Limits...
Cooperatively transcriptional and epigenetic regulation of sonic hedgehog overexpression drives malignant potential of breast cancer.

Duan ZH, Wang HC, Zhao DM, Ji XX, Song M, Yang XJ, Cui W - Cancer Sci. (2015)

Bottom Line: Comprehensive understanding of the regulation mechanism of Shh in cancer cells is necessary to find an effective approach to selectively block its tumorigenic function.Moreover, in vitro data demonstrated that both NF-κB activation and hypomethylation in promoter region were positively associated with the overexpression of Shh.Furthermore, the biological function data indicated that overexpressed Shh enhanced the self-renewal capacity and migration ability of breast cancer cells, which could be augmented by promoter demethylation and NF-κB activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Life Science and Biopharmaceutical of Shenyang Pharmaceutical University, Shenyang, China.

Show MeSH

Related in: MedlinePlus

The effects of promoter methylation and nuclear factor-kappa B (NF-κB) on the transcription of Sonic hedgehog (Shh) gene in MDA-MB-231 cells. (a) Position and promoter activities of the Shh deletion pGL3 vector. Schematic representation of the promoter/luciferase constructs. Promoter activities of the cloned 5′-untranscribed sequences of the Shh gene and deletion constructs. The promoter activities were measured using the Dual-Luciferase Reporter Assay System (Promega) after transfection into MDA-MB-231(MM231) cells. The bars represent average luciferase activities compared with the control pGL3 vector. (b) The promoter activity of Shh-P2 reporter after being treated with TNF-α or/and PDTC in MM231 cells. (c) The effect of TNF-α or/and PDTC on Shh mRNA expression in MM231 cells. The mRNA were analyzed for Shh levels by quantitative PCR using specific primers as described in the Materials and Methods. GAPDH was used as control. (d) The effect of TNF-α or/and 5-Aza on Shh mRNA expression in MM231 cells. (e) ChIP assays confirmed the binding of NF-κB(p65) to the promoter regions upstream of Shh gene. Promoter 2 located in −83 to +80 upstream of Shh gene. (f) The effects of 5-Aza and TNF-α on NF-κB DNA binding activity. MM231 cells were stimulated with TNF-α (20 ng/mL) or 5-Aza (20 μM) for 24 h. Nuclear extracts from MM231 cells were assayed for NF-κB p65 activation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4556399&req=5

fig03: The effects of promoter methylation and nuclear factor-kappa B (NF-κB) on the transcription of Sonic hedgehog (Shh) gene in MDA-MB-231 cells. (a) Position and promoter activities of the Shh deletion pGL3 vector. Schematic representation of the promoter/luciferase constructs. Promoter activities of the cloned 5′-untranscribed sequences of the Shh gene and deletion constructs. The promoter activities were measured using the Dual-Luciferase Reporter Assay System (Promega) after transfection into MDA-MB-231(MM231) cells. The bars represent average luciferase activities compared with the control pGL3 vector. (b) The promoter activity of Shh-P2 reporter after being treated with TNF-α or/and PDTC in MM231 cells. (c) The effect of TNF-α or/and PDTC on Shh mRNA expression in MM231 cells. The mRNA were analyzed for Shh levels by quantitative PCR using specific primers as described in the Materials and Methods. GAPDH was used as control. (d) The effect of TNF-α or/and 5-Aza on Shh mRNA expression in MM231 cells. (e) ChIP assays confirmed the binding of NF-κB(p65) to the promoter regions upstream of Shh gene. Promoter 2 located in −83 to +80 upstream of Shh gene. (f) The effects of 5-Aza and TNF-α on NF-κB DNA binding activity. MM231 cells were stimulated with TNF-α (20 ng/mL) or 5-Aza (20 μM) for 24 h. Nuclear extracts from MM231 cells were assayed for NF-κB p65 activation.

Mentions: To elucidate the characteristics of Shh promoter region, a series of pGL3-Shh-promter-luciferase vectors were constructed, and the activities of reporter genes were measured after being transfected in MDA-MB-231 cells for 24 h. Our results indicated that pGL3-Shh-P1 reporter and pGL3-Shh-P2 reporter displayed similar activity, suggesting that the transcription factor Sp1 binding site has no significant effect on activity of the Shh promoter (Fig.3a). In contrast, the deletion of the NF-κB binding site (pGL3-Shh-P3 reporter) resulted in an obvious decrease (Fig.3a), indicating that the NF-κB binding site is crucial to maintain transcription activity of the Shh promoter.


Cooperatively transcriptional and epigenetic regulation of sonic hedgehog overexpression drives malignant potential of breast cancer.

Duan ZH, Wang HC, Zhao DM, Ji XX, Song M, Yang XJ, Cui W - Cancer Sci. (2015)

The effects of promoter methylation and nuclear factor-kappa B (NF-κB) on the transcription of Sonic hedgehog (Shh) gene in MDA-MB-231 cells. (a) Position and promoter activities of the Shh deletion pGL3 vector. Schematic representation of the promoter/luciferase constructs. Promoter activities of the cloned 5′-untranscribed sequences of the Shh gene and deletion constructs. The promoter activities were measured using the Dual-Luciferase Reporter Assay System (Promega) after transfection into MDA-MB-231(MM231) cells. The bars represent average luciferase activities compared with the control pGL3 vector. (b) The promoter activity of Shh-P2 reporter after being treated with TNF-α or/and PDTC in MM231 cells. (c) The effect of TNF-α or/and PDTC on Shh mRNA expression in MM231 cells. The mRNA were analyzed for Shh levels by quantitative PCR using specific primers as described in the Materials and Methods. GAPDH was used as control. (d) The effect of TNF-α or/and 5-Aza on Shh mRNA expression in MM231 cells. (e) ChIP assays confirmed the binding of NF-κB(p65) to the promoter regions upstream of Shh gene. Promoter 2 located in −83 to +80 upstream of Shh gene. (f) The effects of 5-Aza and TNF-α on NF-κB DNA binding activity. MM231 cells were stimulated with TNF-α (20 ng/mL) or 5-Aza (20 μM) for 24 h. Nuclear extracts from MM231 cells were assayed for NF-κB p65 activation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556399&req=5

fig03: The effects of promoter methylation and nuclear factor-kappa B (NF-κB) on the transcription of Sonic hedgehog (Shh) gene in MDA-MB-231 cells. (a) Position and promoter activities of the Shh deletion pGL3 vector. Schematic representation of the promoter/luciferase constructs. Promoter activities of the cloned 5′-untranscribed sequences of the Shh gene and deletion constructs. The promoter activities were measured using the Dual-Luciferase Reporter Assay System (Promega) after transfection into MDA-MB-231(MM231) cells. The bars represent average luciferase activities compared with the control pGL3 vector. (b) The promoter activity of Shh-P2 reporter after being treated with TNF-α or/and PDTC in MM231 cells. (c) The effect of TNF-α or/and PDTC on Shh mRNA expression in MM231 cells. The mRNA were analyzed for Shh levels by quantitative PCR using specific primers as described in the Materials and Methods. GAPDH was used as control. (d) The effect of TNF-α or/and 5-Aza on Shh mRNA expression in MM231 cells. (e) ChIP assays confirmed the binding of NF-κB(p65) to the promoter regions upstream of Shh gene. Promoter 2 located in −83 to +80 upstream of Shh gene. (f) The effects of 5-Aza and TNF-α on NF-κB DNA binding activity. MM231 cells were stimulated with TNF-α (20 ng/mL) or 5-Aza (20 μM) for 24 h. Nuclear extracts from MM231 cells were assayed for NF-κB p65 activation.
Mentions: To elucidate the characteristics of Shh promoter region, a series of pGL3-Shh-promter-luciferase vectors were constructed, and the activities of reporter genes were measured after being transfected in MDA-MB-231 cells for 24 h. Our results indicated that pGL3-Shh-P1 reporter and pGL3-Shh-P2 reporter displayed similar activity, suggesting that the transcription factor Sp1 binding site has no significant effect on activity of the Shh promoter (Fig.3a). In contrast, the deletion of the NF-κB binding site (pGL3-Shh-P3 reporter) resulted in an obvious decrease (Fig.3a), indicating that the NF-κB binding site is crucial to maintain transcription activity of the Shh promoter.

Bottom Line: Comprehensive understanding of the regulation mechanism of Shh in cancer cells is necessary to find an effective approach to selectively block its tumorigenic function.Moreover, in vitro data demonstrated that both NF-κB activation and hypomethylation in promoter region were positively associated with the overexpression of Shh.Furthermore, the biological function data indicated that overexpressed Shh enhanced the self-renewal capacity and migration ability of breast cancer cells, which could be augmented by promoter demethylation and NF-κB activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Life Science and Biopharmaceutical of Shenyang Pharmaceutical University, Shenyang, China.

Show MeSH
Related in: MedlinePlus