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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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Luciferase activity of MM1 cells transfected with miR-155 promoter fragments.A, MM1 cells were co-transfected with miR-155 promoter fragments and either STAT3-siRNA or scrambled siRNA (control). Luciferase activity was assessed after 48 hours. STAT3-siRNA markedly reduced the luciferase activity of the −728 bp fragment but not the shorter −653 and −574 bp fragments. Means ± standard deviations of relative luciferase activity units from at least 3 different experiments are shown. B, Schematic diagram of the reporter gene of the human mir-155 promoter. Fragment −728 bp contains the putative STAT3 binding sites 1 and 2, fragment −635 bp contains the putative STAT3 binding site 2, and fragment −574 bp does not contain a STAT3 binding site. C, MM1 cells were transfected with the unmutated or the t-to-a mutated −728 bp fragment. Incubation of MM1 cells with 50 ng/ml IL-6 significantly enhanced the luciferase activity of MM1 cells transfected with the unmutated but not with the mutated −728 bp fragment. As shown, the luciferase activity of IL-6-stimulated cells transfected with the unmutated −728 bp fragment was significantly higher (P<0.0006) than that of MM1 cells transfected with the mutated fragment. Means ± standard deviations of relative luciferase activity units from 3 different experiments are depicted.
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pone-0064678-g002: Luciferase activity of MM1 cells transfected with miR-155 promoter fragments.A, MM1 cells were co-transfected with miR-155 promoter fragments and either STAT3-siRNA or scrambled siRNA (control). Luciferase activity was assessed after 48 hours. STAT3-siRNA markedly reduced the luciferase activity of the −728 bp fragment but not the shorter −653 and −574 bp fragments. Means ± standard deviations of relative luciferase activity units from at least 3 different experiments are shown. B, Schematic diagram of the reporter gene of the human mir-155 promoter. Fragment −728 bp contains the putative STAT3 binding sites 1 and 2, fragment −635 bp contains the putative STAT3 binding site 2, and fragment −574 bp does not contain a STAT3 binding site. C, MM1 cells were transfected with the unmutated or the t-to-a mutated −728 bp fragment. Incubation of MM1 cells with 50 ng/ml IL-6 significantly enhanced the luciferase activity of MM1 cells transfected with the unmutated but not with the mutated −728 bp fragment. As shown, the luciferase activity of IL-6-stimulated cells transfected with the unmutated −728 bp fragment was significantly higher (P<0.0006) than that of MM1 cells transfected with the mutated fragment. Means ± standard deviations of relative luciferase activity units from 3 different experiments are depicted.

Mentions: Because low levels of activated STAT3 were detected in MM1 cells [21] and luciferase activity was high in MM1 cells transfected with miR-155 promoter transcripts, we co-transfected MM1 cells with miR-155 promoter fragments of different lengths with either STAT3-siRNA or scrambled siRNA (control) and assessed the relative luciferase activity 48 hours after transfection. Luciferase activity analysis revealed that co-transfection with STAT3-siRNA downregulated the luciferase activity of the −728 bp fragment but not the −653 bp or −574 bp fragments (Fig. 2A). The −653 bp fragment contained the putative STAT3 binding site 2, but the −574 bp fragment does not contain any STAT3 binding site (Fig. 2B), which suggests that the STAT3 binding site 1 drives STAT3-induced luciferase activity in MM1 cells. To further delineate these findings, we assessed the luciferase activity of untreated and IL-6-treated MM1 cells 48 hours after transfection with the −728 bp fragment (Fig. 2C). IL-6, known to induce phosphorylation of STAT3 [21], significantly enhanced the luciferase activity of MM1 cells transfected with the −728 bp fragment but not the luciferase activity of cells transfected with a mutated miR-155 −728 bp fragment (Fig. 2C), suggesting that the luciferase activity of site 1 is specific and driven by STAT3.


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)

Luciferase activity of MM1 cells transfected with miR-155 promoter fragments.A, MM1 cells were co-transfected with miR-155 promoter fragments and either STAT3-siRNA or scrambled siRNA (control). Luciferase activity was assessed after 48 hours. STAT3-siRNA markedly reduced the luciferase activity of the −728 bp fragment but not the shorter −653 and −574 bp fragments. Means ± standard deviations of relative luciferase activity units from at least 3 different experiments are shown. B, Schematic diagram of the reporter gene of the human mir-155 promoter. Fragment −728 bp contains the putative STAT3 binding sites 1 and 2, fragment −635 bp contains the putative STAT3 binding site 2, and fragment −574 bp does not contain a STAT3 binding site. C, MM1 cells were transfected with the unmutated or the t-to-a mutated −728 bp fragment. Incubation of MM1 cells with 50 ng/ml IL-6 significantly enhanced the luciferase activity of MM1 cells transfected with the unmutated but not with the mutated −728 bp fragment. As shown, the luciferase activity of IL-6-stimulated cells transfected with the unmutated −728 bp fragment was significantly higher (P<0.0006) than that of MM1 cells transfected with the mutated fragment. Means ± standard deviations of relative luciferase activity units from 3 different experiments are depicted.
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Related In: Results  -  Collection

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pone-0064678-g002: Luciferase activity of MM1 cells transfected with miR-155 promoter fragments.A, MM1 cells were co-transfected with miR-155 promoter fragments and either STAT3-siRNA or scrambled siRNA (control). Luciferase activity was assessed after 48 hours. STAT3-siRNA markedly reduced the luciferase activity of the −728 bp fragment but not the shorter −653 and −574 bp fragments. Means ± standard deviations of relative luciferase activity units from at least 3 different experiments are shown. B, Schematic diagram of the reporter gene of the human mir-155 promoter. Fragment −728 bp contains the putative STAT3 binding sites 1 and 2, fragment −635 bp contains the putative STAT3 binding site 2, and fragment −574 bp does not contain a STAT3 binding site. C, MM1 cells were transfected with the unmutated or the t-to-a mutated −728 bp fragment. Incubation of MM1 cells with 50 ng/ml IL-6 significantly enhanced the luciferase activity of MM1 cells transfected with the unmutated but not with the mutated −728 bp fragment. As shown, the luciferase activity of IL-6-stimulated cells transfected with the unmutated −728 bp fragment was significantly higher (P<0.0006) than that of MM1 cells transfected with the mutated fragment. Means ± standard deviations of relative luciferase activity units from 3 different experiments are depicted.
Mentions: Because low levels of activated STAT3 were detected in MM1 cells [21] and luciferase activity was high in MM1 cells transfected with miR-155 promoter transcripts, we co-transfected MM1 cells with miR-155 promoter fragments of different lengths with either STAT3-siRNA or scrambled siRNA (control) and assessed the relative luciferase activity 48 hours after transfection. Luciferase activity analysis revealed that co-transfection with STAT3-siRNA downregulated the luciferase activity of the −728 bp fragment but not the −653 bp or −574 bp fragments (Fig. 2A). The −653 bp fragment contained the putative STAT3 binding site 2, but the −574 bp fragment does not contain any STAT3 binding site (Fig. 2B), which suggests that the STAT3 binding site 1 drives STAT3-induced luciferase activity in MM1 cells. To further delineate these findings, we assessed the luciferase activity of untreated and IL-6-treated MM1 cells 48 hours after transfection with the −728 bp fragment (Fig. 2C). IL-6, known to induce phosphorylation of STAT3 [21], significantly enhanced the luciferase activity of MM1 cells transfected with the −728 bp fragment but not the luciferase activity of cells transfected with a mutated miR-155 −728 bp fragment (Fig. 2C), suggesting that the luciferase activity of site 1 is specific and driven by STAT3.

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