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Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma.

Kong LM, Liao CG, Chen L, Yang HS, Zhang SH, Zhang Z, Bian HJ, Xing JL, Chen ZN - J. Cell. Mol. Med. (2010)

Bottom Line: Significantly higher expression of CD147 and significantly lower promoter methylation level were observed in HCC cell lines compared to normal cell lines and tissues control.Moreover, HCC patients with unmethylated CD147 promoter had a significantly higher recurrence rate (88.1%versus 58.3%; P < 0.05) and death rate (83.3%versus 50.0%; P < 0.05) than patients with methylated CD147 promoter.In conclusions, promoter hypomethylation up-regulates CD147 expression primarily through increasing Sp1 binding and associates with poor prognosis in HCC patients.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cancer Biology, Cell Engineering Research Centre & Department of Cell Biology, Fourth Military Medical University, Xi'an, China.

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Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro. (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro. The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. *P < 0.05.
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fig05: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro. (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro. The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. *P < 0.05.

Mentions: To directly examine if Sp1 binds to the putative binding site within the critical CD147 promoter region, EMSA was performed with biotin-labelled oligonucleotides spanning the Sp1-binding sites as probe. Shifted complexes were observed when the labelled wild-type probe was used (Fig. 5A, lane 2), while these complexes disappeared when 100-fold molar excess of unlabelled wild-type probe was added (Fig. 5A, lane 3). EMSA was also performed by using mutant probes in order to understand the contribution of the Sp1-binding sites to the observed binding patterns. The mutation in the Sp1-binding sites dramatically decreased the level of complex formation (Fig. 5A, lanes 4). To further confirm the specific binding of Sp1 to the CD147 minimal promoter, supershift assay was performed with anti-Sp1 antibody. The protein–DNA complex of interest was supershifted by anti-Sp1 antibody (Fig. 5A, lane 6). However, no supershift was observed when control IgG was used (Fig. 5A, lane 5). We also observed that Sp1 could not bind to the methylated CD147 gene promoter probe (Fig. 5A, lane 7). The EMSA results demonstrated that Sp1 specifically bound to the Sp1-binding sites in the CD147 gene promoter and this binding could be interfered by methylation status.


Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma.

Kong LM, Liao CG, Chen L, Yang HS, Zhang SH, Zhang Z, Bian HJ, Xing JL, Chen ZN - J. Cell. Mol. Med. (2010)

Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro. (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro. The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. *P < 0.05.
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getmorefigures.php?uid=PMC4373337&req=5

fig05: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro. (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro. The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. *P < 0.05.
Mentions: To directly examine if Sp1 binds to the putative binding site within the critical CD147 promoter region, EMSA was performed with biotin-labelled oligonucleotides spanning the Sp1-binding sites as probe. Shifted complexes were observed when the labelled wild-type probe was used (Fig. 5A, lane 2), while these complexes disappeared when 100-fold molar excess of unlabelled wild-type probe was added (Fig. 5A, lane 3). EMSA was also performed by using mutant probes in order to understand the contribution of the Sp1-binding sites to the observed binding patterns. The mutation in the Sp1-binding sites dramatically decreased the level of complex formation (Fig. 5A, lanes 4). To further confirm the specific binding of Sp1 to the CD147 minimal promoter, supershift assay was performed with anti-Sp1 antibody. The protein–DNA complex of interest was supershifted by anti-Sp1 antibody (Fig. 5A, lane 6). However, no supershift was observed when control IgG was used (Fig. 5A, lane 5). We also observed that Sp1 could not bind to the methylated CD147 gene promoter probe (Fig. 5A, lane 7). The EMSA results demonstrated that Sp1 specifically bound to the Sp1-binding sites in the CD147 gene promoter and this binding could be interfered by methylation status.

Bottom Line: Significantly higher expression of CD147 and significantly lower promoter methylation level were observed in HCC cell lines compared to normal cell lines and tissues control.Moreover, HCC patients with unmethylated CD147 promoter had a significantly higher recurrence rate (88.1%versus 58.3%; P < 0.05) and death rate (83.3%versus 50.0%; P < 0.05) than patients with methylated CD147 promoter.In conclusions, promoter hypomethylation up-regulates CD147 expression primarily through increasing Sp1 binding and associates with poor prognosis in HCC patients.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cancer Biology, Cell Engineering Research Centre & Department of Cell Biology, Fourth Military Medical University, Xi'an, China.

Show MeSH
Related in: MedlinePlus