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Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

Sayar N, Karahan G, Konu O, Bozkurt B, Bozdogan O, Yulug IG - Clin Epigenetics (2015)

Bottom Line: TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues.Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells.TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Bilkent University, Faculty of Science, TR-06800 Ankara, Turkey.

ABSTRACT

Background: CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation.

Results: Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells.

Conclusions: TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

No MeSH data available.


Related in: MedlinePlus

Expression and promoter methylation analyses of TAGLN gene in BC cell lines. a Box-plot analysis based on qRT-PCR results showing strong downregulation of TAGLN expression in BC cell lines compared to NTB cell lines. Log2 expression levels relative to GAPDH reference gene are shown. Horizontal dashed lines represent median values. Error bars: standard error of means (SEM). **P < 0.01; Mann–Whitney Test. b qRT-PCR analysis of AZA-treated BC and NTB cell lines indicating high levels of induction in TAGLN expression levels. Values represent the log2 ratios of expression levels in AZA-treated cell lines to those in their DMSO-treated cells. c QUMA software analyses based on bisulfite sequencing of BC and NTB cell lines showing hypermethylation in TAGLN promoter in BC cells. Percent values represent the average methylation of 5 clones for each cell line. Full circles: methylated; Empty circles: unmethylated. Sequence numbers of CpGs are shown above circles. d QUMA analysis showing significantly higher methylation in BC cells compared to NTB; Mann–Whitney test. Methylated DNA ratio: black area; unmethylated ratio: white area in the pie chart. e Scatter plot showing the spearman correlation of TAGLN methylation status with log2(expression) levels in BC and NTB cell lines
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Fig2: Expression and promoter methylation analyses of TAGLN gene in BC cell lines. a Box-plot analysis based on qRT-PCR results showing strong downregulation of TAGLN expression in BC cell lines compared to NTB cell lines. Log2 expression levels relative to GAPDH reference gene are shown. Horizontal dashed lines represent median values. Error bars: standard error of means (SEM). **P < 0.01; Mann–Whitney Test. b qRT-PCR analysis of AZA-treated BC and NTB cell lines indicating high levels of induction in TAGLN expression levels. Values represent the log2 ratios of expression levels in AZA-treated cell lines to those in their DMSO-treated cells. c QUMA software analyses based on bisulfite sequencing of BC and NTB cell lines showing hypermethylation in TAGLN promoter in BC cells. Percent values represent the average methylation of 5 clones for each cell line. Full circles: methylated; Empty circles: unmethylated. Sequence numbers of CpGs are shown above circles. d QUMA analysis showing significantly higher methylation in BC cells compared to NTB; Mann–Whitney test. Methylated DNA ratio: black area; unmethylated ratio: white area in the pie chart. e Scatter plot showing the spearman correlation of TAGLN methylation status with log2(expression) levels in BC and NTB cell lines

Mentions: TAGLN gene expression was analyzed with qRT-PCR and found to be significantly downregulated in 15 different breast carcinoma cell lines compared to the non-tumorigenic cell lines (P = 0.0034, Fig. 2a). The expression of TAGLN was restored in all analyzed cell lines upon AZA treatment, except for NTB cell line MCF12A (Fig. 2b). Expression levels of TAGLN were concordant in microarray and qRT-PCR experiments, when fold induction levels upon AZA treatment were compared in MCF7, MDA-MB-231, and MCF12A cell lines.Fig. 2


Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

Sayar N, Karahan G, Konu O, Bozkurt B, Bozdogan O, Yulug IG - Clin Epigenetics (2015)

Expression and promoter methylation analyses of TAGLN gene in BC cell lines. a Box-plot analysis based on qRT-PCR results showing strong downregulation of TAGLN expression in BC cell lines compared to NTB cell lines. Log2 expression levels relative to GAPDH reference gene are shown. Horizontal dashed lines represent median values. Error bars: standard error of means (SEM). **P < 0.01; Mann–Whitney Test. b qRT-PCR analysis of AZA-treated BC and NTB cell lines indicating high levels of induction in TAGLN expression levels. Values represent the log2 ratios of expression levels in AZA-treated cell lines to those in their DMSO-treated cells. c QUMA software analyses based on bisulfite sequencing of BC and NTB cell lines showing hypermethylation in TAGLN promoter in BC cells. Percent values represent the average methylation of 5 clones for each cell line. Full circles: methylated; Empty circles: unmethylated. Sequence numbers of CpGs are shown above circles. d QUMA analysis showing significantly higher methylation in BC cells compared to NTB; Mann–Whitney test. Methylated DNA ratio: black area; unmethylated ratio: white area in the pie chart. e Scatter plot showing the spearman correlation of TAGLN methylation status with log2(expression) levels in BC and NTB cell lines
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Fig2: Expression and promoter methylation analyses of TAGLN gene in BC cell lines. a Box-plot analysis based on qRT-PCR results showing strong downregulation of TAGLN expression in BC cell lines compared to NTB cell lines. Log2 expression levels relative to GAPDH reference gene are shown. Horizontal dashed lines represent median values. Error bars: standard error of means (SEM). **P < 0.01; Mann–Whitney Test. b qRT-PCR analysis of AZA-treated BC and NTB cell lines indicating high levels of induction in TAGLN expression levels. Values represent the log2 ratios of expression levels in AZA-treated cell lines to those in their DMSO-treated cells. c QUMA software analyses based on bisulfite sequencing of BC and NTB cell lines showing hypermethylation in TAGLN promoter in BC cells. Percent values represent the average methylation of 5 clones for each cell line. Full circles: methylated; Empty circles: unmethylated. Sequence numbers of CpGs are shown above circles. d QUMA analysis showing significantly higher methylation in BC cells compared to NTB; Mann–Whitney test. Methylated DNA ratio: black area; unmethylated ratio: white area in the pie chart. e Scatter plot showing the spearman correlation of TAGLN methylation status with log2(expression) levels in BC and NTB cell lines
Mentions: TAGLN gene expression was analyzed with qRT-PCR and found to be significantly downregulated in 15 different breast carcinoma cell lines compared to the non-tumorigenic cell lines (P = 0.0034, Fig. 2a). The expression of TAGLN was restored in all analyzed cell lines upon AZA treatment, except for NTB cell line MCF12A (Fig. 2b). Expression levels of TAGLN were concordant in microarray and qRT-PCR experiments, when fold induction levels upon AZA treatment were compared in MCF7, MDA-MB-231, and MCF12A cell lines.Fig. 2

Bottom Line: TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues.Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells.TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Bilkent University, Faculty of Science, TR-06800 Ankara, Turkey.

ABSTRACT

Background: CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation.

Results: Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells.

Conclusions: TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

No MeSH data available.


Related in: MedlinePlus