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Methylation Status of SP1 Sites within miR-23a-27a-24-2 Promoter Region Influences Laryngeal Cancer Cell Proliferation and Apoptosis.

Wang Y, Zhang ZX, Chen S, Qiu GB, Xu ZM, Fu WN - Biomed Res Int (2016)

Bottom Line: Here we found a CG-rich region spanning two SP1 sites in the cluster promoter region.We also found that construct with two SP1 sites had highest luciferase activity and SP1 specifically bound the gene cluster promoter in vitro.We conclude that demethylated SP1 sites in miR-23a-27a-24-2 cluster upregulate the cluster expression, leading to proliferation promotion and early apoptosis inhibition in laryngeal cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, China Medical University, Shenyang 110122, China.

ABSTRACT
DNA methylation plays critical roles in regulation of microRNA expression and function. miR-23a-27a-24-2 cluster has various functions and aberrant expression of the cluster is a common event in many cancers. However, whether DNA methylation influences the cluster expression and function is not reported. Here we found a CG-rich region spanning two SP1 sites in the cluster promoter region. The SP1 sites in the cluster were demethylated and methylated in Hep2 cells and HEK293 cells, respectively. Meanwhile, the cluster was significantly upregulated and downregulated in Hep2 cells and HEK293 cells, respectively. The SP1 sites were remethylated and the cluster was significantly downregulated in Hep2 cells into which methyl donor, S-adenosyl-L-methionine, was introduced. Moreover, S-adenosyl-L-methionine significantly increased Hep2 cell viability and repressed Hep2 cell early apoptosis. We also found that construct with two SP1 sites had highest luciferase activity and SP1 specifically bound the gene cluster promoter in vitro. We conclude that demethylated SP1 sites in miR-23a-27a-24-2 cluster upregulate the cluster expression, leading to proliferation promotion and early apoptosis inhibition in laryngeal cancer cells.

No MeSH data available.


Related in: MedlinePlus

Activities of miR-23a-27a-24-2 cluster promote and binding of SP1 to the cluster CG-rich region. (a) Relative luciferases of different constructs in miR-23a-27a-24-2 cluster are promoted. (b) Binding of SP1 to the cluster CG-rich region in vitro. ∗ indicates P < 0.05.
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fig3: Activities of miR-23a-27a-24-2 cluster promote and binding of SP1 to the cluster CG-rich region. (a) Relative luciferases of different constructs in miR-23a-27a-24-2 cluster are promoted. (b) Binding of SP1 to the cluster CG-rich region in vitro. ∗ indicates P < 0.05.

Mentions: Luciferase reporter assay result indicated that construct overlapping two SP1 sites had strongest luciferase activity and even that harbouring one SP1 site showed significantly higher luciferase ability compared to the controls (Figure 3(a)), implying that the SP1 sites probably regulate miR-23a-27a-24-2 cluster expression. EMSA result displayed that SP1-labled wild type probe had strong binding ability to nuclear proteins compared to the probe-free group. By addition of anti-SP1 antibody, a shifting band was detected. SP1-unlabeled probe reduced the binding and SP1-mutant probe had no effect on the binding (Figure 3(b)). The findings confirmed that both SP1 sites are import cis-acting elements in miR-23a-27a-24-2 cluster regulation.


Methylation Status of SP1 Sites within miR-23a-27a-24-2 Promoter Region Influences Laryngeal Cancer Cell Proliferation and Apoptosis.

Wang Y, Zhang ZX, Chen S, Qiu GB, Xu ZM, Fu WN - Biomed Res Int (2016)

Activities of miR-23a-27a-24-2 cluster promote and binding of SP1 to the cluster CG-rich region. (a) Relative luciferases of different constructs in miR-23a-27a-24-2 cluster are promoted. (b) Binding of SP1 to the cluster CG-rich region in vitro. ∗ indicates P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4821919&req=5

fig3: Activities of miR-23a-27a-24-2 cluster promote and binding of SP1 to the cluster CG-rich region. (a) Relative luciferases of different constructs in miR-23a-27a-24-2 cluster are promoted. (b) Binding of SP1 to the cluster CG-rich region in vitro. ∗ indicates P < 0.05.
Mentions: Luciferase reporter assay result indicated that construct overlapping two SP1 sites had strongest luciferase activity and even that harbouring one SP1 site showed significantly higher luciferase ability compared to the controls (Figure 3(a)), implying that the SP1 sites probably regulate miR-23a-27a-24-2 cluster expression. EMSA result displayed that SP1-labled wild type probe had strong binding ability to nuclear proteins compared to the probe-free group. By addition of anti-SP1 antibody, a shifting band was detected. SP1-unlabeled probe reduced the binding and SP1-mutant probe had no effect on the binding (Figure 3(b)). The findings confirmed that both SP1 sites are import cis-acting elements in miR-23a-27a-24-2 cluster regulation.

Bottom Line: Here we found a CG-rich region spanning two SP1 sites in the cluster promoter region.We also found that construct with two SP1 sites had highest luciferase activity and SP1 specifically bound the gene cluster promoter in vitro.We conclude that demethylated SP1 sites in miR-23a-27a-24-2 cluster upregulate the cluster expression, leading to proliferation promotion and early apoptosis inhibition in laryngeal cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, China Medical University, Shenyang 110122, China.

ABSTRACT
DNA methylation plays critical roles in regulation of microRNA expression and function. miR-23a-27a-24-2 cluster has various functions and aberrant expression of the cluster is a common event in many cancers. However, whether DNA methylation influences the cluster expression and function is not reported. Here we found a CG-rich region spanning two SP1 sites in the cluster promoter region. The SP1 sites in the cluster were demethylated and methylated in Hep2 cells and HEK293 cells, respectively. Meanwhile, the cluster was significantly upregulated and downregulated in Hep2 cells and HEK293 cells, respectively. The SP1 sites were remethylated and the cluster was significantly downregulated in Hep2 cells into which methyl donor, S-adenosyl-L-methionine, was introduced. Moreover, S-adenosyl-L-methionine significantly increased Hep2 cell viability and repressed Hep2 cell early apoptosis. We also found that construct with two SP1 sites had highest luciferase activity and SP1 specifically bound the gene cluster promoter in vitro. We conclude that demethylated SP1 sites in miR-23a-27a-24-2 cluster upregulate the cluster expression, leading to proliferation promotion and early apoptosis inhibition in laryngeal cancer cells.

No MeSH data available.


Related in: MedlinePlus