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Molecular Subtype-Specific Expression of MicroRNA-29c in Breast Cancer Is Associated with CpG Dinucleotide Methylation of the Promoter.

Poli E, Zhang J, Nwachukwu C, Zheng Y, Adedokun B, Olopade OI, Han YJ - PLoS ONE (2015)

Bottom Line: MicroRNA-29c (miR-29c) has been shown to be significantly down-regulated in basal-like breast tumors and to be involved in cell invasion and sensitivity to chemotherapy.Bisulfite sequencing of CpG sites in the miR-29c promoter region showed higher methylation in basal-like breast cancer cell lines compared to luminal subtype cells with a significant inverse correlation between expression and methylation of miR-29c.Analysis of primary breast tumors using The Cancer Genome Atlas (TCGA) dataset confirmed significantly higher levels of methylation of the promoter in basal-like breast tumors compared to all other subtypes.

View Article: PubMed Central - PubMed

Affiliation: Center for Clinical Cancer Genetics and Global Health, Section of Hematology/Oncology, Department of Medicine, University of Chicago, Chicago, IL, United States of America.

ABSTRACT
Basal-like breast cancer is a molecularly distinct subtype of breast cancer that is highly aggressive and has a poor prognosis. MicroRNA-29c (miR-29c) has been shown to be significantly down-regulated in basal-like breast tumors and to be involved in cell invasion and sensitivity to chemotherapy. However, little is known about the genetic and regulatory factors contributing to the altered expression of miR-29c in basal-like breast cancer. We here report that epigenetic modifications at the miR-29c promoter, rather than copy number variation of the gene, may drive the lower expression of miR-29c in basal-like breast cancer. Bisulfite sequencing of CpG sites in the miR-29c promoter region showed higher methylation in basal-like breast cancer cell lines compared to luminal subtype cells with a significant inverse correlation between expression and methylation of miR-29c. Analysis of primary breast tumors using The Cancer Genome Atlas (TCGA) dataset confirmed significantly higher levels of methylation of the promoter in basal-like breast tumors compared to all other subtypes. Furthermore, inhibition of CpG methylation with 5-aza-CdR increases miR-29c expression in basal-like breast cancer cells. Flourescent In Situ Hybridization (FISH) revealed chromosomal abnormalities at miR-29c loci in breast cancer cell lines, but with no correlation between copy number variation and expression of miR-29c. Our data demonstrated that dysregulation of miR-29c in basal-like breast cancer cells may be in part driven by methylation at CpG sites. Epigenetic control of the miR-29c promoter by epigenetic modifiers may provide a potential therapeutic target to overcome the aggressive behavior of these cancers.

No MeSH data available.


Related in: MedlinePlus

Analyses and Identification of MiR-29c Promoter.(a). UCSC genome browser shows location of the miR-29c/29b2 gene at chromosome 1q32. Two primary transcripts are shown with GenBank ID, EU154352 and EU154352. Potential promoter region located 20 kb (region A) or 1Kb (region B) upstream of the gene with 6 or 1 CAGE tags, respectively, were indicated with red boxes. The enhancer (H3K4me1) and promoter (HeK4Me3) markers of histone modifications from ENCODE are also shown. (b). ChIP against RNA Polymerase II (Pol II) revealed enrichment of Pol II at the 20kb-upstream promoter (region A). PCR were performed using two primer sets (primer set 1 and 2) targeting at each promoter. ChIP without any antibody (No Ab) or with an antibody against Mouse IgG was used as a negative control. (c) Luciferase activity was measured in MDA-MB-231 cells transfected with pGL3-Basic luciferase reporter vectors or miR-29c promoter-luciferase constructs. The means±SD from three experiments are shown.
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pone.0142224.g002: Analyses and Identification of MiR-29c Promoter.(a). UCSC genome browser shows location of the miR-29c/29b2 gene at chromosome 1q32. Two primary transcripts are shown with GenBank ID, EU154352 and EU154352. Potential promoter region located 20 kb (region A) or 1Kb (region B) upstream of the gene with 6 or 1 CAGE tags, respectively, were indicated with red boxes. The enhancer (H3K4me1) and promoter (HeK4Me3) markers of histone modifications from ENCODE are also shown. (b). ChIP against RNA Polymerase II (Pol II) revealed enrichment of Pol II at the 20kb-upstream promoter (region A). PCR were performed using two primer sets (primer set 1 and 2) targeting at each promoter. ChIP without any antibody (No Ab) or with an antibody against Mouse IgG was used as a negative control. (c) Luciferase activity was measured in MDA-MB-231 cells transfected with pGL3-Basic luciferase reporter vectors or miR-29c promoter-luciferase constructs. The means±SD from three experiments are shown.

Mentions: To determine if epigenetic modifications at the miR-29c promoter drives the lower expression of miR-29c in basal-like breast cancer, we first identified and characterized the miR-29c promoter in breast cancer cells. Using CAGE tag data, which is based on preparation and sequencing of concatemers of DNA tags derived from the initial 20 nucleotides from the 5′ end of mRNA, we were able to identify two areas of upstream of the miR-29b-2/29c gene that were potential TSS of miR-29c/29b2 (Fig 2A). Region A (~20 kb upstream of miR-29c) had six CAGE tags, while region B (~1kb upstream) had one CAGE tag. When we analyzed chromatin signatures of transcriptional promoters and enhancers [30, 31] from ENCODE dataset, we found the presence of a marker of histone modification (H3K4me1) in the regions both regions A and B. In contrast, an active promoter marker of histone modification (H3K4me3) was only identified in region A (Fig 2A). To validate in silico analysis experimentally, we performed ChIP assays using antibodies against RNA polymerase II and mouse IgG, followed by PCR with two independent primer sets targeting at each region. Enrichment of RNA polymerase II was observed on the region A (lanes 6 & 7, Fig 2B) with no enrichment in the absence of antibody (lanes 2–3, Fig 2B) or in the presence of mouse IgG controls (lanes 4–5, Fig 2B). Region B showed weak enrichment of RNA polymerase II only with primer set 1(lane 13, Fig 2B).


Molecular Subtype-Specific Expression of MicroRNA-29c in Breast Cancer Is Associated with CpG Dinucleotide Methylation of the Promoter.

Poli E, Zhang J, Nwachukwu C, Zheng Y, Adedokun B, Olopade OI, Han YJ - PLoS ONE (2015)

Analyses and Identification of MiR-29c Promoter.(a). UCSC genome browser shows location of the miR-29c/29b2 gene at chromosome 1q32. Two primary transcripts are shown with GenBank ID, EU154352 and EU154352. Potential promoter region located 20 kb (region A) or 1Kb (region B) upstream of the gene with 6 or 1 CAGE tags, respectively, were indicated with red boxes. The enhancer (H3K4me1) and promoter (HeK4Me3) markers of histone modifications from ENCODE are also shown. (b). ChIP against RNA Polymerase II (Pol II) revealed enrichment of Pol II at the 20kb-upstream promoter (region A). PCR were performed using two primer sets (primer set 1 and 2) targeting at each promoter. ChIP without any antibody (No Ab) or with an antibody against Mouse IgG was used as a negative control. (c) Luciferase activity was measured in MDA-MB-231 cells transfected with pGL3-Basic luciferase reporter vectors or miR-29c promoter-luciferase constructs. The means±SD from three experiments are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4634951&req=5

pone.0142224.g002: Analyses and Identification of MiR-29c Promoter.(a). UCSC genome browser shows location of the miR-29c/29b2 gene at chromosome 1q32. Two primary transcripts are shown with GenBank ID, EU154352 and EU154352. Potential promoter region located 20 kb (region A) or 1Kb (region B) upstream of the gene with 6 or 1 CAGE tags, respectively, were indicated with red boxes. The enhancer (H3K4me1) and promoter (HeK4Me3) markers of histone modifications from ENCODE are also shown. (b). ChIP against RNA Polymerase II (Pol II) revealed enrichment of Pol II at the 20kb-upstream promoter (region A). PCR were performed using two primer sets (primer set 1 and 2) targeting at each promoter. ChIP without any antibody (No Ab) or with an antibody against Mouse IgG was used as a negative control. (c) Luciferase activity was measured in MDA-MB-231 cells transfected with pGL3-Basic luciferase reporter vectors or miR-29c promoter-luciferase constructs. The means±SD from three experiments are shown.
Mentions: To determine if epigenetic modifications at the miR-29c promoter drives the lower expression of miR-29c in basal-like breast cancer, we first identified and characterized the miR-29c promoter in breast cancer cells. Using CAGE tag data, which is based on preparation and sequencing of concatemers of DNA tags derived from the initial 20 nucleotides from the 5′ end of mRNA, we were able to identify two areas of upstream of the miR-29b-2/29c gene that were potential TSS of miR-29c/29b2 (Fig 2A). Region A (~20 kb upstream of miR-29c) had six CAGE tags, while region B (~1kb upstream) had one CAGE tag. When we analyzed chromatin signatures of transcriptional promoters and enhancers [30, 31] from ENCODE dataset, we found the presence of a marker of histone modification (H3K4me1) in the regions both regions A and B. In contrast, an active promoter marker of histone modification (H3K4me3) was only identified in region A (Fig 2A). To validate in silico analysis experimentally, we performed ChIP assays using antibodies against RNA polymerase II and mouse IgG, followed by PCR with two independent primer sets targeting at each region. Enrichment of RNA polymerase II was observed on the region A (lanes 6 & 7, Fig 2B) with no enrichment in the absence of antibody (lanes 2–3, Fig 2B) or in the presence of mouse IgG controls (lanes 4–5, Fig 2B). Region B showed weak enrichment of RNA polymerase II only with primer set 1(lane 13, Fig 2B).

Bottom Line: MicroRNA-29c (miR-29c) has been shown to be significantly down-regulated in basal-like breast tumors and to be involved in cell invasion and sensitivity to chemotherapy.Bisulfite sequencing of CpG sites in the miR-29c promoter region showed higher methylation in basal-like breast cancer cell lines compared to luminal subtype cells with a significant inverse correlation between expression and methylation of miR-29c.Analysis of primary breast tumors using The Cancer Genome Atlas (TCGA) dataset confirmed significantly higher levels of methylation of the promoter in basal-like breast tumors compared to all other subtypes.

View Article: PubMed Central - PubMed

Affiliation: Center for Clinical Cancer Genetics and Global Health, Section of Hematology/Oncology, Department of Medicine, University of Chicago, Chicago, IL, United States of America.

ABSTRACT
Basal-like breast cancer is a molecularly distinct subtype of breast cancer that is highly aggressive and has a poor prognosis. MicroRNA-29c (miR-29c) has been shown to be significantly down-regulated in basal-like breast tumors and to be involved in cell invasion and sensitivity to chemotherapy. However, little is known about the genetic and regulatory factors contributing to the altered expression of miR-29c in basal-like breast cancer. We here report that epigenetic modifications at the miR-29c promoter, rather than copy number variation of the gene, may drive the lower expression of miR-29c in basal-like breast cancer. Bisulfite sequencing of CpG sites in the miR-29c promoter region showed higher methylation in basal-like breast cancer cell lines compared to luminal subtype cells with a significant inverse correlation between expression and methylation of miR-29c. Analysis of primary breast tumors using The Cancer Genome Atlas (TCGA) dataset confirmed significantly higher levels of methylation of the promoter in basal-like breast tumors compared to all other subtypes. Furthermore, inhibition of CpG methylation with 5-aza-CdR increases miR-29c expression in basal-like breast cancer cells. Flourescent In Situ Hybridization (FISH) revealed chromosomal abnormalities at miR-29c loci in breast cancer cell lines, but with no correlation between copy number variation and expression of miR-29c. Our data demonstrated that dysregulation of miR-29c in basal-like breast cancer cells may be in part driven by methylation at CpG sites. Epigenetic control of the miR-29c promoter by epigenetic modifiers may provide a potential therapeutic target to overcome the aggressive behavior of these cancers.

No MeSH data available.


Related in: MedlinePlus