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Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells.

Mishra DR, Chaudhary S, Krishna BM, Mishra SK - PLoS ONE (2015)

Bottom Line: Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms.These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter.Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Laboratory, Gene function and regulation Group, Institute of Life Sciences, Bhubaneswar, Odisha, India.

ABSTRACT
Cytosolic inorganic pyrophosphatase plays an important role in the cellular metabolism by hydrolyzing inorganic pyrophosphate (PPi) formed as a by-product of various metabolic reactions. Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms. In humans, the expression of inorganic pyrophosphatase (PPA1) is deregulated in different types of cancer and is involved in the migration and invasion of gastric cancer cells and proliferation of ovarian cancer cells. However, the transcriptional regulation of the gene encoding PPA1 is poorly understood. To gain insights into PPA1 gene regulation, a 1217 bp of its 5'-flanking region was cloned and analyzed. The 5'-deletion analysis of the promoter revealed a 266 bp proximal promoter region exhibit most of the transcriptional activity and upon sequence analysis, three putative Sp1 binding sites were found to be present in this region. Binding of Sp1 to the PPA1 promoter was confirmed by Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay. Importance of these binding sites was verified by site-directed mutagenesis and overexpression of Sp1 transactivates PPA1 promoter activity, upregulates protein expression and increases chromatin accessibility. p300 binds to the PPA1 promoter and stimulates Sp1 induced promoter activity. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor induces PPA1 promoter activity and protein expression and HAT activity of p300 was important in regulation of PPA1 expression. These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter. Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

No MeSH data available.


Related in: MedlinePlus

Effect of PPA1 knockdown on the colony formation and cell viability in MCF7 cells.(A) Representative picture of colony formation assay in MCF7 cells. PPA1 shRNA and scramble shRNA transfected cells were seeded in 12 well plates and colonies were visualized by crystal violet staining after 2 weeks of puromycin selection. (B) Number of colonies of MCF7 cells transfected with scramble shRNA or, PPA1 shRNA from triplicate wells, p˂0.05. (C) Effect of PPA1 shRNA on the cell survival. MCF7 cells transfected with scramble shRNA or PPA1 shRNA were seeded in 24 well plates in triplicate and selected with puromycin. Cell viability was analyzed by the MTT assay at 24 h and 48 h of puromycin selection. Data represents mean ± S.E.M of two independent experiments, p˂0.05. (D) Western blot showing knockdown of PPA1 after 48 h of transfection of PPA1 shRNA in MCF7 cells.
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pone.0124864.g006: Effect of PPA1 knockdown on the colony formation and cell viability in MCF7 cells.(A) Representative picture of colony formation assay in MCF7 cells. PPA1 shRNA and scramble shRNA transfected cells were seeded in 12 well plates and colonies were visualized by crystal violet staining after 2 weeks of puromycin selection. (B) Number of colonies of MCF7 cells transfected with scramble shRNA or, PPA1 shRNA from triplicate wells, p˂0.05. (C) Effect of PPA1 shRNA on the cell survival. MCF7 cells transfected with scramble shRNA or PPA1 shRNA were seeded in 24 well plates in triplicate and selected with puromycin. Cell viability was analyzed by the MTT assay at 24 h and 48 h of puromycin selection. Data represents mean ± S.E.M of two independent experiments, p˂0.05. (D) Western blot showing knockdown of PPA1 after 48 h of transfection of PPA1 shRNA in MCF7 cells.

Mentions: To study the role of PPA1 knockdown in MCF7 cell growth or survival, colony formation assay was carried out. As shown in Fig 6A, knockdown of PPA1 significantly decreased the colony forming ability compared to control cells. Further, down regulation of PPA1 on cell viability was studied by MTT assay and as shown in Fig 6C, knockdown of PPA1 significantly reduced cell viability compared to control cells after 48 h of puromycin selection. Results of both the experiments were in agreement with each other.


Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells.

Mishra DR, Chaudhary S, Krishna BM, Mishra SK - PLoS ONE (2015)

Effect of PPA1 knockdown on the colony formation and cell viability in MCF7 cells.(A) Representative picture of colony formation assay in MCF7 cells. PPA1 shRNA and scramble shRNA transfected cells were seeded in 12 well plates and colonies were visualized by crystal violet staining after 2 weeks of puromycin selection. (B) Number of colonies of MCF7 cells transfected with scramble shRNA or, PPA1 shRNA from triplicate wells, p˂0.05. (C) Effect of PPA1 shRNA on the cell survival. MCF7 cells transfected with scramble shRNA or PPA1 shRNA were seeded in 24 well plates in triplicate and selected with puromycin. Cell viability was analyzed by the MTT assay at 24 h and 48 h of puromycin selection. Data represents mean ± S.E.M of two independent experiments, p˂0.05. (D) Western blot showing knockdown of PPA1 after 48 h of transfection of PPA1 shRNA in MCF7 cells.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0124864.g006: Effect of PPA1 knockdown on the colony formation and cell viability in MCF7 cells.(A) Representative picture of colony formation assay in MCF7 cells. PPA1 shRNA and scramble shRNA transfected cells were seeded in 12 well plates and colonies were visualized by crystal violet staining after 2 weeks of puromycin selection. (B) Number of colonies of MCF7 cells transfected with scramble shRNA or, PPA1 shRNA from triplicate wells, p˂0.05. (C) Effect of PPA1 shRNA on the cell survival. MCF7 cells transfected with scramble shRNA or PPA1 shRNA were seeded in 24 well plates in triplicate and selected with puromycin. Cell viability was analyzed by the MTT assay at 24 h and 48 h of puromycin selection. Data represents mean ± S.E.M of two independent experiments, p˂0.05. (D) Western blot showing knockdown of PPA1 after 48 h of transfection of PPA1 shRNA in MCF7 cells.
Mentions: To study the role of PPA1 knockdown in MCF7 cell growth or survival, colony formation assay was carried out. As shown in Fig 6A, knockdown of PPA1 significantly decreased the colony forming ability compared to control cells. Further, down regulation of PPA1 on cell viability was studied by MTT assay and as shown in Fig 6C, knockdown of PPA1 significantly reduced cell viability compared to control cells after 48 h of puromycin selection. Results of both the experiments were in agreement with each other.

Bottom Line: Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms.These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter.Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Laboratory, Gene function and regulation Group, Institute of Life Sciences, Bhubaneswar, Odisha, India.

ABSTRACT
Cytosolic inorganic pyrophosphatase plays an important role in the cellular metabolism by hydrolyzing inorganic pyrophosphate (PPi) formed as a by-product of various metabolic reactions. Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms. In humans, the expression of inorganic pyrophosphatase (PPA1) is deregulated in different types of cancer and is involved in the migration and invasion of gastric cancer cells and proliferation of ovarian cancer cells. However, the transcriptional regulation of the gene encoding PPA1 is poorly understood. To gain insights into PPA1 gene regulation, a 1217 bp of its 5'-flanking region was cloned and analyzed. The 5'-deletion analysis of the promoter revealed a 266 bp proximal promoter region exhibit most of the transcriptional activity and upon sequence analysis, three putative Sp1 binding sites were found to be present in this region. Binding of Sp1 to the PPA1 promoter was confirmed by Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay. Importance of these binding sites was verified by site-directed mutagenesis and overexpression of Sp1 transactivates PPA1 promoter activity, upregulates protein expression and increases chromatin accessibility. p300 binds to the PPA1 promoter and stimulates Sp1 induced promoter activity. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor induces PPA1 promoter activity and protein expression and HAT activity of p300 was important in regulation of PPA1 expression. These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter. Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

No MeSH data available.


Related in: MedlinePlus