe attenuates this binding. The addition of anti-phosphoserine–STAT3 and anti-STAT3 antibodies revealed a similar effect, suggesting that STAT3 binds the miR-155 promoter at binding site 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672147&req=5

pone-0064678-g003: STAT3 binds to the miR-155 promoter.A, Left panel: ChIP of CLL cell nuclear protein revealed that STAT3 co-immunoprecipitated with the DNA of the STAT3 target genes STAT3, c-Myc, p21/WAF1, and VEGF-C, but not that of the control gene RPL30. Right panel: Anti-STAT3 antibodies co-immunoprecipitated with the DNA of probe 1 but not that of probes 2 or 3, suggesting that STAT3 binds to the putative STAT3 binding site 1. In both experiments, immunoglobulin G1 (IgG) was used as an isotypic control. B, ChIP of CLL cells incubated without or with 30 ng/ml rhIL-6. Using primer 1, we found that the amount of STAT3 DNA that was co-immunoprecipitated with anti-STAT3 antibodies from CLL cells incubated with IL-6 was higher than that from control cells, suggesting that IL-6 enhances the binding of STAT3 to the miR-155 promoter. C. EMSA performed using the cells of two CLL patients demonstrated that CLL cell nuclear protein binds to miR-155– biotinylated (labeled) DNA probe 1 and that excess cold, unlabeled probe attenuates this binding. No significant binding was obtained with labeled probe 2. D, EMSA performed using the cells of the same two CLL patients demonstrated that CLL cell nuclear protein binds to miR-155–labeled DNA probe 1 and that excess cold, unlabeled probe attenuates this binding. The addition of anti-phosphoserine–STAT3 and anti-STAT3 antibodies revealed a similar effect, suggesting that STAT3 binds the miR-155 promoter at binding site 1.
Mentions: Because the luciferase activity analysis suggested that STAT3 binds to site 1 in MM1 cells, we sought to determine whether STAT3 binds to site 1 of the miR-155 promoter in CLL cells. ChIP revealed that anti-STAT3 antibodies co-immunoprecipitated the DNA of the STAT3-regulated genes STAT3, c-Myc, VEGF-C, and p21/WAF1 but not the DNA of the control gene RPL30 (Figure 3A, left panel). Anti-STAT3 antibodies also co-immunoprecipitated the DNA of primer 1, which harbored the STAT3 binding site 1, but not the DNA of primer 2, which harbored the STAT3 binding site 2, or the DNA of primer 3, the negative control (Fig. 3A, right panel), suggesting that STAT3 binds to site 1 of the miR-155 promoter.

Bottom Line: Of the two putative binding sites, STAT3-siRNA reduced the luciferase activity of the construct containing the 700-709 bp STAT3 binding site, suggesting that this site is involved in STAT3-induced transcription.Finally, STAT3-small hairpin RNA downregulated miR-155 gene expression, suggesting that constitutively activated STAT3 binds to the miR-155 gene promoter.Together, these results suggest that STAT3 activates miR-155 in CLL cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.

ABSTRACT
MicroRNA (miR) abnormalities play a key role in the pathogenesis of chronic lymphocytic leukemia (CLL). High levels of miR-155 have been detected in human neoplasms, and overexpression of miR-155 has been found to induce lymphoma in mice. High levels of miR-155 were detected in CLL cells and STAT3, which is known to induce miR-21 and miR-181b-1 expression, is constitutively activated in CLL. Given these findings, we hypothesized that STAT3 induces miR-155. Sequence analysis revealed that the miR-155 promoter harbors two putative STAT3 binding sites. Therefore, truncated miR-155 promoter constructs and STAT3 small interfering RNA (siRNA) were co-transfected into MM1 cells. Of the two putative binding sites, STAT3-siRNA reduced the luciferase activity of the construct containing the 700-709 bp STAT3 binding site, suggesting that this site is involved in STAT3-induced transcription. Electrophoretic mobility shift assay confirmed that STAT3 bound to the miR-155 promoter in CLL cells, and chromatin immunoprecipitation and luciferase assay confirmed that STAT3 bound to the 700-709 bp but not the 615-624 bp putative STAT3 binding site in CLL cells. Finally, STAT3-small hairpin RNA downregulated miR-155 gene expression, suggesting that constitutively activated STAT3 binds to the miR-155 gene promoter. Together, these results suggest that STAT3 activates miR-155 in CLL cells.

Show MeSH
Related in: MedlinePlus