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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

Analyses of AZA affected genes in BC cell lines. a The outline of the gene selection process. The numbers on the left and right represent the probe sets obtained after the filtering process for gene selection in MCF7 and MDA-MB-231 cell lines, respectively. b Number of probe sets significantly up/downregulated by AZA treatment, determined by the selection process given in (a). c, d DAVID analyses of Pathways affected upon AZA treatment in MCF7 cells (c) and MDA-MB 231 cells (d). Bars represent the percentage of up- and downregulated genes involved in the indicated pathways while the lines represent the P values for enrichment. Dashed lines indicate P = 0.05 thresholds
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Fig1: Analyses of AZA affected genes in BC cell lines. a The outline of the gene selection process. The numbers on the left and right represent the probe sets obtained after the filtering process for gene selection in MCF7 and MDA-MB-231 cell lines, respectively. b Number of probe sets significantly up/downregulated by AZA treatment, determined by the selection process given in (a). c, d DAVID analyses of Pathways affected upon AZA treatment in MCF7 cells (c) and MDA-MB 231 cells (d). Bars represent the percentage of up- and downregulated genes involved in the indicated pathways while the lines represent the P values for enrichment. Dashed lines indicate P = 0.05 thresholds

Mentions: We treated the MCF7 and MDA-MB-231 BC cells and a NTB cell line MCF12A with AZA or corresponding DMSO controls before subjecting them to microarray analysis to identify new TSG targets for promoter DNA hypermethylation in breast cancer. Quality control analyses proved our data to be of high quality, and replicates of each experiment correlated well with each other (data not shown). Expressions of large groups of genes were altered upon AZA treatment by 1.5-fold and above, even at very stringent significance thresholds (Additional file 1 and Additional file 4: Table S1). A selection procedure was applied to identify the genes that are altered upon AZA treatment in MCF7 and MDA-MB-231 cells, but not in MCF 12A cells (Additional file 2 and Fig. 1a). Gene lists, generated using this procedure, were used for further analyses (Additional file 3). Probe sets commonly or differentially altered in MCF7 and MDA-MB-231 cell lines were also determined (Fig. 1b). We found that 17 and 22 % of the probe sets induced in MCF7 or MDA-MB-231 cells, respectively, were induced in common while the remaining genes were cell-line specific.Fig. 1


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)

Analyses of AZA affected genes in BC cell lines. a The outline of the gene selection process. The numbers on the left and right represent the probe sets obtained after the filtering process for gene selection in MCF7 and MDA-MB-231 cell lines, respectively. b Number of probe sets significantly up/downregulated by AZA treatment, determined by the selection process given in (a). c, d DAVID analyses of Pathways affected upon AZA treatment in MCF7 cells (c) and MDA-MB 231 cells (d). Bars represent the percentage of up- and downregulated genes involved in the indicated pathways while the lines represent the P values for enrichment. Dashed lines indicate P = 0.05 thresholds
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4587865&req=5

Fig1: Analyses of AZA affected genes in BC cell lines. a The outline of the gene selection process. The numbers on the left and right represent the probe sets obtained after the filtering process for gene selection in MCF7 and MDA-MB-231 cell lines, respectively. b Number of probe sets significantly up/downregulated by AZA treatment, determined by the selection process given in (a). c, d DAVID analyses of Pathways affected upon AZA treatment in MCF7 cells (c) and MDA-MB 231 cells (d). Bars represent the percentage of up- and downregulated genes involved in the indicated pathways while the lines represent the P values for enrichment. Dashed lines indicate P = 0.05 thresholds
Mentions: We treated the MCF7 and MDA-MB-231 BC cells and a NTB cell line MCF12A with AZA or corresponding DMSO controls before subjecting them to microarray analysis to identify new TSG targets for promoter DNA hypermethylation in breast cancer. Quality control analyses proved our data to be of high quality, and replicates of each experiment correlated well with each other (data not shown). Expressions of large groups of genes were altered upon AZA treatment by 1.5-fold and above, even at very stringent significance thresholds (Additional file 1 and Additional file 4: Table S1). A selection procedure was applied to identify the genes that are altered upon AZA treatment in MCF7 and MDA-MB-231 cells, but not in MCF 12A cells (Additional file 2 and Fig. 1a). Gene lists, generated using this procedure, were used for further analyses (Additional file 3). Probe sets commonly or differentially altered in MCF7 and MDA-MB-231 cell lines were also determined (Fig. 1b). We found that 17 and 22 % of the probe sets induced in MCF7 or MDA-MB-231 cells, respectively, were induced in common while the remaining genes were cell-line specific.Fig. 1

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