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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 activates miR-155 in CLL cells. A,CLL cells were incubated for 20 minutes with 30 ng/ml rhIL-6. Following incubation, cells were collected at different times, and total RNAs were prepared. qRT-PCR (upper panel) and RT-PCR (lower panel) revealed that IL-6 upregulated miR-155 expression levels in a time-dependent manner. The U6 gene was used as an internal control for miRNA expression. B, Transfecting CLL cells with STAT3-siRNA at a transfection efficiency of 35% downregulated the expressions of miR-155 and the STAT3-regulated genes Bcl-2, c-Myc, Cyclin D1, p21/WAF1, and VEGF-C.
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pone-0064678-g004: STAT3 activates miR-155 in CLL cells. A,CLL cells were incubated for 20 minutes with 30 ng/ml rhIL-6. Following incubation, cells were collected at different times, and total RNAs were prepared. qRT-PCR (upper panel) and RT-PCR (lower panel) revealed that IL-6 upregulated miR-155 expression levels in a time-dependent manner. The U6 gene was used as an internal control for miRNA expression. B, Transfecting CLL cells with STAT3-siRNA at a transfection efficiency of 35% downregulated the expressions of miR-155 and the STAT3-regulated genes Bcl-2, c-Myc, Cyclin D1, p21/WAF1, and VEGF-C.

Mentions: We next sought to determine whether STAT3 activates miR-155 in CLL cells. Because IL-6 has been found to increase the levels of serine pSTAT3 and induce the phosphorylation of STAT3 on tyrosine residues [15], we incubated CLL cells with 30 mg/ml rhIL-6 for 20 minutes and analyzed their miR-155 expression levels at different times following incubation using RT-PCR and relative qRT-PCR. Both experiments revealed that the miR-155 expression levels 2 hours after incubation were almost 4-fold higher than those at baseline and that miR expression returned to baseline levels at 16 hours, suggesting that IL-6 activates miR-155 expression (Fig. 4A). We then transfected CLL cells with STAT3-siRNA and used relative qRT-PCR to quantitate STAT3-regulated gene mRNA levels. We found that STAT3-shRNA markedly downregulated the expression levels of miR-155 as well as those of the STAT3-regulated genes Bcl-2, c-Myc, cyclin D1, p21/WAF1, and VEGF-C (Fig. 4B).


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 activates miR-155 in CLL cells. A,CLL cells were incubated for 20 minutes with 30 ng/ml rhIL-6. Following incubation, cells were collected at different times, and total RNAs were prepared. qRT-PCR (upper panel) and RT-PCR (lower panel) revealed that IL-6 upregulated miR-155 expression levels in a time-dependent manner. The U6 gene was used as an internal control for miRNA expression. B, Transfecting CLL cells with STAT3-siRNA at a transfection efficiency of 35% downregulated the expressions of miR-155 and the STAT3-regulated genes Bcl-2, c-Myc, Cyclin D1, p21/WAF1, and VEGF-C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672147&req=5

pone-0064678-g004: STAT3 activates miR-155 in CLL cells. A,CLL cells were incubated for 20 minutes with 30 ng/ml rhIL-6. Following incubation, cells were collected at different times, and total RNAs were prepared. qRT-PCR (upper panel) and RT-PCR (lower panel) revealed that IL-6 upregulated miR-155 expression levels in a time-dependent manner. The U6 gene was used as an internal control for miRNA expression. B, Transfecting CLL cells with STAT3-siRNA at a transfection efficiency of 35% downregulated the expressions of miR-155 and the STAT3-regulated genes Bcl-2, c-Myc, Cyclin D1, p21/WAF1, and VEGF-C.
Mentions: We next sought to determine whether STAT3 activates miR-155 in CLL cells. Because IL-6 has been found to increase the levels of serine pSTAT3 and induce the phosphorylation of STAT3 on tyrosine residues [15], we incubated CLL cells with 30 mg/ml rhIL-6 for 20 minutes and analyzed their miR-155 expression levels at different times following incubation using RT-PCR and relative qRT-PCR. Both experiments revealed that the miR-155 expression levels 2 hours after incubation were almost 4-fold higher than those at baseline and that miR expression returned to baseline levels at 16 hours, suggesting that IL-6 activates miR-155 expression (Fig. 4A). We then transfected CLL cells with STAT3-siRNA and used relative qRT-PCR to quantitate STAT3-regulated gene mRNA levels. We found that STAT3-shRNA markedly downregulated the expression levels of miR-155 as well as those of the STAT3-regulated genes Bcl-2, c-Myc, cyclin D1, p21/WAF1, and VEGF-C (Fig. 4B).

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