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Upregulation of endogenous HMOX1 expression by a computer-designed artificial transcription factor.

Guo H, Tian Y, Lu H, Wei Y, Ying D - J. Biomed. Biotechnol. (2010)

Bottom Line: In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included.We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression.The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Third Military Medical University, Sha-Ping-Ba District, Chongqing, China.

ABSTRACT
Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases.

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Related in: MedlinePlus

Effect of the ATF on endogenous HMOX1 expression. Quantitative real-time RT-PCR was performed; the candidate vectors were pcDNA3.1-AZP-p65-flag (AZP-p65), pcDNA3.1-NLS- AZP-p65-flag (NLS-AZP-p65), PUC57-AZP (AZP), pcDNA3.1-NLS-eGFP-p65-flag (p65), and pcDNA3.1-NLS-azp*-p65-flag (NLS-azp*-p65). For each of them, four dose gradients (0.5 μg, 1.0 μg, 1.5 μg, and 2.0 μg) were used. The data represent the means ± S.D.
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fig6: Effect of the ATF on endogenous HMOX1 expression. Quantitative real-time RT-PCR was performed; the candidate vectors were pcDNA3.1-AZP-p65-flag (AZP-p65), pcDNA3.1-NLS- AZP-p65-flag (NLS-AZP-p65), PUC57-AZP (AZP), pcDNA3.1-NLS-eGFP-p65-flag (p65), and pcDNA3.1-NLS-azp*-p65-flag (NLS-azp*-p65). For each of them, four dose gradients (0.5 μg, 1.0 μg, 1.5 μg, and 2.0 μg) were used. The data represent the means ± S.D.

Mentions: For ATF vectors, HMOX1 mRNA levels increased with the vector dose, and a dose-response relationship existed. At 0.5 μg, 1.0 μg, 1.5 μg, and 2.0 μg, HMOX1 mRNA of the pcDNA3.1-AZP-p65-flag group was 3.19-, 4.06-, 4.75-, and 5.91-fold above control, while that of the pcDNA3.1-NLS-AZP-p65-flag group was 4.71-, 5.90-, 9.12-, and 10.28-fold above control (Figure 6). Here, the “fold above control” represented ratios of the HMOX1 mRNA level of the ECV304 cells transfected with different candidate vectors to that of the ECV304 cells transfected with pcDNA3.1 (control). Different from the dual-luciferase reporter assay results, the ATF with NLS seemed to be more efficient than that without NLS in upregulating endogenous HMOX1 expression. This may be attributed to the differences in the nature of the two assays. In contrast, the three control vectors failed to raise HMOX-1 mRNA levels at any dose gradient. These indicated that the HMOX1 enhancer-specific ATF could upregulate endogenous HMOX1 expression, as expected. The ATF with NLS was more efficient than that without NLS.


Upregulation of endogenous HMOX1 expression by a computer-designed artificial transcription factor.

Guo H, Tian Y, Lu H, Wei Y, Ying D - J. Biomed. Biotechnol. (2010)

Effect of the ATF on endogenous HMOX1 expression. Quantitative real-time RT-PCR was performed; the candidate vectors were pcDNA3.1-AZP-p65-flag (AZP-p65), pcDNA3.1-NLS- AZP-p65-flag (NLS-AZP-p65), PUC57-AZP (AZP), pcDNA3.1-NLS-eGFP-p65-flag (p65), and pcDNA3.1-NLS-azp*-p65-flag (NLS-azp*-p65). For each of them, four dose gradients (0.5 μg, 1.0 μg, 1.5 μg, and 2.0 μg) were used. The data represent the means ± S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2913762&req=5

fig6: Effect of the ATF on endogenous HMOX1 expression. Quantitative real-time RT-PCR was performed; the candidate vectors were pcDNA3.1-AZP-p65-flag (AZP-p65), pcDNA3.1-NLS- AZP-p65-flag (NLS-AZP-p65), PUC57-AZP (AZP), pcDNA3.1-NLS-eGFP-p65-flag (p65), and pcDNA3.1-NLS-azp*-p65-flag (NLS-azp*-p65). For each of them, four dose gradients (0.5 μg, 1.0 μg, 1.5 μg, and 2.0 μg) were used. The data represent the means ± S.D.
Mentions: For ATF vectors, HMOX1 mRNA levels increased with the vector dose, and a dose-response relationship existed. At 0.5 μg, 1.0 μg, 1.5 μg, and 2.0 μg, HMOX1 mRNA of the pcDNA3.1-AZP-p65-flag group was 3.19-, 4.06-, 4.75-, and 5.91-fold above control, while that of the pcDNA3.1-NLS-AZP-p65-flag group was 4.71-, 5.90-, 9.12-, and 10.28-fold above control (Figure 6). Here, the “fold above control” represented ratios of the HMOX1 mRNA level of the ECV304 cells transfected with different candidate vectors to that of the ECV304 cells transfected with pcDNA3.1 (control). Different from the dual-luciferase reporter assay results, the ATF with NLS seemed to be more efficient than that without NLS in upregulating endogenous HMOX1 expression. This may be attributed to the differences in the nature of the two assays. In contrast, the three control vectors failed to raise HMOX-1 mRNA levels at any dose gradient. These indicated that the HMOX1 enhancer-specific ATF could upregulate endogenous HMOX1 expression, as expected. The ATF with NLS was more efficient than that without NLS.

Bottom Line: In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included.We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression.The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Third Military Medical University, Sha-Ping-Ba District, Chongqing, China.

ABSTRACT
Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases.

Show MeSH
Related in: MedlinePlus